To characterize the genetic diversity and evolutionary patterns of four Weining chicken strains from Guizhou Province, China, we generated genome-wide single nucleotide polymorphisms (SNPs) using dd-RAD sequencing. We found that Weining chicken exhibited moderate genetic diversity, with observed heterozygosity (Ho = 0.267) lower than expected heterozygosity (He = 0.312) and a positive inbreeding coefficient (FIS ≈ 0.14), indicating some degree of inbreeding across the population. Among strains, HM showed the highest diversity while HT had the lowest. Analyses of population structure, phylogeny, principal component analysis, and admixture consistently revealed weak genetic differentiation (FST < 0.06) and frequent gene flow among strains. Kinship analysis demonstrated mostly low pairwise relatedness, with a minority of individuals exhibiting closer kinship. Selection signature scans identified multiple candidate regions; genes within these regions were significantly enriched for biological processes and pathways related to immune response (e.g., IL-17 signaling) and metabolic regulation (e.g., MAPK signaling), which were consistently highlighted in both GO and KEGG enrichment analyses. These results demonstrate that Weining chicken has moderate genetic diversity, low population differentiation, and evidence of inbreeding and gene flow, with candidate selection signals in immune and metabolic pathways (e.g., IL-17 and MAPK signaling), providing a quantitative basis for conservation and breeding programs.

ABSTRACT The brittle star of genus Gymnolophus Brock, 1888 (Ophiuroidea: Ophiotrichidae Ljungman, 1867) is commonly found in epizoic relationships with crinoid hosts, such as Comaster schlegelii (Crinoidea: Comatulidae Fleming, 1828), benefiting from food and protection. Here we describe a new hexamerous epizoic species, Gymnolophus sexradia sp. nov., from the South China Sea. We provide a detailed description of the ossicle structure, based on micro‐CT scanning, and sequences of 1464 nuclear exons and the complete mitogenome of the new species. The specimen features a hexamerous body plan and six arms which may indicate fissiparity. The shape and articulation of the arm vertebrae, along with the lateral arm plates and overlapping split dorsal arm plates, enhance arm flexibility in the horizontal direction, facilitating its grasping behavior toward the crinoid host. Phylogenetic analysis of nuclear exons clusters the new species with Ophiomaza cacaotica (Lyman, 1871) and Gymnolophus obscura (Ljungman, 1867). The complete mitogenome consists of 15,669 bp and 37 genes with a conserved gene order identical to that of Ophiactis savignyi and other studied mitogenomes in Ophiotrichidae. Notably, 14 residues in four mitochondrial genes were identified as positively selected, which may reflect adaptations linked to the epizoic lifestyle with crinoid hosts.

A remarkable example of symbiosis involves the pygmy seahorse (Hippocampus bargibanti). It lives obligatorily on gorgonian corals, mimicking their polyps with pink coloration and skin protuberances. Unique for seahorses, pygmy seahorses retain juvenile paedomorphic stunted snouts, resembling the coral's polyps. We analyzed the tiny seahorse's genome revealing the genomic bases of several adaptations to their mutualistic life including substantial reductions in conserved noncoding elements that are associated with genes in the vicinity of those CNEs that are known to play a role in growth and metamorphosis- related pathways. Comparative RNA- and ATAC- Seq analyses during their ontogeny suggest that their stunted snout might result from craniofacial remodeling associated with hoxa2b defunctionalization. This is consistent also with findings from in situ hybridization and CRISPR experiments. Their immune system shows extremely low numbers of MHC genes and additional considerable losses of other immune- related genes. This is likely facilitated by the host coral's antimicrobial metabolites and by the earlier evolution of male pregnancy that requires immunotolerance.

Recurring outbreaks of crown-of-thorns starfish (COTS) are one of the greatest threats to corals and have caused catastrophic damage to coral reef ecosystems in recent decades. As obligate corallivores, COTS can tolerate toxic coral mucus, and the sharp spines covering their bodies provide protection from predators. While both of these characteristics are known to contribute to the vitality of COTS and aggravate the impact of COTS outbreaks, the genetic mechanisms underlying their adaptation remain unclear. In this study, we generated a chromosome-level COTS genome and suggested the importance of chromosome fusion in coral-feeding adaptation. In addition, high expression levels of EP4 receptors in the COTS spine apex indicated their potential ability to use the coral prostaglandin PGE2, which might further activate the skeletogenic gene regulatory network responsible for the growth of the spine and body plan of COTS. A comparison of the historical population sizes of COTS from different locations revealed a unique trend in the Coral Triangle, suggesting a potentially higher risk of outbreaks in this region. Our results provide a molecular basis for the adaptive evolution of COTS and a better understanding of its interactions in coral reef ecosystems.

Seahorses exhibit a unique reproduction mode of male pregnancy. Successful gestation plays a crucial role in the seahorse reproduction process and the aquaculture industry. However, the substantial paternal-embryo differences in seahorses remain largely unexplored. In the present study, transcriptomic and metabolic differences between pregnant males and embryos during gestation were explored to investigate the roles of key genes and metabolites in pregnant males and embryos in seahorses. Higher levels of essential amino acids and polyunsaturated fatty acids in embryos compared to fathers may indicate their vital role in embryonic development. The high expression of immune-related genes (CCLs and CCRs) and downregulation of antioxidants in fathers may provide immune protection and reduce the immunological rejection of embryos during gestation. The transcript-metabolite correlation network during pregnancy further indicated the role of key genes and metabolites during embryonic eye (N-acetyl-L-histidine, PAX6, and FZT4) and tail (WNT5 and WNT8) development. The findings of the present study not only provide valuable data on differences between pregnant males and embryos in seahorse, but also lay foundation for future research on seahorse aquaculture.

Symbiosis is one of the most important evolutionary processes shaping biodiversity. Phylogenetic and morphological analyses can enhance our understanding of the origin and evolution of symbiosis. The symbiotic relationship between Trapezioidea crabs and branching corals, with high species diversity and flexible symbiosis, is iconic coral/crustacea symbiosis in the marine ecosystem. In this study, the evolutionary history of Trapezioidea crabs was investigated by constructing a phylogenetic relationship based on the whole mitogenome sequences of 66 Brachyura species, including 17 newly sequenced Trapezioidea species. Trapezioidea crabs were split at least into two phylogenetically distant lineages originated at different times. The two lineages, both including facultative and obligate symbionts, convergently evolved smooth and flattened trapezoidal carapaces favorable for specific coral-living. The significantly larger cheliped in obligate species indicated a deeper degree of coral dependence compared with facultative and other associate species.

The current view of hematopoiesis considers leukocytes on a continuum with distinct developmental origins, and which exert non-overlapping functions. However, there is less known about the function and phenotype of ontogenetically distinct neutrophil populations. In this work, using a photoconvertible transgenic zebrafish line; Tg(mpx:Dendra2), we selectively label rostral blood island-derived and caudal hematopoietic tissue-derived neutrophils in vivo during steady state or upon injury. By comparing the migratory properties and single-cell expression profiles of both neutrophil populations at steady state we show that rostral neutrophils show higher csf3b expression and migration capacity than caudal neutrophils. Upon injury, both populations share a core transcriptional profile as well as subset-specific transcriptional signatures. Accordingly, both rostral and caudal neutrophils are recruited to the wound independently of their distance to the injury. While rostral neutrophils respond uniformly, caudal neutrophils respond heterogeneously. Collectively, our results reveal that co-existing neutrophils populations with ontogenically distinct origin display functional differences.

Male pregnancy in syngnathids (seahorses, pipefishes, and sea dragons) is an evolutionary innovation in the animal kingdom. Paternal immune resistance to the fetus is a critical challenge, particularly in seahorses with fully enclosed brood pouches and sophisticated placentas. In this study, comparative genomic analysis revealed that all syngnathid species lost three vertebrate-conserved Toll-like receptors (TLR1, TLR2, and TLR9), of which all play essential roles in immune protection and immune tolerance in the uterus and placenta. Quantitative real-time PCR (qRT-PCR) analysis showed that the TLR paralog genes including TLR18, TLR25, and TLR21 were highly expressed in the placenta inside the seahorse brood pouch and changed dynamically during the breeding cycle, suggesting the potentially important role of the TLRs during male pregnancy. Furthermore, the immune challenge test in vitro showed a remarkable expression response from all three TLR genes to specific pathogenic antigens, confirming their immune function in seahorse brood pouches. Notably, the altered antigen recognition spectrum of these genes appeared to functionally compensate in part for the lost TLRs, in contrast to that observed in other species. Therefore, we suggest that gene loss and co-option of TLRs may be a typical evolutionary strategy for facilitating paternal immunological adaptation during male pregnancy.

The recent whole-genome duplication (WGD) in goldfish (Carassius auratus) approximately 14 million years ago makes it a valuable model for studying gene evolution during the early stages after WGD. We analyzed the transcriptome of the goldfish retina at the level of single-cell (scRNA-seq) and open chromatin regions (scATAC-seq). We identified a group of genes that have undergone dosage selection, accounting for 5% of the total 11,444 ohnolog pairs. We also identified 306 putative sub/neo-functionalized ohnolog pairs that are likely to be under cell-type-specific genetic variation at single-cell resolution. Diversification in the expression patterns of several ohnolog pairs was observed in the retinal cell subpopulations. The single-cell level transcriptome analysis in this study uncovered the early stages of evolution in retinal cell of goldfish after WGD. Our results provide clues for understanding the relationship between the early stages of gene evolution after WGD and the evolution of diverse vertebrate retinal functions.

The data that support the findings of this study are available upon reasonable request to the corresponding author. Materials and methods and other supplementary figures and tables of this study have been shared, and the link to the repository is https://doi.org/10.6084/m9.figshare.17430203.v5. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Seahorse is a specialized animal due to its male pregnant strategy in the brood pouch, which can evidently provide immune protection, nutrition, and osmoregulation to the offspring. Secretory phospholipase A2 group IB (sPLA2-IB), which encodes a secreted phospholipase A2 that functions for metabolic regulation and antibacterial defense, plays a vital role for immune regulation in teleost, but its potential immune functions in seahorse remain unknown. Here, we aimed to evaluate the characteristics and functional profiles of sPLA2-IBs in lined seahorse (Hippocampus erectus), which is one of the most important breeding seahorse species in the world. Results showed that most teleost species have only one sPLA2-IB gene, while the seahorses presented two tandem repetitive copies (sPLA2-IBa and sPLA2-IBb). This finding showed that the highest expression levels of sPLA2-IBs in seahorses were in the liver, and significant up-regulation could be induced by lipopolysaccharide (LPS), polyinosinic-polycytidylic acid (Poly[I:C]), as well as the seahorse's common pathogen Vibrio parahaemolyticus. Interestingly, the expression levels of sPLA2-IBs in the brood pouch showed an upward trend during pregnancy, compared with that in non-pregnancy stage, and peaked in the late pregnancy stage, implying that seahorse sPLA2-IBs in brood pouch might function for immune defense during pregnancy. In addition, seahorse sPLA2-IBa was significantly highly expressed in early developing embryos in brood pouch, but sPLA2-IBb expression dramatically increased in new-born juveniles, which implies the functional divergence between the two sPLA2-IBs by different ontogenetic expression. In conclusion, this study indicated that seahorse sPLA2-IBs play an important role in the immunoprotection during male pregnancy, and functional co-option of seahorse sPLA2-IBs might be conducive to improving the survival rate of its offspring.

Zebrafish, a popular organism for studying embryonic development and for modeling human diseases, has so far lacked a systematic functional annotation program akin to those in other animal models. To address this, we formed the international DANIO-CODE consortium and created a central repository to store and process zebrafish developmental functional genomic data. Our data coordination center ( https://danio-code.zfin.org ) combines a total of 1,802 sets of unpublished and re-analyzed published genomic data, which we used to improve existing annotations and show its utility in experimental design. We identified over 140,000 cis-regulatory elements throughout development, including classes with distinct features dependent on their activity in time and space. We delineated the distinct distance topology and chromatin features between regulatory elements active during zygotic genome activation and those active during organogenesis. Finally, we matched regulatory elements and epigenomic landscapes between zebrafish and mouse and predicted functional relationships between them beyond sequence similarity, thus extending the utility of zebrafish developmental genomics to mammals.

Diffusing fluid from methane seepage in cold seep field creates zones with physicochemical gradients and divergent ecosystems like the mussel beds and clam beds. Three species of brittle stars (Ophiuroidea) were discovered in the Haima cold seep fields, of which Ophiophthalmus serratus and Histampica haimaensis were found on top of or within mussel beds and clam beds, whereas Amphiura sp. was only collected from muds in the clam bed assemblage. Here, we evaluated the genetic signatures of micro-environmental adaptation of brittle stars to cold seep through the comparison of mitogenomes. This study provided two complete mitogenome sequences of O. serratus and Amphiura sp. and compared with those of H. haimaensis and other non-seep species. We found that the split events of the seep and non-seep species were as ancient as the Cretaceous period (∼148–98 Mya). O. serratus and H. haimaensis display rapid residue mutation and mitogenome rearrangements compared to their shallow or deep-sea relatives, in contrast, Amphiura sp. only show medium, regardless of nucleotide mutation rate or mitogenome rearrangement, which may correlate with their adaptation to one or two micro-ecosystems. Furthermore, we identified 10 positively selected residues in ND4 in the Amphiura sp. lineage, suggesting important roles of the dehydrogenase complex in Amphiura sp. adaptive to the cold seep environment. Our results shed light on the different evolutionary strategies during colonization in different micro-environments.

Using adult zebrafish inner ears as a model for sensorineural regeneration, we ablated the mechanosensory receptors and characterized the single-cell epigenome and transcriptome at consecutive time points during hair cell regeneration. We utilized deep learning on the regeneration-induced open chromatin sequences and identified cell-specific transcription factor (TF) motif patterns. Enhancer activity correlated with gene expression and identified potential gene regulatory networks. A pattern of overlapping Sox- and Six-family TF gene expression and binding motifs was detected, suggesting a combinatorial program of TFs driving regeneration and cell identity. Pseudotime analysis of single-cell transcriptomic data suggested that support cells within the sensory epithelium changed cell identity to a "progenitor" cell population that could differentiate into hair cells. We identified a 2.6 kb DNA enhancer upstream of the sox2 promoter that, when deleted, showed a dominant phenotype that resulted in a hair-cell-regeneration-specific deficit in both the lateral line and adult inner ear.

Viviparity has originated independently more than 150 times in vertebrates, while the male pregnancy only emerged in Syngnathidae fishes, such as seahorses. The typical male pregnancy seahorses have closed sophisticated brood pouch that act as both uterus and placenta, representing an excellent model system for studying the evolutionary process of paternal immune protection. Phylogenetic analysis indicated that the hampII gene family has multiple tandem duplicated genes and shows independent lineage-specific expansion in seahorses, and they had the highest ratio of nonsynonymous substitutions to synonymous substitutions (dN/dS) in the seahorse phylogenetic branch. The expression levels of hampIIs in the brood pouch placenta were significantly higher during pregnancy than non-pregnancy. Both LPS stimulation test in vivo and cytotoxicity test in vitro proved the immunological protection function of hampIIs against pathogen infection in seahorse. Besides, seahorse hampII peptides exhibit weaker antibacterial function, but stronger agglutination and free endotoxin inhibition. We assumed that the modified immunological function seemed to be a trade-off between the resistance to microbial attack and offspring protection. In brief, this study suggests that the rapid co-option of hampIIs contributes to the evolutionary adaption to paternal immune care during male pregnancy.

ABSTRACTIn anamniote embryos the major wave of zygotic genome activation (ZGA) starts during the mid-blastula transition. This major wave of ZGA is facilitated by several mechanisms, including dilution of repressive maternal factors and accumulation of activating transcription factors during the fast cell division cycles preceding the mid-blastula transition. However, a set of genes escape global genome repression and are activated substantially earlier, during what is called, the minor wave of genome activation. While the mechanisms underlying the major wave of genome activation have been studied extensively, the minor wave of genome activation is little understood. In zebrafish the earliest expressed RNA polymerase II (Pol II) transcribed genes are activated in a pair of large transcription bodies depleted of chromatin, abundant in elongating Pol II and nascent RNAs (Hadzhiev et al., 2019; Hilbert et al., 2021). This transcription body includes the miR-430 gene cluster required for maternal mRNA clearance. Here we explored the genomic, chromatin organisation and cis-regulatory mechanisms of the minor wave of genome activation occurring in the transcription body. By long read genome sequencing we identified a remarkable cluster of miR-430 genes with over 300 promoters and spanning 0.6 Mb, which represent the highest promoter density of the genome. We demonstrate that the miR-430 gene cluster is required for the formation of the transcription body and acts as a transcription organiser for minor wave activation of a set of zinc finger genes scattered on the same chromosome arm, which share promoter features with the miR-430 cluster. These promoter features are shared among minor wave genes overall and include the TATA-box and sharp transcription start site profile. Single copy miR-430 promoter transgene reporter experiments indicate the importance of promoter-autonomous mechanisms regulating escape from global repression of the early embryo. These results together suggest that formation of the transcription body in the early embryo is the result of high promoter density coupled to a minor wave-specific core promoter code for transcribing key minor wave ZGA genes, which are required for the overhaul of the transcriptome during early embryonic development.

Zebrafish, a popular model for embryonic development and for modelling human diseases, has so far lacked a systematic functional annotation programme akin to those in other animal models. To address this, we formed the international DANIO-CODE consortium and created the first central repository to store and process zebrafish developmental functional genomic data. Our Data Coordination Center ( https://danio-code.zfin.org ) combines a total of 1,802 sets of unpublished and reanalysed published genomics data, which we used to improve existing annotations and show its utility in experimental design. We identified over 140,000 cis-regulatory elements in development, including novel classes with distinct features dependent on their activity in time and space. We delineated the distinction between regulatory elements active during zygotic genome activation and those active during organogenesis, identifying new aspects of how they relate to each other. Finally, we matched regulatory elements and epigenomic landscapes between zebrafish and mouse and predict functional relationships between them beyond sequence similarity, extending the utility of zebrafish developmental genomics to mammals.

Goldfish is an ornamental fish with diverse phenotypes. However, the limited genomic resources of goldfish hamper our understanding of the genetic basis for its phenotypic diversity. To provide enriched genomic resources and infer possible mechanisms underlying skin pigmentation, we performed a large-scale transcriptomic sequencing on 13 adult goldfish tissues, larvae at one- and three-days post hatch, and skin tissues with four different color pigmentation. A total of 25.52 Gb and 149.80 Gb clean data were obtained using the PacBio and Illumina platforms, respectively. Onto the goldfish reference genome, we mapped 137,674 non-redundant transcripts, of which 5.54% was known isoforms and 78.53% was novel isoforms of the reference genes, and the remaining 21,926 isoforms are novel isoforms of additional new genes. Both skin-specific and color-specific transcriptomic analyses showed that several significantly enriched genes were known to be involved in melanogenesis, tyrosine metabolism, PPAR signaling pathway, folate biosynthesis metabolism and so on. Thirteen differentially expressed genes across different color skins were associated with melanogenesis and pteridine synthesis including mitf , ednrb , mc1r , tyr , mlph and gch1 , and xanthophore differentiation such as pax7 , slc2a11 and slc2a15 . These transcriptomic data revealed pathways involved in goldfish pigmentation and improved the gene annotation of the reference genome.

Few fishes have evolved elevated body temperatures compared with ambient temperatures, and only in opah (Lampris spp) is the entire body affected. To understand the molecular basis of endothermy, we analyzed the opah genome and identified 23 genes with convergent amino acid substitutions across fish, birds, and mammals, including slc8b1, which encodes the mitochondrial Na+/Ca2+ exchanger and is essential for heart function and metabolic heat production. Among endothermic fishes, 44 convergent genes with suggestive metabolic functions were identified, such as glrx3, encoding a crucial protein for hemoglobin maturation. Numerous genes involved in the production and retention of metabolic heat were also found to be under positive selection. Analyses of opah's unique inner-heat-producing pectoral muscle layer (PMI), an evolutionary key innovation, revealed that many proteins were co-opted from dorsal swimming muscles for thermogenesis and oxidative phosphorylation. Thus, the opah genome provides valuable resources and opportunities to uncover the genetic basis of thermal adaptations in fish.

Calcification of various tissues is a significant health issue associated with aging, cancer and autoimmune diseases. There are both environmental and genetic factors behind this phenomenon and understanding them is essential for the development of efficient therapeutic approaches. Pseudoxanthoma elasticum (PXE) is a rare genetic disease, a prototype for calcification disorders, resulting from the dysfunction of ABCC6, a transport protein found in the membranes of cells. It is identified by excess calcification in a variety of tissues (e.g., eyes, skin, arteries) and currently it has no cure, known treatments target the symptoms only. Preclinical studies of PXE have been successful in mice, proving the usefulness of animal models for the study of the disease. Here, we present a new zebrafish (Danio rerio) model for PXE. By resolving some ambiguous assemblies in the zebrafish genome, we show that there are two functional and one non-functional paralogs for ABCC6 in zebrafish (abcc6a, abcc6b.1, and abcc6b.2, respectively). We created single and double mutants for the functional paralogs and characterized their calcification defects with a combination of techniques. Zebrafish deficient in abcc6a show defects in their vertebral calcification and also display ectopic calcification foci in their soft tissues. Our results also suggest that the impairment of abcc6b.1 does not affect this biological process.