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Anan’s rock agama (Laudakia sacra) is a lizard species endemic to the harsh high-altitude environment of the Qinghai–Tibet Plateau, a region characterized by low oxygen tension and high ultraviolet (UV) radiation. To better understand the genetic mechanisms underlying highland adaptation of ectotherms, we assembled a 1.80-Gb L. sacra genome, which contained 284 contigs with an N50 of 20.19 Mb and a BUSCO score of 93.54%. Comparative genomic analysis indicated that mutations in certain genes, including HIF1A, TIE2, and NFAT family members and genes in the respiratory chain, may be common adaptations to hypoxia among high-altitude animals. Compared with lowland reptiles, MLIP showed a convergent mutation in L. sacra and the Tibetan hot-spring snake (Thermophis baileyi), which may affect their hypoxia adaptation. In L. sacra, several genes related to cardiovascular remodeling, erythropoiesis, oxidative phosphorylation, and DNA repair may also be tailored for adaptation to UV radiation and hypoxia. Of note, ERCC6 and MSH2, two genes associated with adaptation to UV radiation in T. baileyi, exhibited L. sacra-specific mutations that may affect peptide function. Thus, this study provides new insights into the potential mechanisms underpinning high-altitude adaptation in ectotherms and reveals certain genetic generalities for animals’ survival on the plateau.

casper has been a widely used transparent mutant of zebrafish. It possesses a combined loss of reflective iridophores and light-absorbing melanophores, which gives rise to its almost transparent trunk throughout larval and adult stages. Nevertheless, genomic causal mutations of this transparent phenotype are poorly defined. To identify the potential genetic basis of this fascinating morphological phenotype, we constructed genome maps by performing genome sequencing of 28 zebrafish individuals including wild-type AB strain, roy orbison (roy), and casper mutants. A total of 4.3 million high-quality and high-confidence homozygous single nucleotide polymorphisms (SNPs) were detected in the present study. We also identified a 6.0-Mb linkage disequilibrium block specifically in both roy and casper that was composed of 39 functional genes, of which the mpv17 gene was potentially involved in the regulation of iridophore formation and maintenance. This is the first report of high-confidence genomic mutations in the mpv17 gene of roy and casper that potentially leads to defective splicing as one major molecular clue for the iridophore loss. Additionally, comparative transcriptomic analyses of skin tissues from the AB, roy and casper groups revealed detailed transcriptional changes of several core genes that may be involved in melanophore and iridophore degeneration. In summary, our updated genome and transcriptome sequencing of the casper and roy mutants provides novel genetic clues for the iridophore loss. These new genomic variation maps will offer a solid genetic basis for expanding the zebrafish mutant database and in-depth investigation into pigmentation of animals.

Chinese white dolphin (Sousa chinensis), also known as the Indo-Pacific humpback dolphin, has been classified as “Vulnerable” on the IUCN Red List of Threatened Species. It is a special cetacean species that lives in tropical and subtropical nearshore waters, with significant differences from other cetaceans. Here, we sequenced and assembled a draft genome of the Chinese white dolphin with a total length of 2.3 Gb and annotation of 18,387 protein-coding genes. Genes from certain expanded families are potentially involved in DNA replication and repairing, suggesting that they may be related to adaptation of this marine mammal to nearshore environments. We also discovered that its historical population had undergone a remarkable bottleneck incident before the Mindel glaciation. In addition, a comparative genomic survey on antihypertensive peptides (AHTPs) among five representative mammals with various residential habitats (such as remarkable differences in exogenous ion concentrations and sea depth) revealed that these small bioactive peptides were highly conserved among these examined mammals, and they had the most abundant hits in collagen subunit proteins, especially for two putative AHTP peptides Gly-Leu-Pro (GLP) and Leu-Gly-Pro (LGP). Our genome assembly will be a valuable resource for further genetic researches on adaptive ecology and conservation biology of cetaceans, and for in-depth investigations into bioactive peptides in aquatic and terrestrial mammals for development of peptide-based drugs to treat various human cardiovascular diseases.

Limbs originated from paired fish fins are an important innovation in Gnathostomata. Many studies have focused on limb development-related genes, of which the T-box transcription factor 4 gene (tbx4) has been considered as one of the most essential factors in the regulation of the hindlimb development. We previously confirmed pelvic fin loss in tbx4-knockout zebrafish. Here, we report a high-quality genome assembly of the Japanese eel (Anguilla japonica), which is an economically important fish without pelvic fins. The assembled genome is 1.13 Gb in size, with a scaffold N50 of 1.03 Mb. In addition, we collected 24 tbx4 sequences from 22 teleost fishes to explore the correlation between tbx4 and pelvic fin evolution. However, we observed complete exon structures of tbx4 in several pelvic-fin-loss species such as Ocean sunfish (Mola mola) and ricefield eel (Monopterus albus). More interestingly, an inversion of a special tbx4 gene cluster (brip1-tbx4-tbx2b- bcas3) occurred twice independently, which coincides with the presence of fin spines. A nonsynonymous mutation (M82L) was identified in the nuclear localization sequence (NLS) of the Japanese eel tbx4. We also examined variation and loss of hindlimb enhancer B (HLEB), which may account for pelvic fin loss in Tetraodontidae and Diodontidae. In summary, we generated a genome assembly of the Japanese eel, which provides a valuable genomic resource to study the evolution of fish tbx4 and helps elucidate the mechanism of pelvic fin loss in teleost fishes. Our comparative genomic studies, revealed for the first time a potential correlation between the tbx4 gene cluster and the evolutionary development of toxic fin spines. Because fin spines in teleosts are usually venoms, this tbx4 gene cluster may facilitate the genetic engineering of toxin-related marine drugs.

Blue tilapia (Oreochromis aureus) has been an economically important fish in Asian countries. It can grow and reproduce in both freshwater and brackish water conditions, whereas it is also considered as a significant invasive species around the world. This species has been widely used as the hybridization parent(s) for tilapia breeding with a major aim to produce novel strains. However, available genomic resources are still limited for this important tilapia species. Here, we for the first time sequenced and assembled a draft genome for a seawater cultured blue tilapia (0.92 Gb), with 97.8% completeness and a scaffold N50 of 1.1 Mb, which suggests a relatively high quality of this genome assembly. We also predicted 23,117 protein-coding genes in the blue tilapia genome. Comparisons of predicted antimicrobial peptides between the blue tilapia and its close relative Nile tilapia proved that these immunological genes are highly similar with a genome-wide scattering distribution. As a valuable genetic resource, our blue tilapia genome assembly will benefit for biomedical researches and practical molecular breeding for high resistance to various diseases, which have been a critical problem in the aquaculture of tilapias.

The Yenyuan stream salamander (Batrachuperus yenyuanensis) has been previously evaluated with regards to phylogeny, population genetics, and hematology, but genomic information is sparse due to the giant genome size of salamanders which contain highly repetitive sequences, thus resulting in the lack of a complete reference genome. This study evaluates the encoding genetic sequences and provides the first transcriptome assembly of Yenyuan stream salamander based on mixed samples from the liver, spermary, muscle and spleen tissues. Using this transcriptome assembly and available encoding sequences from other vertebrates, the gene families, phylogenetic status, and species divergence time were compared or estimated. A total of 13,750 encoding sequences were successfully obtained from the transcriptome assembly of Yenyuan stream salamander, estimated to contain 40.1% of the unigenes represented in tetrapod databases. A total of 88.79% of these genes could be annotated to a biological function by current databases. Through gene family clustering, we found multiple possible isoforms of the Scribble gene—whose function is related to regeneration—based on sequence similarity. Meanwhile, we constructed a robust phylogenetic tree based on 56 single-copy orthologues, which indicates that based on phylogenetic position, the Yenyuan stream salamander presents the closest relationship with the Chinese giant salamander (Andrias davidianus) of the investigated vertebrates. Based on the fossil-calibrated phylogeny, we estimated that the lineage divergence between the ancestral Yenyuan stream salamander and the Chinese giant salamander may have occurred during the Cretaceous period (~78.4 million years ago). In conclusion, this study not only provides a candidate gene that is valuable for exploring the remarkable capacity of regeneration in the future, but also gives an interesting insight into the understanding of Yenyuan stream salamander by this first transcriptome assembly.

Angiotensin-converting enzymes, ACE and ACE2, are two main elements in the renin⁻angiotensin system, with a crucial role in the regulation of blood pressure in vertebrates. Previous studies paid much attention to their physiological functions in model organisms, whereas the studies on other animals and related evolution have been sparse. Our present study performed a comprehensive genomic investigation on ace and ace2 genes in vertebrates. We successfully extracted the nucleotide sequences of ace and ace2 genes from high-quality genome assemblies of 36 representative vertebrates. After construction of their evolutionary tree, we observed that most of the phylogenetic positions are consistent with the species tree; however, certain differences appear in coelacanths and frogs, which may suggest a very slow evolutionary rate in the initial evolution of ace and ace2 in vertebrates. We further compared evolutionary rates within the entire sequences of ace and ace2, and determined that ace2 evolved slightly faster than ace. Meanwhile, we counted that the exon numbers of ace and ace2 in vertebrates are usually 25 and 18 respectively, while certain species may occur exon fusion or disruption to decrease or increase their exon numbers. Interestingly, we found three homologous regions between ace and ace2, suggesting existence of gene duplication during their evolutionary process. In summary, this report provides novel insights into vertebrate ace and ace2 genes through a series of genomic and molecular comparisons.

Antihypertensive peptides (AHTPs) are a group of small peptides with the main role to block key enzymes or receptors in the angiotensin genesis pathway. A great number of AHTPs have been isolated or digested from natural food resources; however, comprehensive studies on comparisons of AHTPs in various species from the perspective of big data are rare. Here, we established a simplified local AHTP database, and performed in situ mapping for high throughput identification of AHTPs with high antihypertensive activity from high-quality whole proteome datasets of 18 fish species. In the 35 identified AHTPs with reported high activity, we observed that Gly-Leu-Pro, Leu-Pro-Gly, and Val-Ser-Val are the major components of fish proteins, and AHTP hit numbers in various species demonstrated a similar distributing pattern. Interestingly, Atlantic salmon (Salmo salar) is in possession of far more abundant AHTPs compared with other fish species. In addition, collagen subunit protein is the largest group with more matching AHTPs. Further exploration of two collagen subunits (col4a5 and col8a1) in more fish species suggested that the hit pattern of these conserved proteins among teleost is almost the same, and their phylogeny is consistent with the evolution of these fish species. In summary, our present study provides basic information for the relationship of AHTPs with fish proteins, which sheds light on rapid discovery of marine drugs or food additives from fish protein hydrolysates to alleviate hypertension.

Bile salt hydrolase (BSH) is a well-known enzyme that has been commonly characterized in probiotic bacteria, as it has cholesterol-lowering effects. However, its molecular investigations are scarce. Here, we build a local database of BSH sequences from Lactobacillaceae (BSH–SDL), and phylogenetic analysis and homology searches were employed to elucidate their comparability and distinctiveness among species. Evolutionary study demonstrates that BSH sequences in BSH–SDL are divided into five groups, named BSH A, B, C, D and E here, which can be the genetic basis for BSH classification and nomenclature. Sequence analysis suggests the differences between BSH-active and BSH-inactive proteins clearly, especially on site 82. In addition, a total of 551 BSHs from 107 species are identified from 451 genomes of 158 Lactobacillaceae species. Interestingly, those bacteria carrying various copies of BSH A or B can be predicted to be potential cholesterol-lowering probiotics, based on the results of phylogenetic analysis and the subtypes that those previously reported BSH-active probiotics possess. In summary, this study elaborates the molecular basis of BSH in Lactobacillaceae systematically, and provides a novel methodology as well as a consistent standard for the identification of the BSH subtype. We believe that high-throughput screening can be efficiently applied to the selection of promising candidate BSH-active probiotics, which will advance the development of healthcare products in cholesterol metabolism.

Melatonin is a pleiotropic molecule with various important physiological roles in vertebrates. l-aromatic amino acid decarboxylase (AAAD) is the second enzyme for melatonin synthesis. By far, a clear-cut gene function of AAAD in the biosynthesis of melatonin has been unclear in vertebrates. Here, we provide novel insights into the evolution of AAAD based on 77 vertebrate genomes. According to our genome-wide alignments, we extracted a total of 151 aaad nucleotide sequences. A phylogenetic tree was constructed on the basis of these sequences and corresponding protein alignments, indicating that tetrapods and diploid bony fish genomes contained one aaad gene and a new aaad-like gene, which formed a novel AAAD family. However, in tetraploid teleosts, there were two copies of the aaad gene due to whole genome duplication. A subsequent synteny analysis investigated 81 aaad sequences and revealed their collinearity and systematic evolution. Interestingly, we discovered that platypus (Ornithorhynchus anatinus), Atlantic cod (Guadus morhua), Mexican tetra (Astyanax mexicanus), and a Sinocyclocheilus cavefish (S. anshuiensis) have long evolutionary branches in the phylogenetic topology. We also performed pseudogene identification and selection pressure analysis; however, the results revealed a deletion of 37 amino acids in Atlantic cod and premature stop codons in the cave-restricted S. anshuiensis and A. mexicanus, suggesting weakening or disappearing rhythms in these cavefishes. Selective pressure analysis of aaad between platypus and other tetrapods showed that rates of nonsynonymous (Ka) and synonymous (Ks) substitutions were higher when comparing the platypus to other representative tetrapods, indicating that, in this semiaquatic mammal, the aaad gene experienced selection during the process of evolution. In summary, our current work provides novel insights into aaad genes in vertebrates from a genome-wide view.

The family of secretory calcium-binding phosphoproteins (SCPPs) have been considered vital to skeletal tissue mineralization. However, most previous SCPP studies focused on phylogenetically distant animals but not on those closely related species. Here we provide novel insights into the coevolution of SCPP genes and fish scales in 10 species from Otophysi. According to their scale phenotypes, these fishes can be divided into three groups, i.e., scaled, sparsely scaled, and scaleless. We identified homologous SCPP genes in the genomes of these species and revealed an absence of some SCPP members in some genomes, suggesting an uneven evolutionary history of SCPP genes in fishes. In addition, most of these SCPP genes, with the exception of SPP1, individually form one or two gene cluster(s) on each corresponding genome. Furthermore, we constructed phylogenetic trees using maximum likelihood method to estimate their evolution. The phylogenetic topology mostly supports two subclasses in some species, such as Cyprinus carpio, Sinocyclocheilus anshuiensis, S. grahamin, and S. rhinocerous, but not in the other examined fishes. By comparing the gene structures of recently reported candidate genes, SCPP1 and SCPP5, for determining scale phenotypes, we found that the hypothesis is suitable for Astyanax mexicanus, but denied by S. anshuiensis, even though they are both sparsely scaled for cave adaptation. Thus, we conclude that, although different fish species display similar scale phenotypes, the underlying genetic changes however might be diverse. In summary, this paper accelerates the recognition of the SCPP family in teleosts for potential scale evolution.