Seahorses have a specialized morphology that includes a toothless tubular mouth, a body covered with bony plates, a male brood pouch, and the absence of caudal and pelvic fins. Here we report the sequencing and de novo assembly of the genome of the tiger tail seahorse, Hippocampus comes.
Comparative genomic analysis identifies higher protein and nucleotide evolutionary rates in H. Comes compared with other teleost fish genomes. We identified an astacin metalloprotease gene family that has undergone expansion and is highly expressed in the male brood pouch. We also find that the H. Comes genome lacks enamel matrix protein-coding proline/glutamine-rich secretory calcium-binding phosphoprotein genes, which might have led to the loss of mineralized teeth.
Tbx4, a regulator of hindlimb development, is also not found in H. Comes genome. Knockout of tbx4 in zebrafish showed a ‘pelvic fin-loss’ phenotype similar to that of seahorses. Members of the teleost family Syngnathidae (seahorses, pipefishes and seadragons) , comprising approximately 300 species, display a complex array of morphological innovations and reproductive behaviours. This includes specialized morphological phenotypes such as an elongated snout with a small terminal mouth, fused jaws, absent pelvic and caudal fins, and an extended body covered with an armour of bony plates instead of scales. Syngnathids are also unique among vertebrates due to their ‘male pregnancy’, whereby males nourish developing embryos in a brood pouch until hatching and parturition occurs.
In addition, members of the subfamily Hippocampinae (seahorses) exhibit other derived features such as the lack of a caudal fin, a characteristic prehensile tail, and a vertical body axis. To understand the genetic basis of the specialized morphology and reproductive system of seahorses, we sequenced the genome of the tiger tail seahorse, H. Comes, and carried out comparative genomic analyses with the genome sequences of other ray-finned fishes (Actinopterygii). The genome of a male H. Comes individual was sequenced using the Illumina HiSeq 2000 platform. After filtering low-quality and duplicate reads, 132.13 Gb (approximately 190-fold coverage of the estimated 695 Mb genome) of reads from libraries with insert sizes ranging from 170 bp to 20 kb were retained for assembly. The filtered reads were assembled using SOAPdenovo (version 2.04) to yield a 501.6 Mb assembly with an N50 contig size and N50 scaffold size of 34.7 kb and 1.8 Mb, respectively.
Total RNA from combined soft tissues of H. Comes was sequenced using RNA-sequencing (RNA-seq) and assembled de novo. Comes genome assembly is of high quality, as 99% of the de novo assembled transcripts (76,757 out of 77,040) could be mapped to the assembly; and 243 out of 248 core eukaryotic genes mapping approach (CEGMA) genes are complete in the assembly.We predicted 23,458 genes in the genome of H. Comes based on homology and by mapping the RNA-seq data of H.
Comes and a closely related species, the lined seahorse, Hippocampus erectus, to the genome assembly (see Methods and ). More than 97% of the predicted genes (22,941 genes) either have homologues in public databases (Swissprot, Trembl and the Kyoto Encyclopedia of Genes and Genomes (KEGG)) or are supported by assembled RNA-seq transcripts. Analysis of gene family evolution using a maximum likelihood framework identified an expansion of 25 gene families (261 genes; 1.11%) and contraction of 54 families (96 genes; 0.41%) in the H. Comes lineage ( and ). Transposable elements comprise around 24.8% (124.5 Mb) of the H. Comes genome, with class II DNA transposons being the most abundant class (9%; 45 Mb). Only one wave of transposable element expansion was identified, with no evidence for a recent transposable element burst (Kimura divergence ≤ 5).
The phylogenetic relationships between H. Comes and other teleosts were determined using a genome-wide set of 4,122 one-to-one orthologous genes. The phylogenetic analysis showed that H. Comes is a sister group to other percomorph fishes analysed (stickleback, Gasterosteus aculeatus; medaka, Oryzias latipes; Nile tilapia, Oreochromis niloticus; fugu, Takifugu rubripes; and platyfish, Xiphophorus maculatus) with the exception of blue-spotted mudskipper ( Boleophthalmus pectinirostris), a member of the family Gobiidae. Our inference, which placed the mudskipper as the outgroup, differs from that of a previous phylogenetic analysis based on fewer protein-coding genes that had placed syngnathids as an outgroup. Estimated divergence times of H. Comes and other teleosts calculated using MCMCTree suggest that H.
Comes diverged from the other percomorphs approximately 103.8 million years ago, during the Cretaceous period. Interestingly, the branch length of H. Comes is longer than that of other teleosts, suggesting a higher protein evolutionary rate compared to other teleosts analysed in this study. This result was found to be statistically significant by both relative rate test and two cluster analysis. To determine whether the neutral nucleotide substitution rate of H. Comes is also higher, we generated a neutral tree on the basis of fourfold degenerate sites and calculated the pairwise distance of each teleost to the spotted gar (an outgroup).
The pairwise distance of H. Comes was again higher compared with other teleosts, indicating that the neutral evolutionary rate of H. Comes is also higher than that of other teleosts. The reasons for this higher molecular evolutionary rate in H. Comes are unclear.
Gene loss or loss of function can contribute to evolutionary novelties and can be positively selected for. We identified several genes that are not found in the H.
Comes genome but are found in other sequenced teleost genomes.Secretory calcium-binding phosphoprotein (SCPP) genes encode extracellular matrix proteins that are involved in the formation of mineralized tissues such as bone, dentin, enamel and enameloid. Bony vertebrate genomes encode multiple SCPP genes that can be divided into two groups, the acidic and the proline/glutamine (P/Q)-rich SCPP genes.
Acidic SCPPs regulate the mineralization of collagen scaffolds in bone and dentin whereas the P/Q-rich SCPPs are primarily involved in enamel or enameloid formation. Analysis of the H.
Comes genome and the transcriptomes of H. Erectus showed that both contain two acidic SCPP genes, scpp1 and spp1. However, no intact P/Q-rich gene could be identified. The only P/Q-rich gene present in the H.
Comes genome assembly, scpp5, is represented by only three out of ten exons, indicating that it has become a pseudogene. Seahorses and pipefish (family Syngnathidae) are toothless, a phenomenon known as edentulism. Besides syngnathids, edentulism has occurred convergently in several other vertebrate lineages, the most notable ones being birds, turtles, and some mammals such as baleen whales, pangolins and anteaters. The loss of teeth in birds, turtles and mammals has been attributed to inactivating mutations in one or more P/Q-rich enamel-specific SCPP genes such as Enam, Amel, Ambn and Amtn, and the dentin-specific gene, Dspp. In the case of H. Comes, the complete loss of functional P/Q-rich SCPP genes may explain the loss of mineralized teeth.Animals use their sense of smell, or olfaction, for finding food, mates and avoiding predators.
Olfaction is mediated by olfactory receptors (ORs), which constitute the largest family of G-protein-coupled receptors. We were able to identify in the H. Comes genome a significantly smaller repertoire of OR genes than in other teleosts ( P value.A derived phenotype of seahorse and other syngnathids is the complete lack of pelvic fins. Pelvic fins are homologous to tetrapod hindlimbs and primarily serve a role in body trim and subtle swimming manoeuvres during teleost locomotion.
In addition, pelvic spines have an important role in protection against predators. Pelvic fin loss has occurred independently in several teleost lineages, including Tetraodontidae (for example, pufferfishes), Anguillidae (eels) and Gasterosteidae (some populations of sticklebacks), and is frequently associated with a reduced pressure from predators and/or the evolution of an elongated body plan.
In pufferfish (fugu), pelvic fin loss is associated with a change in the expression pattern of hoxd9a. In freshwater populations of stickleback, the loss of pelvic fins has been demonstrated to be due to deletions in the pelvic fin-specific enhancer of pitx1 (ref. ).Analysis of the H. Comes genome and the transcriptomes of H. Erectus (see, section 2), suggested that tbx4, a transcription factor conserved in jawed vertebrates, is not present in the seahorse genome (, section 9).
To verify this, we carried out degenerate polymerase chain reaction (PCR) using genomic DNA from H. Comes and several other species of syngnathids and some non-syngnathids. While the degenerate primers amplified a fragment of tbx4 from non-syngnathids, they failed to amplify a tbx4 fragment from syngnathid fishes (see, section 9). Tbx4 is a T-box DNA-binding domain-containing transcription factor that acts as a regulator of hindlimb formation in mammals.
Loss of function of this gene in mouse leads to a failure of hindlimb formation, as well as strong pleiotropic defects in lung and placental development. Expression of zebrafish tbx4 specifically in pelvic fins suggests a similar role in appendage patterning in fishes. Given the major role of tbx4 in hindlimb formation in mammals, we hypothesized that its absence in H. Comes might be associated with the loss of pelvic fins. To test this hypothesis, we generated a CRISPR–Cas9 tbx4-knockout mutant zebrafish line. Interestingly, unlike homozygous mouse Tbx4 mutants, which fail to develop a functional allantois, the homozygous zebrafish mutants are viable but completely lack pelvic fins without exhibiting any other gross morphological abnormalities in pectoral or median fins ( and; see also, section 9.3, in particular for additional phenotype analysis). This finding is consistent with the results of a recent study that showed that mutations in tbx4 are associated with the loss of pelvic fins in a naturally occurring zebrafish strain called pelvic finless (see also, section 9.3).
These results show that tbx4 has a role in pelvic fin formation in teleosts and suggests that the loss of pelvic fins in H. Comes may be related to the loss of tbx4. A, Vista plot of conserved elements in the tbx2b- tbx4- brip1 syntenic region in fugu (reference genome), seahorse ( H. Comes), stickleback and zebrafish showing that tbx4 is missing from this locus in seahorse. The blue and red peaks represent conserved exonic and non-coding sequences, respectively.
B, Lateral (top) and ventral view (bottom) of wild-type (WT) and a representative (one out of five) F3 homozygous tbx4-null mutant ( tbx4 −/−) zebrafish. Bottom panel shows a close-up of the pelvic region (dashed lines indicate the approximate zoom region). Scale bar, 1 mm. Pelvic fins are indicated with black or white arrowheads in the wild-type fish. Homozygous tbx4-null mutants entirely lack pelvic fins without showing any other gross morphological defects. Male pregnancy is an evolutionary innovation unique to syngnathids.
In teleosts, the C6AST subfamily of astacin metalloproteases—such as high choriolytic enzyme (HCE) and low choriolytic enzyme (LCE)—are involved in lysing the chorion surrounding the egg, leading to hatching of embryos. A member of this subfamily, patristacin ( pastn), was found to be highly expressed in the brood pouch of pregnant males of the Gulf pipefish, Syngnathus scovelli, leading to the suggestion that this gene may have a role in the evolution of male pregnancy. A pastn gene was also found to be highly expressed in the brood pouch of the male big belly seahorse, H. Abdominalis, during mid- and late pregnancy, suggesting a shared role for this gene in male pregnancy in syngnathids.The H. Comes genome contains six pastn genes ( pastn1 to pastn6; ) organized in a cluster.
To examine their expression patterns in the brood pouch, we carried out RNA-seq analysis at different stages of brood pouch development (see, section 2) in H. Erectus, as this species is easy to obtain and breed in the laboratory. Erectus exhibit very similar reproductive cycles and their coding sequences are highly similar (average identity of 93.3%; determined by aligning H. Erectus RNA-seq transcripts to the H. Comes genome assembly).
We identified orthologues for five of the H. Comes pastn genes ( pastn1, pastn2, pastn3, pastn5 and pastn6) in the RNA-seq transcripts of H. Quantitative reverse transcription PCR (qRT–PCR) analysis of these genes showed that some of them are expressed at significantly higher levels in early- and late-pregnant stages. For example, pastn2 is expressed at significantly higher levels in early- and late-pregnant stages compared to the non-pregnant stage, whereas pastn1 and pastn3 are expressed at significantly higher levels during the late-pregnant stage compared to non-pregnant stage. This expression pattern suggests a role for these pastn genes in brood pouch development and/or hatching of embryos within the brood pouch prior to parturition. A, Astacin gene loci in various ray-finned fish genomes showing expansion of pastn genes in seahorse ( H.
Comes) and c6ast genes in platyfish. Chr, chromosome.
B, The phylogeny of the astacin gene family in ray-finned fishes. Only pastn or c6ast genes shown in a are labelled. Shows an expanded version of the tree with all the genes labelled.
Danio Aequipinnatus Genome Body Size Evolution Chart
C, Expression patterns of pastn genes in relation to 18S ribosomal RNA genes in the brood pouch of male H. Erectus determined by qRT–PCR. All data are expressed as mean ± standard error of mean ( n = 5) and evaluated by one-way analysis of variance (ANOVA) followed by Tukey’s honestly significant difference test for adjusting P values from multiple comparisons (see Methods and for details of methods). The average duration of pregnancy (from fertilization to parturition) is 17 days. The y axis represents expression level in relation to 18S rRNA genes. Pastn1 is expressed at low levels at the non-pregnant stage, which is not clearly visible in the figure due to the large scale used. Corel draw x8 crackeado 2019. Non-pregnant: no embryos in the brood pouch; early pregnant: 2–4 days post-fertilization; late pregnant: 12–14 days post-fertilization.