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.

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.

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.

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.

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.

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.

Taxonomy on three novel species of Sphaeromyxa Thélohan 1892 (Myxozoa, Bivalvulida, Sphaeromyxidae) with insight into the evolution of the genus.

Three new myxosporeans of the genus Sphaeromyxa Thélohan 1892 were discovered from the coastal waters off Xiamen in the East China Sea and characterized based on morphological and SSU rDNA data. Sphaeromyxa photopectoralis sp. n. was described from Photopectoralis bindus, and Sphaeromyxa sebastisca sp. n. was described infecting both Sebastiscus marmoratus (type-host) and Scorpaenopsis cirrosa. These two species are morphologically consistent with the "balbianii" group, possessing straight myxospores and truncated ends, but are distinct from one another genetically and by myxospore dimensions. A third myxosporean infecting Siganus fuscescens was described as Sphaeromyxa xiamenensis sp. n., and this species is morphologically consistent with the "incurvata" group, bearing arcuate myxospores with rounded ends. The molecular phylogeny and estimated rRNA secondary structure suggest that marine sphaeromyxids are probably derived from freshwater myxidiids, and "incurvata" and "balbianii" groups might each represent independent evolutionary lineages. The present study also shows that S. limocapitis phylogenetically nested in "incurvata" group.

Adaptive Evolution of the Eda Gene and Scales Loss in Schizothoracine Fishes in Response to Uplift of the Tibetan Plateau.

Schizothoracine is the predominant wild fish subfamily of the Tibetan plateau (TP). Their scales, pharyngeal teeth and barbels have gradually regressed with increasing altitude. Schizothoracine have been divided into three groups: primitive, specialized and highly specialized. Ectodysplasin-A (Eda) has been considered as a major gene that contributes to the development of skin appendages. The present study cloned the Eda genes of 51 Schizothoracine fish species which represent the three groups and five Barbinae species. Phylogenetic analyses indicated that Eda may have acted as the genetic trigger for scale loss in the Schizothoracine. Furthermore, 14 single nucleotide polymorphisms (SNPs) and two deletions (18 bp and 6 bp in size), were also detected in the Eda coding sequence of the highly specialized group compared to the primitive group. The same SNPs and two indels result in four non-synonymous and two G-X-Y and 1 XY motif indels, which possibly contribute to significant structure changes in the Eda gene. The domain including (G-X-Y)n motif in the Eda gene is relatively conserved amongst teleosts. Based on the above results, we hypothesize that the evolution of Eda gene might be associated with the scale loss in Schizothoracine fishes in response to the phased uplift of the TP.

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.

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/.

First comparative study of primate morphological and molecular evolutionary rates including muscle data: implications for the tempo and mode of primate and human evolution.

Here we provide the first report about the rates of muscle evolution derived from Bayesian and parsimony cladistic analyses of primate higher-level phylogeny, and compare these rates with published rates of molecular evolution. It is commonly accepted that there is a 'general molecular slow-down of hominoids', but interestingly the rates of muscle evolution in the nodes leading and within the hominoid clade are higher than those in the vast majority of other primate clades. The rate of muscle evolution at the node leading to Homo (1.77) is higher than that at the nodes leading to Pan (0.89) and particularly to Gorilla (0.28). Notably, the rates of muscle evolution at the major euarchontan and primate nodes are different, but within each major primate clade (Strepsirrhini, Platyrrhini, Cercopithecidae and Hominoidea) the rates at the various nodes, and particularly at the nodes leading to the higher groups (i.e. including more than one genera), are strikingly similar. We explore the implications of these new data for the tempo and mode of primate and human evolution.

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.

Comparative analyses of DNA methylation and sequence evolution using Nasonia genomes.

The functional and evolutionary significance of DNA methylation in insect genomes remains to be resolved. Nasonia is well situated for comparative analyses of DNA methylation and genome evolution, since the genomes of a moderately distant outgroup species as well as closely related sibling species are available. Using direct sequencing of bisulfite-converted DNA, we uncovered a substantial level of DNA methylation in 17 of 18 Nasonia vitripennis genes and a strong correlation between methylation level and CpG depletion. Notably, in the sex-determining locus transformer, the exon that is alternatively spliced between the sexes is heavily methylated in both males and females, whereas other exons are only sparsely methylated. Orthologous genes of the honeybee and Nasonia show highly similar relative levels of CpG depletion, despite ~190 My divergence. Densely and sparsely methylated genes in these species also exhibit similar functional enrichments. We found that the degree of CpG depletion is negatively correlated with substitution rates between closely related Nasonia species for synonymous, nonsynonymous, and intron sites. This suggests that mutation rates increase with decreasing levels of germ line methylation. Thus, DNA methylation is prevalent in the Nasonia genome, may participate in regulatory processes such as sex determination and alternative splicing, and is correlated with several aspects of genome and sequence evolution.

Microsatellite development for an endangered bream Megalobrama pellegrini (Teleostei, Cyprinidae) using 454 sequencing.

Megalobrama pellegrini is an endemic fish species found in the upper Yangtze River basin in China. This species has become endangered due to the construction of the Three Gorges Dam and overfishing. However, the available genetic data for this species is limited. Here, we developed 26 polymorphic microsatellite markers from the M. pellegrini genome using next-generation sequencing techniques. A total of 257,497 raw reads were obtained from a quarter-plate run on 454 GS-FLX titanium platforms and 49,811 unique sequences were generated with an average length of 404 bp; 24,522 (49.2%) sequences contained microsatellite repeats. Of the 53 loci screened, 33 were amplified successfully and 26 were polymorphic. The genetic diversity in M. pellegrini was moderate, with an average of 3.08 alleles per locus, and the mean observed and expected heterozygosity were 0.47 and 0.51, respectively. In addition, we tested cross-species amplification for all 33 loci in four additional breams: M. amblycephala, M. skolkovii, M. terminalis, and Sinibrama wui. The cross-species amplification showed a significant high level of transferability (79%-97%), which might be due to their dramatically close genetic relationships. The polymorphic microsatellites developed in the current study will not only contribute to further conservation genetic studies and parentage analyses of this endangered species, but also facilitate future work on the other closely related species.

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.

Phylogeography of the endemic Gymnocypris chilianensis (Cyprinidae): sequential westward colonization followed by allopatric evolution in response to cyclical Pleistocene glaciations on the Tibetan Plateau.

The schizothoracine Gymnocypris chilianensis is restricted to the Shiyang, Ruoshui and Shule Rivers, listed from east to west, along the northeast edge of the Tibetan Plateau. This distribution provides a valuable system to test hypotheses about postglacial colonization. We used mitochondrial DNA sequence data (a control region and the cytochrome b gene; 1894 bp) to assess the phylogeographic structure of this species based on 278 specimens sampled from throughout the species' entire geographical range. We found three lineages corresponding geographically to the three rivers, suggesting three independent glacial differentiation centers within the northeast edge of the Tibetan Plateau. The phylogenetic analysis suggested that the Shiyang River population forms a lineage that separated from the other populations of G. chilianensis at the basal phylogenetic split within this species. The molecular data further demonstrated a clear pattern of decreasing genetic diversity from the eastern Shiyang River towards the central Ruoshui River and western Shule River lineages, a pattern consistent with sequential western colonization. We therefore propose a phylogeographic scenario for G. chilianensis of a gradual westerly expansion from the Shiyang River population along the northeast edge of the Tibetan Plateau, with subsequent allopatric evolution at approximately 0.37 and 0.05 million years ago (Ma), through at least two glacial maxima. Together with the genetic evidence reported in other species, our findings suggest that this common biogeographic pattern emphasizes the importance of the northeastern edge region of the Tibetan Plateau as a hotspot of genetic diversity for some taxa.

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.