Tracing the genetic footprints of vertebrate landing in non-teleost ray-finned fishes.

Rich fossil evidence suggests that many traits and functions related to terrestrial evolution were present long before the ancestor of lobe- and ray-finned fishes. Here, we present genome sequences of the bichir, paddlefish, bowfin, and alligator gar, covering all major early divergent lineages of ray-finned fishes. Our analyses show that these species exhibit many mosaic genomic features of lobe- and ray-finned fishes. In particular, many regulatory elements for limb development are present in these fishes, supporting the hypothesis that the relevant ancestral regulation networks emerged before the origin of tetrapods. Transcriptome analyses confirm the homology between the lung and swim bladder and reveal the presence of functional lung-related genes in early ray-finned fishes. Furthermore, we functionally validate the essential role of a jawed vertebrate highly conserved element for cardiovascular development. Our results imply the ancestors of jawed vertebrates already had the potential gene networks for cardio-respiratory systems supporting air breathing.

African lungfish genome sheds light on the vertebrate water-to-land transition.

Lungfishes are the closest extant relatives of tetrapods and preserve ancestral traits linked with the water-to-land transition. However, their huge genome sizes have hindered understanding of this key transition in evolution. Here, we report a 40-Gb chromosome-level assembly of the African lungfish (Protopterus annectens) genome, which is the largest genome assembly ever reported and has a contig and chromosome N50 of 1.60 Mb and 2.81 Gb, respectively. The large size of the lungfish genome is due mainly to retrotransposons. Genes with ultra-long length show similar expression levels to other genes, indicating that lungfishes have evolved high transcription efficacy to keep gene expression balanced. Together with transcriptome and experimental data, we identified potential genes and regulatory elements related to such terrestrial adaptation traits as pulmonary surfactant, anxiolytic ability, pentadactyl limbs, and pharyngeal remodeling. Our results provide insights and key resources for understanding the evolutionary pathway leading from fishes to humans.

Genomic insights into the seawater adaptation in Cyprinidae.

BACKGROUND: Cyprinidae, the largest fish family, encompasses approximately 367 genera and 3006 species. While they exhibit remarkable adaptability to diverse aquatic environments, it is exceptionally rare to find them in seawater, with the Far Eastern daces being of few exceptions. Therefore, the Far Eastern daces serve as a valuable model for studying the genetic mechanisms underlying seawater adaptation in Cyprinidae. RESULTS: Here, we sequenced the chromosome-level genomes of two Far Eastern daces (Pseudaspius brandtii and P. hakonensis), the two known cyprinid fishes found in seawater, and performed comparative genomic analyses to investigate their genetic mechanism of seawater adaptation. Demographic history reconstruction of the two species reveals that their population dynamics are correlated with the glacial-interglacial cycles and sea level changes. Genomic analyses identified Pseudaspius-specific genetic innovations related to seawater adaptation, including positively selected genes, rapidly evolving genes, and conserved non-coding elements (CNEs). Functional assays of Pseudaspius-specific variants of the prolactin (prl) gene showed enhanced cell adaptation to greater osmolarity. Functional assays of Pseudaspius specific CNEs near atg7 and usp45 genes suggest that they exhibit higher promoter activity and significantly induced at high osmolarity. CONCLUSIONS: Our results reveal the genome-wide evidence for the evolutionary adaptation of cyprinid fishes to seawater, offering valuable insights into the molecular mechanisms supporting the survival of migratory fish in marine environments. These findings are significant as they contribute to our understanding of how cyprinid fishes navigate and thrive in diverse aquatic habitats, providing useful implications for the conservation and management of marine ecosystems.

Multi-omics Investigation of Freeze Tolerance in the Amur Sleeper, an Aquatic Ectothermic Vertebrate.

Freeze tolerance, the ability of an organism to survive internal ice formation, is a striking survival strategy employed by some ectotherms living in cold environments. However, the genetic bases of this remarkable adaptation are largely unknown. The Amur sleeper (Perccottus glenii), the only known freeze-tolerant fish species, can overwinter with its entire body frozen in ice. Here, we sequenced the chromosome-level genome of the Amur sleeper and performed comparative genomic, transcriptomic, and metabolomic analyses to investigate its strategies for surviving freezing. Evolutionary analysis suggested that the Amur sleeper diverged from its closest non-cold-hardy relative about 15.07 million years ago and has experienced a high rate of protein evolution. Transcriptomic and metabolomic data identified a coordinated and tissue-specific regulation of genes and metabolites involved in hypometabolism, cellular stress response, and cryoprotectant accumulation involved in freezing and thawing. Several genes show evidence of accelerated protein sequence evolution or family size expansion were found as adaptive responses to freezing-induced stresses. Specifically, genetic changes associated with cytoskeleton stability, cryoprotectant synthesis, transmembrane transport, and neuroprotective adaptations were identified as potentially key innovations that aid in freezing survival. Our work provides valuable resources and opportunities to unveil the molecular adaptations supporting freeze tolerance in ectothermic vertebrates.

The Role of Introgression During the Radiation of Endemic Fishes Adapted to Living at Extreme Altitudes in the Tibetan Plateau.

Recent genomic analyses of evolutionary radiations suggest that ancient introgression may facilitate rapid diversification and adaptive radiation. The loach genus Triplophysa, a genus with most species endemic to Tibetan Plateau, shows ecological diversity and rapid evolution and represents a potential example of adaptive radiation linked to the uplift of the Tibetan Plateau. Here, we interrogate the complex evolutionary history of Triplophysa fishes through the analysis of whole-genome sequences. By reconstructing the phylogeny of Triplophysa, quantifying introgression across this clade, and simulating speciation and migration processes, we confirm that extensive gene flow events occurred across disparate Triplophysa species. Our results suggest that introgression plays a more substantial role than incomplete lineage sorting in underpinning phylogenetic discordance in Triplophysa. The results also indicate that genomic regions affected by ancient gene flow exhibit characteristics of lower recombination rates and nucleotide diversity and may associate with selection. Simulation analysis of Triplophysa tibetana suggests that the species may have been affected by the Gonghe Movement in the third uplift of the Tibetan Plateau, resulting in founder effects and a subsequent reduction in Ne.

Phylogenomics of the Ancient and Species-Depauperate Gars Tracks 150 Million Years of Continental Fragmentation in the Northern Hemisphere.

Reconstructing deep-time biogeographic histories is limited by the comparatively recent diversification of most extant lineages. Ray-finned fishes, which include nearly half of all living vertebrates, are no exception. Although most lineages of ray-finned fishes radiated around the Cretaceous-Paleogene boundary, a handful of ancient, species-poor clades still persist. These lineages can illuminate very old biogeographic trends, but their low species richness can also limit the reconstruction of these patterns. The seven extant species of gars distributed in freshwater habitats in North America and Cuba are an old clade with a fossil record spanning over 150 million years of Earth history. Using a genomic data set of DNA sequences of 1105 exons for the seven living species and an updated morphological matrix of all extant and extinct taxa, we infer the phylogenetic relationships of gars and test how divergence times and biogeographic reconstructions are influenced by sequential and joint estimation and the effect on these inferences when using different taxon sets based on fossil completeness. Our analyses consistently show that the two extant gar genera Atractosteus and Lepisosteus diverged approximately 105 million years ago and many of the inferred divergences in the gar time-calibrated phylogeny closely track major Mesozoic tectonic events, including the separation of the Americas, the expansion of the early Atlantic, and the Cretaceous reorganization of North American river systems. The crown clades Atractosteus and Lepisosteus originated in the Cenozoic of eastern North America, implying that this region has served as both the origin of extant gar diversity and the refugium of this iconic ancient lineage. These results exemplify how combining phylogenomics with the fossil record provides congruence around the evolutionary history of ancient clades like gars and can reveal long-lost biogeographic patterns. [Lepisosteidae; fossilized birth death; biogeography; phylogenomics; paleontology.].

Genomic and functional evidence reveals convergent evolution in fishes on the Tibetan Plateau.

High-altitude environments are strong drivers of adaptive evolution in endemic organisms. However, little is known about the genetic mechanisms of convergent adaptation among different lineages, especially in fishes. There are three independent fish groups on the Tibetan Plateau: Tibetan Loaches, Schizothoracine fishes and Glyptosternoid fishes; all are well adapted to the harsh environmental conditions. They represent an excellent example of convergent evolution but with an unclear genetic basis. We used comparative genomic analyses between Tibetan fishes and fishes from low altitudes and detected genomic signatures of convergent evolution in fishes on the Tibetan Plateau. The Tibetan fishes exhibited genome-wide accelerated evolution in comparison with a control set of fishes from low altitudes. A total of 368 positively selected genes were identified in Tibetan fishes, which were enriched in functional categories related to energy metabolism and hypoxia response. Widespread parallel amino acid substitutions were detected among the Tibetan fishes and a subset of these substitutions occurred in positively selected genes associated with high-altitude adaptation. Functional assays suggested that von Hippel-Lindau (VHL) tumour suppressor genes from Tibetan fishes enhance hypoxia-inducible factor (HIF) activity convergently under hypoxia compared to low-altitude fishes. The results provide genomic and functional evidence supporting convergent genetic mechanisms for high-altitude adaptation in fishes on the Tibetan Plateau.

Comparative mitochondrial genome analysis of Neodontobutis hainanensis and Perccottus glenii reveals conserved genome organization and phylogeny.

To investigate the molecular evolution of mitochondrial genomes among the family Odontobutidae, the complete mitochondrial genomes of Neodontobutis hainanensis and Perccottus glenii were sequenced and compared with seven odontobutids species. The genome organization, base composition, codon usage, and gene arrangement of N. hainanensis exhibited high similarity to P. glenii compared to those of other Odontobutidae species. Reconstructed phylogenetic analyses of Odontobutidae strongly supported that Neodontobutis and Perccottus formed a unifying group sister to Odontobutis. Our molecular dating time revealed that the two species diverged approximately 21.7 Ma during Miocene, later than that of Odontobutis. Selection analyses showed stronger selective constraints in mitochondrial genes for P. glenii. However, two positively selected sites in NADH4 and NADH6 genes were respectively detected in N. hainanensis and P. glenii, indicating that they might evolve different metabolic performance in response to the contrasting environments.

FishDB: an integrated functional genomics database for fishes.

BACKGROUND: Hundreds of genomes and transcriptomes of fish species have been sequenced in recent years. However, fish scholarship currently lacks a comprehensive, integrated, and up-to-date collection of fish genomic data. RESULTS: Here we present FishDB, the first database for fish multi-level omics data, available online at http://fishdb.ihb.ac.cn . The database contains 233 fish genomes, 201 fish transcriptomes, 5841 fish mitochondrial genomes, 88 fish gene sets, 16,239 miRNAs of 65 fishes, 1,330,692 piRNAs and 4852 lncRNAs of Danio rerio, 59,040 Mb untranslated regions (UTR) of 230 fishes, and 31,918 Mb coding sequences (CDS) of 230 fishes. Among these, we newly generated a total of 11 fish genomes and 53 fish transcriptomes. CONCLUSIONS: This release contains over 410,721.67 Mb sequences and provides search functionality, a BLAST server, JBrowse, and PrimerServer modules.

Respiratory Toxicity of Azoxystrobin, Pyraclostrobin and Coumoxystrobin on Chlorella vulgaris.

Azoxystrobin (AZ), pyraclostrobin (PYR) and coumoxystrobin (COU) exert negative impacts on Chlorella vulgaris. Thus, in this study, C. vulgaris was used to assess the respiratory toxicity of AZ, PYR and COU by determining the acute toxicity, complex III activity and ATP viability. The 96 h-EC50 values of AZ, PYR and COU for C. vulgaris were 1.85, 2.21 and 1.62 mg/L, respectively. AZ, PYR and COU exerted significant effects on complex III activity and ATP viability after exposure to 0.71, 1.01 and 1.08 mg/L of the fungicides. The binding potentials of AZ, PYR and COU toward ubiquinone were - 10.44, - 9.31 and - 12.98 kcal/mol, respectively, which had adverse effects on amino acids. These results provided new insight into the potential acute respiratory toxicity mechanisms of these strobilurin fungicides in algae.

Comparative iTRAQ proteomics revealed proteins associated with lobed fin regeneration in Bichirs.

BACKGROUND: Polypterus senegalus can fully regenerate its pectoral lobed fins, including a complex endoskeleton, with remarkable precision. However, despite the enormous potential of this species for use in medical research, its regeneration mechanisms remain largely unknown. METHODS: To identify the differentially expressed proteins (DEPs) during the early stages of lobed fin regeneration in P. senegalus, we performed a differential proteomic analysis using isobaric tag for relative and absolute quantitation (iTRAQ) approach based quantitative proteome from the pectoral lobed fins at 3 time points. Furthermore, we validated the changes in protein expression with multiple-reaction monitoring (MRM) analysis. RESULTS: The experiment yielded a total of 3177 proteins and 15,091 unique peptides including 1006 non-redundant (nr) DEPs. Of these, 592 were upregulated while 349 were downregulated after lobed fin amputation when compared to the original tissue. Bioinformatics analyses showed that the DEPs were mainly associated with Ribosome and RNA transport, metabolic, ECM-receptor interaction, Golgi and endoplasmic reticulum, DNA replication, and Regulation of actin cytoskeleton. CONCLUSIONS: To our knowledge, this is the first proteomic research to investigate alterations in protein levels and affected pathways in bichirs' lobe-fin/limb regeneration. In addition, our study demonstrated a highly dynamic regulation during lobed fin regeneration in P. senegalus. These results not only provide a comprehensive dataset on differentially expressed proteins during the early stages of lobe-fin/limb regeneration but also advance our understanding of the molecular mechanisms underlying lobe-fin/limb regeneration.

Correlated expression of retrocopies and parental genes in zebrafish.

Previous studies of the function and evolution of retrocopies in plants, Drosophila and non-mammalian chordates provided new insights into the origin of novel genes. However, little is known about retrocopies and their parental genes in teleosts, and it remains obscure whether there is any correlation between them. The present study aimed to characterize the spatial and temporal expression profiles of retrogenes and their parental genes based on RNA-Seq data from Danio rerio embryos and tissues from adult. Using a modified pipeline, 306 retrocopies were identified in the zebrafish genome, most of which exhibited ancient retroposition, and 76 of these showed a Ks < 2.0. Expression of a retrocopy is generally expected to present no correlation with its parental gene, as regulatory regions are not part of the retroposition event. Here, this assumption was tested based on RNA-Seq data from eight stages and thirteen tissue types of zebrafish. However, the result suggested that retrocopies displayed correlated expression with their parental genes. The level of correlation was found to decrease during embryogenesis, but to increase slightly within a tissue using Ks as the proxy for the divergence time. Tissue specificity was also observed: retrocopies were found to be expressed at a more specific level compared with their parental genes. Unlike Drosophila, which has sex chromosomes, zebrafish do not show testis-biased expression. Our study elaborated temporal and spatial patterns of expression of retrocopies in zebrafish, examined the correlation between retrocopies and parental genes and analyzed potential source of regulated elements of retrocopies, which lay a foundation for further functional study of retrocopies.

Phylogenomic analyses reveal a molecular signature linked to subterranean adaptation in rodents.

BACKGROUND: Genome-wide signatures of convergent evolution are widely expected but rarely revealed in animals. Subterranean rodent genome and transcriptome data produced by next-generation sequencing facilitate the use of phylogenetic methods to infer non-synonymous and synonymous substitution rates within coding regions, which can reveal changes at the molecular level that are correlated with the dramatic shift from a terrestrial to subterranean habitat. RESULTS: Our study used previously sequenced genome or transcriptome data of two subterranean rodents, the blind mole rat and naked mole rat, and their terrestrial relatives, the mouse and guinea pig, to investigate the genetic basis of rodent subterranean adaptation. An analysis of 4996 orthologous genes revealed that the substitution pace of coding sequences was significantly slower in the blind mole rat than in the mouse, and slower in the naked mole rat than in the guinea pig. The dN/dS ratio was significantly higher in the blind mole rat than in the mouse and in the naked mole rat than in the guinea pig. These patterns are most likely related to the longer generation time and lower effective population size of subterranean rodents caused by subterranean ecological constraints. We also identified some genes and gene ontology (GO) categories that might be candidates for adaptation to subterranean life. CONCLUSIONS: Our study reveals a case of subterranean convergent evolution in rodents that is correlated with change in the pace and mode of molecular evolution observed at the genome scale. We believe that this genomic signature could have also evolved in other cases of subterranean convergence. Additionally, the genes that displayed the most radical changes in their patterns of evolution and their associated GO categories provide a strong basis for further comparative and functional studies, and potentially reveal molecular signatures of adaptation to subterranean life.

Characterization of transcriptome changes in saline stress adaptation on Leuciscus merzbacheri using PacBio Iso-Seq and RNA-Seq.

Leuciscus merzbacheri is a native fish species found exclusively in the Junggar Basin in Xinjiang. It exhibits remarkable adaptability, thriving in varying water conditions such as the saline waters, the semi-saline water, and the freshwater. Despite its significant economic and ecological value, the underlying mechanisms of its remarkable salinity tolerance remain elusive. Our study marks the first time the full-length transcriptome of L. merzbacheri has been reported, utilizing RNA-Seq and PacBio Iso-Seq technologies. We found that the average length of the full-length transcriptome is 1,780 bp, with an N50 length of 2,358 bp. We collected RNA-Seq data from gill, liver, and kidney tissues of L. merzbacheri from both saline water and freshwater environments and conducted comparative analyses across these tissues. Further analysis revealed significant enrichment in several key functional gene categories and signalling pathways related to stress response and environmental adaptation. The findings provide a valuable genetic resource for further investigation into saline-responsive candidate genes, which will deepen our understanding of teleost adaptation to extreme environmental stress. This knowledge is crucial for the future breeding and conservation of native fish species.

The genomic signatures of evolutionary stasis.

Evolutionary stasis characterizes lineages that seldom speciate and show little phenotypic change over long stretches of geological time. Although lineages that appear to exhibit evolutionary stasis are often called living fossils, no single mechanism is thought to be responsible for their slow rates of morphological evolution and low species diversity. Some analyses of molecular evolutionary rates in a handful of living fossil lineages have indicated that these clades exhibit slow rates of genomic change. Here, we investigate mechanisms of evolutionary stasis using a dataset of 1,105 exons for 481 vertebrate species. We demonstrate that two ancient clades of ray-finned fishes classically called living fossils, gars and sturgeons, exhibit the lowest rates of molecular substitution in protein-coding genes among all jawed vertebrates. Comparably low rates of evolution are observed at fourfold degenerate sites in gars and sturgeons, implying a mechanism of stasis decoupled from selection that we speculate is linked to a highly effective DNA repair apparatus. We show that two gar species last sharing common ancestry over 100 million years ago produce morphologically intermediate and fertile hybrids in the wild. This makes gars the oldest naturally hybridizing divergence among eukaryotes and supports a theoretical prediction that slow rates of nucleotide substitution across the genome slow the accumulation of genetic incompatibilities, enabling hybridization across deeply divergent lineages and slowing the rate of speciation over geological timescales. Our results help establish molecular stasis as a barrier to speciation and phenotypic innovation and provide a mechanism to explain the low species diversity in living fossil lineages.

Comparative transcriptomic evidence of physiological changes and potential relationships in vertebrates under different dormancy states.

Dormancy represents a fascinating adaptive strategy for organisms to survive in unforgiving environments. After a period of dormancy, organisms often exhibit exceptional resilience. This period is typically divided into hibernation and aestivation based on seasonal patterns. However, the mechanisms by which organisms adapt to their environments during dormancy, as well as the potential relationships between different states of dormancy, deserve further exploration. Here, we selected Perccottus glenii and Protopterus annectens as the primary subjects to study hibernation and aestivation, respectively. Based on histological and transcriptomic analysis of multiple organs, we discovered that dormancy involved a coordinated functional response across organs. Enrichment analyses revealed noteworthy disparities between the two dormant species in their responses to extreme temperatures. Notably, similarities in gene expression patterns pertaining to energy metabolism, neural activity, and biosynthesis were noted during hibernation, suggesting a potential correlation between hibernation and aestivation. To further explore the relationship between these two phenomena, we analyzed other dormancy-capable species using data from publicly available databases. This comparative analysis revealed that most orthologous genes involved in metabolism, cell proliferation, and neural function exhibited consistent expression patterns during dormancy, indicating that the observed similarity between hibernation and aestivation may be attributable to convergent evolution. In conclusion, this study enhances our comprehension of the dormancy phenomenon and offers new insights into the molecular mechanisms underpinning vertebrate dormancy.

Genome-wide identification, characterization, and expression analysis of lineage-specific genes within zebrafish.

BACKGROUND: The genomic basis of teleost phenotypic complexity remains obscure, despite increasing availability of genome and transcriptome sequence data. Fish-specific genome duplication cannot provide sufficient explanation for the morphological complexity of teleosts, considering the relatively large number of extinct basal ray-finned fishes. RESULTS: In this study, we performed comparative genomic analysis to discover the Conserved Teleost-Specific Genes (CTSGs) and orphan genes within zebrafish and found that these two sets of lineage-specific genes may have played important roles during zebrafish embryogenesis. Lineage-specific genes within zebrafish share many of the characteristics of their counterparts in other species: shorter length, fewer exon numbers, higher GC content, and fewer of them have transcript support. Chromosomal location analysis indicated that neither the CTSGs nor the orphan genes were distributed evenly in the chromosomes of zebrafish. The significant enrichment of immunity proteins in CTSGs annotated by gene ontology (GO) or predicted ab initio may imply that defense against pathogens may be an important reason for the diversification of teleosts. The evolutionary origin of the lineage-specific genes was determined and a very high percentage of lineage-specific genes were generated via gene duplications. The temporal and spatial expression profile of lineage-specific genes obtained by expressed sequence tags (EST) and RNA-seq data revealed two novel properties: in addition to being highly tissue-preferred expression, lineage-specific genes are also highly temporally restricted, namely they are expressed in narrower time windows than evolutionarily conserved genes and are specifically enriched in later-stage embryos and early larval stages. CONCLUSIONS: Our study provides the first systematic identification of two different sets of lineage-specific genes within zebrafish and provides valuable information leading towards a better understanding of the molecular mechanisms of the genomic basis of teleost phenotypic complexity for future studies.

Surviving under pressure.

Genomic analysis has shed light on how hadal snailfish have adapted to living at depths of several thousand metres.

Aestivation induces widespread transcriptional changes in the African lungfish.

Aestivation is a special ability possessed by some animals to cope with hot and dry environments utilizing dormancy. At a macroscopic level, dormant animals stop moving and eating. At the microscopic level, the expression of a large number of genes in these animals is strictly controlled. However, little is known about what changes occur during aestivation, especially in fish. In this study, we used transcriptome analysis to examine what changes occur in the gills and lungs of the African lungfish (Protopterus annectens) during the maintenance phase of aestivation and speculated on their causes. We found that aestivating transcriptomes were highly similar between gills and lungs. We also found that some genes showed differential expression or alternative splicing, which may be associated with different organs. In addition, differential expression analysis revealed that the lungs maintained significantly higher bioactivity during aestivation, which suggests that the main respiratory organ in aestivating lungfish can transform. Our study provides a reference point for studying the relationship between aestivation and hibernation and further increases understanding of aestivation.

Chromosome-level genome assembly and whole-genome resequencing of topmouth culter (Culter alburnus) provide insights into the intraspecific variation of its semi-buoyant and adhesive eggs.

Topmouth culter (Culter alburnus) is an ecologically and economically important species belonging to the subfamily Culterinae that is native to and widespread in East Asia. Intraspecific variation of semi-buoyant and adhesive eggs in topmouth culter provides an ideal opportunity to investigate the genetic mechanisms of spawning habits underlying the adaptive radiation of cyprinids in East Asia. In this study, we present a chromosome-level genome assembly of topmouth culter and re-sequenced 158 individuals from six locations in China covering three geographical groups and two egg type variations. The topmouth culter genome size was 1.05 Gb, with a contig N50 length of 17.8 Mb and anchored onto 24 chromosomes. Phylogenetic analysis showed that the divergence time of the Culterinae was coinciding with the time of initiation of the Asian monsoon intensification. Gene family evolutionary analysis indicated that the expanded gene families in topmouth culter were associated with dietary adaptation. Population-level genetic analysis indicated clear differentiation among the six populations, which were clustered into three distinct clusters, consistent with their geographical divergence. The historical effective population size of topmouth culter correlated with the Tibetan Plateau uplifting according to the demographic history reconstruction. A selective sweep analysis between adhesive and semi-buoyant egg populations revealed the genes associated with the hydration and adhesiveness of eggs, indicating divergent selection towards different hydrological environments. This study offers a high-resolution genetic resource for further studies on evolutionary adaptation, genetic breeding and conservation of topmouth culter, providing insights into the molecular mechanisms for egg type variation of East Asian cyprinids.