FishTEDB 2.0: an update fish transposable element (TE) database with new functions to facilitate TE research.

We launched the initial version of FishTEDB in 2018, which aimed to establish an open-source, user-friendly, data-rich transposable element (TE) database. Over the past 5 years, FishTEDB 1.0 has gained approximately 10 000 users, accumulating more than 450 000 interactions. With the unveiling of extensive fish genome data and the increasing emphasis on TE research, FishTEDB needs to extend the richness of data and functions. To achieve the above goals, we introduced 33 new fish species to FishTEDB 2.0, encompassing a wide array of fish belonging to 48 orders. To make the updated database more functional, we added a genome browser to visualize the positional relationship between TEs and genes and the estimated TE insertion time in different species. In conclusion, we released a new version of the fish TE database, FishTEDB 2.0, designed to assist researchers in the future study of TE functions and promote the progress of biological theories related to TEs. Database URL: https://www.fishtedb.com/.

Adaptive evolution of mitochondrial genomes in Triplophysa cavefishes.

Extreme conditions in caves pose survival challenges for cave dwellers, who gradually develop adaptive survival features. Cavefishes are one of the most successful animals among cave dwellers. Triplophysa cavefishes are an important group of cavefishes, and they show remarkable adaptability to the extreme environments of caves. However, there is a limited understanding of their adaptation mechanisms. In this study, eight complete mitochondrial genomes of Triplophysa cavefishes were newly obtained, and their genomic characteristics, including the base composition, base bias, and codon usage, were analyzed. Phylogenetic analysis was carried out based on 13 mitochondrial protein-coding genes from 44 Nemacheilidae species. This showed that Triplophysa cavefishes and non-cavefishes separate into two reciprocally monophyletic clades, suggesting a single origin of the cave phenotype. Positive selection analysis strongly suggested that the selection pressure in cavefishes is higher than that in non-cavefishes. Furthermore, the ND5 gene in cavefishes showed evidence of positive selection, which suggests that the gene may play an important role in the adaptation of cavefishes to the cave environment. Protein structure analysis of the ND5 subunit implied that the sites of positive selection in cavefishes might allow them to acquire lower ND5 protein stability, compared to that in non-cavefishes, which might help the accumulation of nonsynonymous (mildly deleterious) mutations. Together, our study revealed the genetic signatures of cave adaptation in Triplophysa cavefishes from the perspective of energy metabolism.

Poaching and illegal trade of Danube sturgeons.

A market study on sturgeon products in the Lower Danube countries (Bulgaria, Romania, Serbia and Ukraine) resulted in a total of 149 samples of caviar and meat, which we subjected to genetic-isotope analyses. The samples comprised 31 samples (21%) of illegal wild-caught origin, 17 samples (11.4%) sold in violation of CITES and EU regulations, and 47 cases (32%) of consumer deception. Although poaching and illegal wildlife trade are often considered a problem in developing countries, these findings bear evidence that a high ratio of poached sturgeon products originates from EU and accession candidate states. Therefore, caviar and sturgeon trade urgently needs improvement to ensure that sturgeon populations will have a future.

Characterization of the male-specific region containing the candidate sex-determining gene in Amur catfish (Silurus asotus) using third-generation- and pool-sequencing data.

Amur catfish (Silurus asotus) is an ecologically and economically important fish species in Asia. Here, we assembled the female and male Amur catfish genomes, with genome sizes of 757.15 and 755.44 Mb, respectively, at the chromosome level using nanopore and Hi-C technologies. Consistent with the known diploid chromosome count, both genomes contained 29 chromosome-size scaffolds covering 98.80 and 98.73 % of the complete haplotypic assembly with scaffold N50 of 28.87 and 27.29 Mb, respectively. The female (n = 40) and male (n = 40) pools were re-sequenced. Comparative analysis of sequencing and re-sequencing data from both sexes confirmed the presence of an XX/XY sex determination system in Amur catfish and revealed Chr5 as the sex chromosome containing an approximately 400 kb Y-specific region (MSY). Gene annotation revealed a male-specific duplicate of amhr2, namely amhr2y, in MSY, which is male-specific in different wild populations and expressed only in the testes. Amur catfish shared partially syntenic MSY and amhr2y genes with the southern catfish (S. meridionalis, Chr24), which were located on different chromosomes. High sequence divergence between amhr2y and amhr2 and high sequence similarity with amhr2y were observed in both species. These results indicate the common origin of the sex-determining (SD) gene and transition of amhr2y in the two Silurus species. Accumulation of repetitive elements in the MSY of both species may be the main driver of the transition of amhr2y. Overall, our study provides valuable catfish genomic resources. Moreover, determination of amhr2y as the candidate SD gene in Amur catfish provides another example of amhr2 as the SD gene in fish.

Molecular evolution of the hemoglobin gene family across vertebrates.

Adaptation to various altitudes and oxygen levels is a major aspect of vertebrate evolution. Hemoglobin is an erythrocyte protein belonging to the globin superfamily, and the α-, ß-globin genes of jawed vertebrates encode tetrameric ((α2ß2) hemoglobin, which contributes to aerobic metabolism by delivering oxygen from the respiratory exchange surfaces into cells. However, there are various gaps in knowledge regarding hemoglobin gene evolution, including patterns in cartilaginous fish and the roles of gene conversion in various taxa. Hence, we evaluated the evolutionary history of the vertebrate hemoglobin gene family by analyses of 97 species representing all classes of vertebrates. By genome-wide analyses, we extracted 879 hemoglobin sequences. Members of the hemoglobin gene family were conserved in birds and reptiles but variable in mammals, amphibians, and teleosts. Gene motifs, structures, and synteny were relatively well-conserved among vertebrates. Our results revealed that purifying selection contributed substantially to the evolution of all vertebrate hemoglobin genes, with mean dN/dS (ω) values ranging from 0.057 in teleosts to 0.359 in reptiles. In general, after the fish-specific genome duplication, the teleost hemoglobin genes showed variation in rates of evolution, and the ß-globin genes showed relatively high ω values after a gene transposition event in amniotes. We also observed that the frequency of gene conversion was high in amniotes, with fewer hemoglobin genes and higher rates of evolution. Collectively, our findings provide detail insight into complex evolutionary processes shaping the vertebrate hemoglobin gene family, involving gene duplication, gene loss, purifying selection, and gene conversion.

Novel genome sequence of Chinese cavefish (Triplophysa rosa) reveals pervasive relaxation of natural selection in cavefish genomes.

All cavefishes, living exclusively in caves across the globe, exhibit similar phenotypic traits, including the characteristic loss of eyes. To understand whether such phenotypic convergence shares similar genomic bases, here we investigated genome-wide evolutionary signatures of cavefish phenotypes by comparing whole-genome sequences of three pairs of cavefishes and their surface fish relatives. Notably, we newly sequenced and generated a whole-genome assembly of the Chinese cavefish Triplophysa rosa. Our comparative analyses revealed several shared features of cavefish genome evolution. Cavefishes had lower mutation rates than their surface fish relatives. In contrast, the ratio of nonsynonymous to synonymous substitutions (ω) was significantly elevated in cavefishes compared to in surface fishes, consistent with the relaxation of purifying selection. In addition, cavefish genomes had an increased mutational load, including mutations that alter protein hydrophobicity profiles, which were considered harmful. Interestingly, however, we found no overlap in positively selected genes among different cavefish lineages, indicating that the phenotypic convergence in cavefishes was not caused by positive selection of the same sets of genes. Analyses of previously identified candidate genes associated with cave phenotypes supported this conclusion. Genes belonging to the lipid metabolism functional ontology were under relaxed purifying selection in all cavefish genomes, which may be associated with the nutrient-poor habitat of cavefishes. Our work reveals previously uncharacterized patterns of cavefish genome evolution and provides comparative insights into the evolution of cave-associated phenotypic traits.

Identification of sex chromosome and sex-determining gene of southern catfish (Silurus meridionalis) based on XX, XY and YY genome sequencing.

Teleosts are important models to study sex chromosomes and sex-determining (SD) genes because they present a variety of sex determination systems. Here, we used Nanopore and Hi-C technologies to generate a high-contiguity chromosome-level genome assembly of a YY southern catfish (Silurus meridionalis). The assembly is 750.0 Mb long, with contig N50 of 15.96 Mb and scaffold N50 of 27.22 Mb. We also sequenced and assembled an XY male genome with a size of 727.2 Mb and contig N50 of 13.69 Mb. We identified a candidate SD gene through comparisons to our previous assembly of an XX individual. By resequencing male and female pools, we characterized a 2.38 Mb sex-determining region (SDR) on Chr24. Analysis of read coverage and comparison of the X and Y chromosome sequences showed a Y specific insertion (approx. 500 kb) in the SDR which contained a male-specific duplicate of amhr2 (named amhr2y). amhr2y and amhr2 shared high-nucleotide identity (81.0%) in the coding region but extremely low identity in the promotor and intron regions. The exclusive expression in the male gonadal primordium and loss-of-function inducing male to female sex reversal confirmed the role of amhr2y in male sex determination. Our study provides a new example of amhr2 as the SD gene in fish and sheds light on the convergent evolution of the duplication of AMH/AMHR2 pathway members underlying the evolution of sex determination in different fish lineages.

Triplophysadaryoae, a new nemacheilid loach species (Teleostei, Nemacheilidae) from the Syr Darya River basin, Central Asia.

Triplophysadaryoae, new species, is described from the Sokh River, a former tributary of Syr Darya that today fails to reach the river, in the Sokh District, an exclave of Uzbekistan, surrounded by Kyrgyzstan. Triplophysadaryoae is distinguished from other species of Triplophysa in Central Asia by a truncate caudal fin with 13 or 14 branched rays, body without obvious mottling, dorsal-fin origin opposite to pelvic-fin insertion, and absence of the posterior chamber of the air bladder. Molecular data suggest that Triplophysadaryoae is closely related to T.ferganaensis from the Shakhimardan stream, a small tributary of Syr Darya in the Yordon village, another exclave of Uzbekistan in Kyrgyzstan. The two species were separated by a Kimura 2-parameter genetic distance of 2.8% in the mitochondrial DNA cytochrome c oxidase subunit I barcode region; they are also distinguished morphologically. A key to the species of Triplophysa in the Syr Darya basin and adjacent regions is provided.

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.

Molecular diversity of Uzbekistan's fishes assessed with DNA barcoding.

Uzbekistan is one of two doubly landlocked countries in the world, where all rivers are endorheic basins. Although fish diversity is relatively poor in Uzbekistan, the fish fauna of the region has not yet been fully studied. The aim of this study was to establish a reliable barcoding reference database for fish in Uzbekistan. A total of 666 specimens, belonging to 59 species within 39 genera, 17 families, and 9 orders, were subjected to polymerase chain reaction amplification in the barcode region and sequenced. The length of the 666 barcodes was 682 bp. The average K2P distances within species, genera, and families were 0.22%, 6.33%, and 16.46%, respectively. The average interspecific distance was approximately 28.8 times higher than the mean intraspecific distance. The Barcode Index Number (BIN) discordance report showed that 666 specimens represented 55 BINs, of which five were singletons, 45 were taxonomically concordant, and five were taxonomically discordant. The barcode gap analysis demonstrated that 89.3% of the fish species examined could be discriminated by DNA barcoding. These results provide new insights into fish diversity in the inland waters of Uzbekistan and can provide a basis for the development of further studies on fish fauna.

Molecular Evolution of clock Genes in Vertebrates.

Circadian rhythms not only influence the overall daily routine of organisms but also directly affect life activities to varying degrees. Circadian locomotor output cycle kaput (Clock), the most critical gene in the circadian rhythm feedback system, plays an important role in the regulation of biological rhythms. Here, we aimed to elucidate the evolutionary history of the clock gene family in a taxonomically diverse set of vertebrates, providing novel insights into the evolution of the clock gene family based on 102 vertebrate genomes. Using genome-wide analysis, we extracted 264 clock sequences. In lobe-finned fishes and some basal non-teleost ray-finned fishes, only two clock isotypes were found (clock1 and clock2). However, the majority of teleosts possess three clock genes (two clock1 genes and one clock2 gene) owing to extra whole-genome duplication. The following syntenic analysis confirmed that clock1a, clock1b, and clock2 are conserved in teleost species. Interestingly, we discovered that osteoglossomorph fishes possess two clock2 genes. Moreover, protein sequence comparisons indicate that CLOCK protein changes among vertebrates were concentrated at the N-terminal and poly Q regions. We also performed a dN/dS analysis, and the results suggest that clock1 and clock2 may show distinct fates for duplicated genes between the lobe-finned and ray-finned fish clades. Collectively, these results provide a genome-wide insight into clock gene evolution in vertebrates.

Environmental DNA metabarcoding primers for freshwater fish detection and quantification: In silico and in tanks.

Environmental DNA (eDNA) techniques refer to utilizing the organisms' DNA extracted from environment samples to genetically identify target species without capturing actual organisms. eDNA metabarcoding via high-throughput sequencing can simultaneously detect multiple fish species from a single water sample, which is a powerful tool for the qualitative detection and quantitative estimates of multiple fish species. However, sequence counts obtained from eDNA metabarcoding may be influenced by many factors, of which primer bias is one of the foremost causes of methodological error. The performance of 18 primer pairs for COI, cytb, 12S rRNA, and 16S rRNA mitochondrial genes, which are all frequently used in fish eDNA metabarcoding, were evaluated in the current study. The ribosomal gene markers performed better than the protein-coding gene markers during in silico screening, resulting in higher taxonomic coverage and appropriate barcode lengths. Four primer pairs-AcMDB07, MiFish-U, Ve16S1, and Ve16S3-designed for various regions of the 12S and 16S rRNA genes were screened for tank metabarcoding in a case study targeting six freshwater fish species. The four primer pairs were able to accurately detect all six species in different tanks, while only MiFish-U, Ve16S1, and Ve16S3 revealed a significant positive relationship between species biomass and read count for the pooled tank data. The positive relationship could not be found in all species within the tanks. Additionally, primer efficiency differed depending on the species while primer preferential species varied in different fish assemblages. This case study supports the potential for eDNA metabarcoding to assess species diversity in natural ecosystems and provides an alternative strategy to evaluate the performance of candidate primers before application of eDNA metabarcoding in natural ecosystems.

Triplophysa ferganaensis, a new loach species from Fergana Valley in Central Asia (Teleostei: Nemacheilidae).

Triplophysa ferganaensis sp. nov. is described from the Shakhimardan stream, a small tributary of the Syr Darya, which does not reach the river in Fergana Valley. It can be distinguished from other valid Triplophysa loaches based on the following combination of characters: body smooth and scaleless, lateral line complete, posterior chamber of air bladder degenerated, inner gill rakers 10-11 on the first-gill arch, outer gill rakers absent, vertebrae 4 + 35-36, 8 + 8 branched caudal-fin rays, caudal peduncle depth 2.1-2.7 times its length, two supratemporal pores, dorsal-fin origin closer to the caudal-fin base than to the snout tip, caudal fin emarginated and pelvic-fin tip reaching the anus. The new species can also be distinguished from its congeners based on the molecular analyses of mitochondrial cytochrome oxidase subunit I (coI) gene sequences. The phylogenetic position of this new species indicates that it is a sister taxon of Triplophysa tenuis.

Triplophysa wulongensis, a new species of cave-dwelling loach (Teleostei, Nemacheilidae) from Chongqing, Southwest China.

We describe a new species of cave-dwelling loach, Triplophysa wulongensis sp. nov., based on specimens collected in a subterranean pool in a cave in Wulong County, Chongqing, Southwest China. The pool is connected to the Wujiang River drainage. Triplophysa wulongensis differs from its congeners by the following combination of characters: eyes present, caudal fin with 18 branched rays; posterior chamber of the air bladder degenerate; stomach U-shaped; intestine without bends or loops immediately posterior to stomach; body smooth and scaleless, and lateral line complete. The mitochondrial cytochrome b sequence differs from those of other published sequences of species of Triplophysa by 14.9-24.9% in K2P distance. Phylogenetic analysis based on cytochrome b gene sequences recovered T. wulongensis as sister taxon to all other cave-dwelling species of Triplophysa.

Morphometric and population genomic evidence for species divergence in the Chimarrichthys fish complex of the Tibetan Plateau.

The uplift of the Tibetan Plateau altered the environmental conditions of the local area substantially. Here, we conducted a comprehensive investigation based on morphometrics, population genomics, and climatic factors to evaluate phenotypic and genome-level variations in a radiation of Chimarrichthys catfish endemic to the Plateau. Discriminant function analysis showed phenotypic differences of Chimarrichthys between rivers with respect to elevation. Genetic structure analysis based on 6606 single nucleotide polymorphisms (SNPs) deduced genetic differences between rivers, and species delimitation indicated that the Chimarrichthys fish complex could be divided into three species. Restriction site-associated DNA tags were mapped to the gene sets of Glyptosternon maculatum, and matches were searched against databases for Gene Ontology annotation. Genomic regions exhibiting marked differences among localities represented a range of biological functions, including growth (gdf11), bone development (bmp8a), cellular response to light stimulus (opn3), regulation of the rhodopsin-mediated signalling pathway (grk1), immune response (rag1 and ung), reproductive process (antxr2), and regulation of intracellular iron levels (ireb2). The tag44126, where gene gdf11 is located, was identified as an outlier exhibiting divergence between rivers with altitude differences, and the SNP is thymine (T) in Dadu and Yalong River (~2700 m), but guanine (G) in Jinsha and Qingyi rivers (~2200 and ~ 684 m), suggesting a possible effect of altitude on its differentiation.

Chromosome-level assembly of southern catfish (silurus meridionalis) provides insights into visual adaptation to nocturnal and benthic lifestyles.

The Southern catfish (Silurus meridionalis) is a nocturnal and benthic freshwater fish endemic to the Yangtze River and its tributaries. In this study, we constructed a chromosome-level draft genome of S. meridionalis using 69.7-Gb Nanopore long reads and 49.5-Gb Illumina short reads. The genome assembly was 741.2 Mb in size with a contig N50 of 13.19 Mb. An additional 116.4 Gb of Bionano and 77.4 Gb of Hi-C data were applied to assemble contigs into scaffolds and further into 29 chromosomes, resulting in a 738.9-Mb genome with a scaffold N50 of 28.04 Mb. A total of 22,965 protein-coding genes were predicted from the genome with 22,519 (98.06%) genes functionally annotated. Comparative genomic and transcriptomic analyses revealed a rod-dominated visual system which was responsible for scotopic vision. The absence of cone opsins SWS1 and SWS2 resulted in the lack of ultraviolet and blue violet sensitivity. Mutations at key amino acid sites of RH1.1, RH1.2 and RH2 resulted in spectral tuning good for dim light vision and narrow colour vision. A higher expression level of rod phototransduction genes than that of cone genes and higher rod-to-cone ratio led to higher optical sensitivity under dim light conditions. In addition, analysis of the genes involved in eye morphogenesis and development revealed the loss of some conserved noncoding elements, which might be associated with the small eyes in catfish. Together, our study provides important clues for the adaptation of the catfish visual system to the nocturnal and benthic lifestyles. The draft genome of S. meridionalis represents a valuable resource for studies of the molecular mechanisms of ecological adaptation.