Chromosome-level genome assembly of hadal snailfish reveals mechanisms of deep-sea adaptation in vertebrates.

As the deepest vertebrate in the ocean, the hadal snailfish (Pseudoliparis swirei), which lives at a depth of 6,000-8,000 m, is a representative case for studying adaptation to extreme environments. Despite some preliminary studies on this species in recent years, including their loss of pigmentation, visual and skeletal calcification genes, and the role of trimethylamine N-oxide in adaptation to high-hydrostatic pressure, it is still unknown how they evolved and why they are among the few vertebrate species that have successfully adapted to the deep-sea environment. Using genomic data from different trenches, we found that the hadal snailfish may have entered and fully adapted to such extreme environments only in the last few million years. Meanwhile, phylogenetic relationships show that they spread into different trenches in the Pacific Ocean within a million years. Comparative genomic analysis has also revealed that the genes associated with perception, circadian rhythms, and metabolism have been extensively modified in the hadal snailfish to adapt to its unique environment. More importantly, the tandem duplication of a gene encoding ferritin significantly increased their tolerance to reactive oxygen species, which may be one of the important factors in their adaptation to high-hydrostatic pressure.

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.

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.

Pseudo-chromosome-length genome assembly for a deep-sea eel Ilyophis brunneus sheds light on the deep-sea adaptation.

High hydrostatic pressure, low temperature, and scarce food supply are the major factors that limit the survival of vertebrates in extreme deep-sea environments. Here, we constructed a high-quality genome of the deep-sea Muddy arrowtooth eel (MAE, Ilyophis brunneus, captured below a depth of 3,500 m) by using Illumina, PacBio, and Hi-C sequencing. We compare it against those of shallow-water eel and other outgroups to explore the genetic basis that underlies the adaptive evolution to deep-sea biomes. The MAE genome was estimated to be 1.47 Gb and assembled into 14 pseudo-chromosomes. Phylogenetic analyses indicated that MAE diverged from its closely related shallow-sea species, European eel, ∼111.9 Mya and experienced a rapid evolution. The genome evolutionary analyses primarily revealed the following: (i) under high hydrostatic pressure, the positively selected gene TUBGCP3 and the expanded family MLC1 may improve the cytoskeleton stability; ACOX1 may enhance the fluidity of cell membrane and maintain transport activity; the expansion of ABCC12 gene family may enhance the integrity of DNA; (ii) positively selected HARS likely maintain the transcription ability at low temperatures; and (iii) energy metabolism under a food-limited environment may be increased by expanded and positively selected genes in AMPK and mTOR signaling pathways.

Monsoon boosted radiation of the endemic East Asian carps.

Major historical events often trigger the rapid flourishing of a few lineages, which in turn shape established biodiversity patterns. How did this process occur and develop? This study provides a window into this issue. The endemic East Asian carps (EEAC) dominated the ichthyofauna of East Asia and exhibited a high degree of adaptation to monsoonal river-lake ecosystems. A series of evidence, including ecogeography, phylogenetics, and macroevolution, suggests that the EEAC is a lineage that arose with the East Asian monsoon and thrived intimately with subsequent monsoon activities. We further deduce the evolution of the EEAC and find that a range of historical events in the monsoon setting (e.g., marine transgression and regression and glacial-interglacial cycle) have further reshaped the distribution patterns of EEAC's members. Comparative genomics analyses reveal that introgressions during the initial period of EEAC radiation and innovations in the regulation of the brain and nervous system may have aided their adaptation to river-lake ecosystems in a monsoon setting, which boosted radiation. Overall, this study strengthens knowledge of the evolutionary patterns of freshwater fishes in East Asia and provides a model case for understanding the impact of major historical events on the evolution of biota.

Sympatric or micro-allopatric speciation in a glacial lake? Genomic islands support neither.

Apparent cases of sympatric speciation may actually be due to micro-allopatric or micro-parapatric speciation. One way to distinguish between these models is to examine the existence and nature of genomic islands of divergence, wherein divergent DNA segments are interspersed with low-divergence segments. Such islands should be rare or absent under micro-allopatric speciation but common in cases of speciation with gene flow. Sympatric divergence of endemic fishes is known from isolated saline, crater, postglacial, and ancient lakes. Two morphologically distinct cyprinid fishes, Gymnocypris eckloni scoliostomus (GS) and G. eckloni eckloni (GE), in a small glacial lake on the Qinghai-Tibet Plateau, Lake Sunmcuo, match the biogeographic criteria of sympatric speciation. In this study, we examined genome-wide variation in 46 individuals from these two groups. The divergence time between the GS and GE lineages was estimated to be 20-60 Kya. We identified 54 large genomic islands (≥100 kb) of speciation, which accounted for 89.4% of the total length of all genomic islands. These islands harboured divergent genes related to olfactory receptors and olfaction signals that may play important roles in food selection and assortative mating in fishes. Although the genomic islands clearly indicated speciation with gene flow and rejected micro-allopatric speciation, they were too large to support the hypothesis of sympatric speciation. Theoretical and recent empirical studies suggested that continual gene flow in sympatry should give rise to many small genomic islands (as small as a few kilobases in size). Thus, the observed pattern is consistent with the extensive evidence on parapatric speciation, in which adjacent habitats facilitate divergent selection but also permit gene flow during speciation. We suggest that many, if not most, of the reported cases of sympatric speciation are likely to be micro-parapatric speciation.

The new chimeric chiron genes evolved essential roles in zebrafish embryonic development by regulating NAD+ levels.

The origination of new genes is important for generating genetic novelties for adaptive evolution and biological diversity. However, their potential roles in embryonic development, evolutionary processes into ancient networks, and contributions to adaptive evolution remain poorly investigated. Here, we identified a novel chimeric gene family, the chiron family, and explored its genetic basis and functional evolution underlying the adaptive evolution of Danioninae fishes. The ancestral chiron gene originated through retroposition of nampt in Danioninae 48-54 million years ago (Mya) and expanded into five duplicates (chiron1-5) in zebrafish 1-4 Mya. The chiron genes (chirons) likely originated in embryonic development and gradually extended their expression in the testis. Functional experiments showed that chirons were essential for zebrafish embryo development. By integrating into the NAD+ synthesis pathway, chirons could directly catalyze the NAD+ rate-limiting reaction and probably impact two energy metabolism genes (nmnat1 and naprt) to be under positive selection in Danioninae fishes. Together, these results mainly demonstrated that the origin of new chimeric chiron genes may be involved in adaptive evolution by integrating and impacting the NAD+ biosynthetic pathway. This coevolution may contribute to the physiological adaptation of Danioninae fishes to widespread and varied biomes in Southeast Asian.

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.

Whole genome sequencing of silver carp (Hypophthalmichthys molitrix) and bighead carp (Hypophthalmichthys nobilis) provide novel insights into their evolution and speciation.

The edible silver carp (Hypophthalmichthys molitrix) and bighead carp (H. nobilis), which are two of the "Four Domesticated Fish" of China, are cultivated intensively worldwide. Here, we constructed 837- and 845-Mb draft genome assemblies for the silver carp and the bighead carp, respectively, including 24,571 and 24,229 annotated protein-coding genes. Genetic maps, anchoring 71.7% and 83.8% of all scaffolds, were obtained for the silver and bighead carp, respectively. Phylogenetic analysis showed that the bighead carp formed a clade with the silver carp, with an estimated divergence time of 3.6 million years ago; the time of divergence between the silver carp and zebrafish was 50.7 million years ago. An East Asian cyprinid genome-specific chromosome fusion took place ~9.2 million years after this clade diverged from the clade containing the common carp and Sinocyclocheilus. KEGG and GO analyses indicated that the expanded gene families in the silver and bighead carp were associated with diseases, the immune system and environmental adaptations. Genomic regions differentiating the silver and bighead carp populations were detected based on the whole-genome sequences of 42 individuals. Genes associated with the divergent regions were associated with reproductive system development and the development of primary female sexual characteristics. Thus, our results provided a novel systematic genomic analysis of the East Asian cyprinids, as well as the evolution and speciation of the silver carp and bighead carp.

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.

Massive Loss of Olfactory Receptors But Not Trace Amine-Associated Receptors in the World's Deepest-Living Fish (Pseudoliparis swirei).

Olfactory receptor repertoires show highly dynamic evolution associated with ecological adaptations in different species. The Mariana snailfish (Pseudoliparis swirei) living below a depth of 6000 m in the Mariana Trench evolved degraded vision and occupies a specific feeding habitat in a dark, low-food environment. However, whether such adaptations involve adaptive changes in the chemosensory receptor repertoire is not known. Here, we conducted a comparative analysis of the olfactory receptor (OR) and trace amine-associated receptor (TAAR) gene repertoires in nine teleosts with a focus on the evolutionary divergence between the Mariana snailfish and its shallow-sea relative, Tanaka's snailfish (Liparis tanakae). We found many fewer functional OR genes and a significantly higher fraction of pseudogenes in the Mariana snailfish, but the numbers of functional TAAR genes in the two species were comparable. Phylogenetic analysis showed that the expansion patterns of the gene families were shared by the two species, but that Mariana snailfish underwent massive gene losses in its OR repertoire. Despite an overall decreased size in OR subfamilies and a reduced number of TAAR subfamilies in the Mariana snailfish, expansion of certain subfamilies was observed. Selective pressure analysis indicated greatly relaxed selective strength in ORs but a slightly enhanced selective strength in TAARs of Mariana snailfish. Overall, our study reveals simplified but specific OR and TAAR repertoires in the Mariana snailfish shaped by natural selection with respect to ecological adaptations in the hadal environment. This is the first study on the chemosensation evolution in vertebrates living in the hadal zone, which could provide new insights into evolutionary adaptation to the hadal environment.

Morphology and genome of a snailfish from the Mariana Trench provide insights into deep-sea adaptation.

It is largely unknown how living organisms-especially vertebrates-survive and thrive in the coldness, darkness and high pressures of the hadal zone. Here, we describe the unique morphology and genome of Pseudoliparis swirei-a recently described snailfish species living below a depth of 6,000 m in the Mariana Trench. Unlike closely related shallow sea species, P. swirei has transparent, unpigmented skin and scales, thin and incompletely ossified bones, an inflated stomach and a non-closed skull. Phylogenetic analyses show that P. swirei diverged from a close relative living near the sea surface about 20 million years ago and has abundant genetic diversity. Genomic analyses reveal that: (1) the bone Gla protein (bglap) gene has a frameshift mutation that may cause early termination of cartilage calcification; (2) cell membrane fluidity and transport protein activity in P. swirei may have been enhanced by changes in protein sequences and gene expansion; and (3) the stability of its proteins may have been increased by critical mutations in the trimethylamine N-oxide-synthesizing enzyme and hsp90 chaperone protein. Our results provide insights into the morphological, physiological and molecular evolution of hadal vertebrates.

DNA barcoding the ichthyofauna of the Yangtze River: Insights from the molecular inventory of a mega-diverse temperate fauna.

Intensification of inland fisheries and aquatic landscape conversion led to a drastic decline of fish populations in the Yangtze River (YR) during the last decades. This situation urges for the development of a large-scale molecular assessment of YR ichthyofauna to further develop standardized methods of molecular identification for conservation and fisheries management purposes. We present here the results of a large-scale campaign to DNA barcode YR freshwater fishes that succeeded in producing 1,424 new DNA barcodes for 123 species. Together with 1,406 sequences mined from BOLD and GenBank, a reference library including 2,830 DNA barcodes for 238 species was compiled. By using four DNA-based species delimitation methods, RESL, ABGD, mPTP and mGMYC, 230 operational taxonomic units (OTUs) were identified and 195 species displayed OTUs that tightly match species boundaries. No barcoding gap was observed; however, and conflicting cases of species and OTU delimitation were identified. A total of 23 species with maximum intraspecific distances above 2% were detected and null genetic distances to the nearest phylogenetic relatives were detected in 11 species. Among those 23 species, 16 were represented by multiple OTUs amounting to 40 OTUs delineated. Several cases of multiple OTUs confined to species boundaries were detected suggesting the presence of overlooked species. A total of 18 OTUs, however, were shared by several species and particularly so for the Qinghai-Tibet plateau endemic species. These results are discussed with reference to previous large-scale DNA barcoding campaign and compared to previous phylogeographic studies in the YR.

Ancient duplications and functional divergence in the interferon regulatory factors of vertebrates provide insights into the evolution of vertebrate immune systems.

Interferon regulatory factors (IRFs) were first discovered as transcription factors that regulate the transcription of human interferon (IFN)-ß. Increasing evidence shows that they might be important players involved in Adaptive immune system (AIS) evolution. Although numbers of IRFs have been identified in chordates, the evolutionary history and functional diversity of this gene family during the early evolution of vertebrates have remained obscure. Using IRF HMM profile and HMMER searches, we identified 148 IRFs in 11 vertebrates and 4 protochordates. For them, we reconstructed the phylogenetic relationships, determined the synteny conservation, investigated the profile of natural selection, and analyzed the expression patterns in four "living fossil" vertebrates: lamprey, elephant shark, coelacanth and bichir. The results from phylogeny and synteny analysis imply that vertebrate IRFs evolved from three predecessors, instead of four as suggested in a previous study, as results from an ancient duplication followed by special expansions and lost during the vertebrate evolution. The profile of natural selection and expression reveals functional dynamics during the process. Together, they suggest that the 2nd whole-genome duplication (2WGD) provided raw materials for innovation in the IRF family, and that the birth of type-I IFN might be an important factor inducing the establishment of IRF-mediated immune networks. As a member involved in the AIS evolution, IRF provide insights into the process and mechanism involved in the complexity and novelties of vertebrate immune systems.

The first complete mitogenome of the South China deep-sea giant isopod Bathynomus sp. (Crustacea: Isopoda: Cirolanidae) allows insights into the early mitogenomic evolution of isopods.

In this study, the complete mitochondrial (mt) genome sequence of the South China deep-sea giant isopod Bathynomus sp. was determined, and this study is the first to explore in detail the mt genome of a deep-sea member of the order Isopoda. This species belongs to the genus Bathynomus, the members of which are saprophagous residents of the deep-sea benthic environment; based on their large size, Bathynomus is included in the "supergiant group" of isopods. The mt genome of Bathynomus sp. is 14,965 bp in length and consists of 13 protein-coding genes, two ribosomal RNA genes, only 18 transfer RNA genes, and a noncoding control region 362 bp in length, which is the smallest control region discovered in Isopoda to date. Although the overall genome organization is typical for metazoans, the mt genome of Bathynomus sp. shows a number of derived characters, such as an inversion of 10 genes when compared to the pancrustacean ground pattern. Rearrangements in some genes (e.g., cob, trnT, nad5, and trnF) are shared by nearly all isopod mt genomes analyzed thus far, and when compared to the putative isopod ground pattern, five rearrangements were found in Bathynomus sp. Two tRNAs exhibit modified secondary structures: The TΨC arm is absent from trnQ, and trnC lacks the DHU. Within the class Malacostraca, trnC arm loss is only found in other isopods. Phylogenetic analysis revealed that Bathynomus sp. (Cymothoida) and Sphaeroma serratum (Sphaeromatidea) form a single clade, although it is unclear whether Cymothoida is monophyletic or paraphyletic. Moreover, the evolutionary rate of Bathynomus sp. (dN/dS [nonsynonymous mutational rate/synonymous mutational rate] = 0.0705) is the slowest measured to date among Cymothoida, which may be associated with its relatively constant deep-sea environment. Overall, our results may provide useful information for understanding the evolution of deep-sea Isopoda species.

Cyprininae phylogeny revealed independent origins of the Tibetan Plateau endemic polyploid cyprinids and their diversifications related to the Neogene uplift of the plateau.

Origin and diversification of the Tibetan polyploid cyprinids (schizothoracins) may help us to explore relationships between diversification of the cyprinids and the Tibetan Plateau uplift. Cyprininae phylogeny was analyzed using mitochondrial and nuclear DNA sequences to trace origins of polyploidy and diversifications of schizothoracins. Ancestral states reconstruction for ploidy levels indicated that the Cyprininae was diploid origin and the schizothoracin clades tetraploid origins. There were two diversification rate shifts along with diversification of the cyprinine fishes in response to the Tibetan uplift. The unusual diversification shifts were located to branches subtending the clades of Tibetan polyploid cyprinids. Our analyses suggested that (i) phylogeny of Cyprininae recovered two independent origins of the Tibetan polyploidy schizothoracins; (ii) diversifications of the schizothoracins were closely related to the Neogene uplift of the Tibetan plateau in the following ways: the relatively ancient Late Oligocene-Middle Miocene adaptive radiation may be associated with the uplift of the southern Tibet and Himalaya; the Middle Miocene-Early Pleistocene lineage-specific diversification broadly coincident with major phase of the Neogene Tibetan uplift; and the most recent Pleistocene diversification shift in Schizothorax closely coincident with the successive Kunlun-Huanghe and Gonghe movements of the Tibetan uplift and the glaciation-induced climate oscillations on the plateau.