Heterologous survey of 130 DNA transposons in human cells highlights their functional divergence and expands the genome engineering toolbox.

Experimental studies on DNA transposable elements (TEs) have been limited in scale, leading to a lack of understanding of the factors influencing transposition activity, evolutionary dynamics, and application potential as genome engineering tools. We predicted 130 active DNA TEs from 102 metazoan genomes and evaluated their activity in human cells. We identified 40 active (integration-competent) TEs, surpassing the cumulative number (20) of TEs found previously. With this unified comparative data, we found that the Tc1/mariner superfamily exhibits elevated activity, potentially explaining their pervasive horizontal transfers. Further functional characterization of TEs revealed additional divergence in features such as insertion bias. Remarkably, in CAR-T therapy for hematological and solid tumors, Mariner2_AG (MAG), the most active DNA TE identified, largely outperformed two widely used vectors, the lentiviral vector and the TE-based vector SB100X. Overall, this study highlights the varied transposition features and evolutionary dynamics of DNA TEs and increases the TE toolbox diversity.

Asymmetric and parallel subgenome selection co-shape common carp domestication.

BACKGROUND: The common carp (Cyprinus carpio) might best represent the domesticated allopolyploid animals. Although subgenome divergence which is well-known to be a key to allopolyploid domestication has been comprehensively characterized in common carps, the link between genetic architecture underlying agronomic traits and subgenome divergence is unknown in the selective breeding of common carps globally. RESULTS: We utilized a comprehensive SNP dataset in 13 representative common carp strains worldwide to detect genome-wide genetic variations associated with scale reduction, vibrant skin color, and high growth rate in common carp domestication. We identified numerous novel candidate genes underlie the three agronomically most desirable traits in domesticated common carps, providing potential molecular targets for future genetic improvement in the selective breeding of common carps. We found that independently selective breeding of the same agronomic trait (e.g., fast growing) in common carp domestication could result from completely different genetic variations, indicating the potential advantage of allopolyploid in domestication. We observed that candidate genes associated with scale reduction, vibrant skin color, and/or high growth rate are repeatedly enriched in the immune system, suggesting that domestication of common carps was often accompanied by the disease resistance improvement. CONCLUSIONS: In common carp domestication, asymmetric subgenome selection is prevalent, while parallel subgenome selection occurs in selective breeding of common carps. This observation is not due to asymmetric gene retention/loss between subgenomes but might be better explained by reduced pleiotropy through transposable element-mediated expression divergence between ohnologs. Our results demonstrate that domestication benefits from polyploidy not only in plants but also in animals.

Prevalent Introgression Underlies Convergent Evolution in the Diversification of Pungitius Sticklebacks.

New mutations and standing genetic variations contribute significantly to repeated phenotypic evolution in sticklebacks. However, less is known about the role of introgression in this process. We analyzed taxonomically and geographically comprehensive genomic data from Pungitius sticklebacks to decipher the extent of introgression and its consequences for the diversification of this genus. Our results demonstrate that introgression is more prevalent than suggested by earlier studies. Although gene flow was generally bidirectional, it was often asymmetric and left unequal genomic signatures in hybridizing species, which might, at least partly, be due to biased hybridization and/or population size differences. In several cases, introgression of variants from one species to another was accompanied by transitions of pelvic and/or lateral plate structures-important diagnostic traits in Pungitius systematics-and frequently left signatures of adaptation in the core gene regulatory networks of armor trait development. This finding suggests that introgression has been an important source of genetic variation and enabled phenotypic convergence among Pungitius sticklebacks. The results highlight the importance of introgression of genetic variation as a source of adaptive variation underlying key ecological and taxonomic traits. Taken together, our study indicates that introgression-driven convergence likely explains the long-standing challenges in resolving the taxonomy and systematics of this small but phenotypically highly diverse group of fish.

Genetic population structure constrains local adaptation in sticklebacks.

Repeated and independent adaptation to specific environmental conditions from standing genetic variation is common. However, if genetic variation is limited, the evolution of similar locally adapted traits may be restricted to genetically different and potentially less optimal solutions or prevented from happening altogether. Using a quantitative trait locus (QTL) mapping approach, we identified the genomic regions responsible for the repeated pelvic reduction (PR) in three crosses between nine-spined stickleback populations expressing full and reduced pelvic structures. In one cross, PR mapped to linkage group 7 (LG7) containing the gene Pitx1, known to control pelvic reduction also in the three-spined stickleback. In the two other crosses, PR was polygenic and attributed to 10 novel QTL, of which 90% were unique to specific crosses. When screening the genomes from 27 different populations for deletions in the Pitx1 regulatory element, these were only found in the population in which PR mapped to LG7, even though the morphological data indicated large-effect QTL for PR in several other populations as well. Consistent with the available theory and simulations parameterized on empirical data, we hypothesize that the observed variability in genetic architecture of PR is due to heterogeneity in the spatial distribution of standing genetic variation caused by >2× stronger population structuring among freshwater populations and >10× stronger genetic isolation by distance in the sea in nine-spined sticklebacks as compared to three-spined sticklebacks.

Substantially adaptive potential in polyploid cyprinid fishes: evidence from biogeographic, phylogenetic and genomic studies.

Whole genome duplication (WGD) is commonly believed to play key roles in vertebrate evolution. However, nowadays polyploidy exists in a few fish, amphibian and reptile groups only, and seems to be an evolutionary dead end in vertebrates. We investigate the evolutionary significance of polyploidization in Cyprinidae-a fish family that contains more polyploid species than any other vertebrate group-with integrated biogeographic, phylogenetic and genomic analyses. First, polyploid species are found to be significantly frequent in areas of higher altitude and lower mean annual temperature compared with diploid species in Cyprinidae. Second, a polyploidy-related diversification rate shift is observed in Cyprinidae. This increased net diversification rate is only seen in three polyploid lineages, and other polyploid lineages have similar net diversification rate as well as diploid lineages in Cyprinidae. Interestingly, significant 'lag times' existed between polyploidization and radiation in Cyprinidae. Multiple polyploid lineages were established approximately 15 Ma through recurrent allopolyploidization events, but the net diversification rate did not start to increase until approximately 5 Ma-long after polyploidization events. Environmental changes associated with the continuous uplift of the Tibetan Plateau and climate change have probably promoted the initial establishment and subsequent radiation of polyploidy in Cyprinidae. Finally, the unique retention of duplicated genes in polyploid cyprinids adapted to harsh environments is found. Taken together, our results suggest that polyploidy in Cyprinidae is far more than an evolutionary dead end, but rather shows substantially adaptive potential. Polyploid cyprinids thus constitute an ideal model system for unveiling largely unexplored consequences of WGD in vertebrates, from genomic evolution to species diversification.

Population transcriptomics reveals weak parallel genetic basis in repeated marine and freshwater divergence in nine-spined sticklebacks.

The degree to which adaptation to similar selection pressures is underlain by parallel versus non-parallel genetic changes is a topic of broad interest in contemporary evolutionary biology. Sticklebacks provide opportunities to characterize and compare the genetic underpinnings of repeated marine-freshwater divergences at both intra- and interspecific levels. While the degree of genetic parallelism in repeated marine-freshwater divergences has been frequently studied in the three-spined stickleback (Gasterosteus aculeatus), much less is known about this in other stickleback species. Using a population transcriptomic approach, we identified both genetic and gene expression variations associated with marine-freshwater divergence in the nine-spined stickleback (Pungitius pungitius). Specifically, we used a genome-wide association study approach, and found that ~1% of the total 173,491 identified SNPs showed marine-freshwater ecotypic differentiation. A total of 861 genes were identified to have SNPs associated with marine-freshwater divergence, but only 12 of these genes have also been reported as candidates associated with marine-freshwater divergence in the three-spined stickleback. Hence, our results indicate a low degree of interspecific genetic parallelism in marine-freshwater divergence. Moreover, 1,578 genes in the brain and 1,050 genes in the liver were differentially expressed between marine and freshwater nine-spined sticklebacks, ~5% of which have also been identified as candidates associated with marine-freshwater divergence in the three-spined stickleback. However, only few of these (e.g., CLDND1) appear to have been involved in repeated marine-freshwater divergence in nine-spined sticklebacks. Taken together, the results indicate a low degree of genetic parallelism in repeated marine-freshwater divergence both at intra- and interspecific levels.

A phylogenomic perspective on diversity, hybridization and evolutionary affinities in the stickleback genus Pungitius.

Hybridization and convergent evolution are phenomena of broad interest in evolutionary biology, but their occurrence poses challenges for reconstructing evolutionary affinities among affected taxa. Sticklebacks in the genus Pungitius are a case in point: evolutionary relationships and taxonomic validity of different species and populations in this circumpolarly distributed species complex remain contentious due to convergent evolution of traits regarded as diagnostic in their taxonomy, and possibly also due to frequent hybridization among taxa. To clarify the evolutionary relationships among different Pungitius species and populations globally, as well as to study the prevalence and extent of introgression among recognized species, genomic data sets of both reference genome-anchored single nucleotide polymorphisms and de novo assembled RAD-tag loci were constructed with RAD-seq data. Both data sets yielded topologically identical and well-supported species trees. Incongruence between nuclear and mitochondrial DNA-based trees was found and suggested possibly frequent hybridization and mitogenome capture during the evolution of Pungitius sticklebacks. Further analyses revealed evidence for frequent nuclear genetic introgression among Pungitius species, although the estimated proportions of autosomal introgression were low. Apart from providing evidence for frequent hybridization, the results challenge earlier mitochondrial and morphology-based hypotheses regarding the number of species and their affinities in this genus: at least seven extant species can be recognized on the basis of genetic data. The results also shed new light on the biogeographical history of the Pungitius-complex, including suggestion of several trans-Arctic invasions of Europe from the Northern Pacific. The well-resolved phylogeny should facilitate the utility of this genus as a model system for future comparative evolutionary studies.

Complex signatures of genomic variation of two non-model marine species in a homogeneous environment.

BACKGROUND: Genomic tools are increasingly being used on non-model organisms to provide insights into population structure and variability, including signals of selection. However, most studies are carried out in regions with distinct environmental gradients or across large geographical areas, in which local adaptation is expected to occur. Therefore, the focus of this study is to characterize genomic variation and selective signals over short geographic areas within a largely homogeneous region. To assess adaptive signals between microhabitats within the rocky shore, we compared genomic variation between the Cape urchin (Parechinus angulosus), which is a low to mid-shore species, and the Granular limpet (Scutellastra granularis), a high shore specialist. RESULTS: Using pooled restriction site associated DNA (RAD) sequencing, we described patterns of genomic variation and identified outlier loci in both species. We found relatively low numbers of outlier SNPs within each species, and identified outlier genes associated with different selective pressures than those previously identified in studies conducted over larger environmental gradients. The number of population-specific outlier loci differed between species, likely owing to differential selective pressures within the intertidal environment. Interestingly, the outlier loci were highly differentiated within the two northernmost populations for both species, suggesting that unique evolutionary forces are acting on marine invertebrates within this region. CONCLUSIONS: Our study provides a background for comparative genomic studies focused on non-model species, as well as a baseline for the adaptive potential of marine invertebrates along the South African west coast. We also discuss the caveats associated with Pool-seq and potential biases of sequencing coverage on downstream genomic metrics. The findings provide evidence of species-specific selective pressures within a homogeneous environment, and suggest that selective forces acting on small scales are just as crucial to acknowledge as those acting on larger scales. As a whole, our findings imply that future population genomic studies should expand from focusing on model organisms and/or studying heterogeneous regions to better understand the evolutionary processes shaping current and future biodiversity patterns, particularly when used in a comparative phylogeographic context.

Complex Genes Are Preferentially Retained After Whole-Genome Duplication in Teleost Fish.

Gene duplication generates new genetic material which, if retained after duplication, may contribute to organismal evolution. A whole-genome duplication occurred in the ancestry of teleost fish and consequently there are many duplicated genes in teleost genomes. Indeed, it has been proposed that the evolutionary diversification of teleost fish may have been stimulated by the fish-specific genome duplication (FSGD). However, it is not clear which factors determine which genes are retained as duplicate copies and which return to a singleton state after duplication. In the present study, gene complexity, in terms of encoded protein length and functional domain number, is compared between duplicate and singleton genes for nine well-annotated teleost genomes. A total of 933 gene families with retained duplicates and 4590 singleton gene families are analysed. Genes with retained duplicates are found to be significantly longer (27.9-38.2%) and to have more functional domains (20.5-26.5%) than singleton genes in all the nine teleost genomes, suggesting that genes encoded longer proteins with and more functional domains were preferentially retained after whole-genome duplication in teleosts. This differential retention of duplicated genes will have increased the genomic complexity of teleost fish after FSGD which, together with differential duplicated gene retention as a lineage-splitting force, may have greatly contributed to the successful diversification of teleost fish.

Deciphering the genomic architecture of the stickleback brain with a novel multilocus gene-mapping approach.

Quantitative traits important to organismal function and fitness, such as brain size, are presumably controlled by many small-effect loci. Deciphering the genetic architecture of such traits with traditional quantitative trait locus (QTL) mapping methods is challenging. Here, we investigated the genetic architecture of brain size (and the size of five different brain parts) in nine-spined sticklebacks (Pungitius pungitius) with the aid of novel multilocus QTL-mapping approaches based on a de-biased LASSO method. Apart from having more statistical power to detect QTL and reduced rate of false positives than conventional QTL-mapping approaches, the developed methods can handle large marker panels and provide estimates of genomic heritability. Single-locus analyses of an F2 interpopulation cross with 239 individuals and 15 198, fully informative single nucleotide polymorphisms (SNPs) uncovered 79 QTL associated with variation in stickleback brain size traits. Many of these loci were in strong linkage disequilibrium (LD) with each other, and consequently, a multilocus mapping of individual SNPs, accounting for LD structure in the data, recovered only four significant QTL. However, a multilocus mapping of SNPs grouped by linkage group (LG) identified 14 LGs (1-6 depending on the trait) that influence variation in brain traits. For instance, 17.6% of the variation in relative brain size was explainable by cumulative effects of SNPs distributed over six LGs, whereas 42% of the variation was accounted for by all 21 LGs. Hence, the results suggest that variation in stickleback brain traits is influenced by many small-effect loci. Apart from suggesting moderately heritable (h2  ≈ 0.15-0.42) multifactorial genetic architecture of brain traits, the results highlight the challenges in identifying the loci contributing to variation in quantitative traits. Nevertheless, the results demonstrate that the novel QTL-mapping approach developed here has distinctive advantages over the traditional QTL-mapping methods in analyses of dense marker panels.

Population genomic evidence for adaptive differentiation in the Baltic Sea herring.

Detecting and estimating the degree of genetic differentiation among populations of highly mobile marine fish having pelagic larval stages is challenging because their effective population sizes can be large, and thus, little genetic drift and differentiation is expected in neutral genomic sites. However, genomic sites subject to directional selection stemming from variation in local environmental conditions can still show substantial genetic differentiation, yet these signatures can be hard to detect with low-throughput approaches. Using a pooled RAD-seq approach, we investigated genomewide patterns of genetic variability and differentiation within and among 20 populations of Atlantic herring in the Baltic Sea (and adjacent Atlantic sites), where previous low-throughput studies and/or studies based on few populations have found limited evidence for genetic differentiation. Stringent quality control was applied in the filtering of 1 791 254 SNPs, resulting in a final data set of 68 182 polymorphic loci. Clear differentiation was identified between Atlantic and Baltic populations in many genomic sites, while differentiation within the Baltic Sea area was weaker and geographically less structured. However, outlier analyses - whether including all populations or only those within the Baltic Sea - uncovered hundreds of directionally selected loci in which variability was associated with either salinity, temperature or both. Hence, our results support the view that although the degree of genetic differentiation among Baltic Sea herring populations is low, there are many genomic regions showing elevated divergence, apparently as a response to temperature- and salinity-related natural selection. As such, the results add to the increasing evidence of local adaptation in highly mobile marine organisms, and those in the young Baltic Sea in particular.

Genomewide scan for adaptive differentiation along altitudinal gradient in the Andrew's toad Bufo andrewsi.

Recent studies of humans, dogs and rodents have started to discover the genetic underpinnings of high altitude adaptations, yet amphibians have received little attention in this respect. To identify possible signatures of adaptation to altitude, we performed a genome scan of 15 557 single nucleotide polymorphisms (SNPs) obtained with restriction site-associated DNA sequencing of pooled samples from 11 populations of Andrew's toad (Bufo andrewsi) from the edge of the Tibetan Plateau, spanning an altitudinal gradient from 1690 to 2768 m.a.s.l. We discovered significant geographic differentiation among all sites, with an average FST   = 0.023 across all SNPs. Apart from clear patterns of isolation by distance, we discovered numerous outlier SNPs showing strong associations with variation in altitude (1394 SNPs), average annual temperature (1859 SNPs) or both (1051 SNPs). Levels and patterns of genetic differentiation in these SNPs were consistent with the hypothesis that they have been subject to directional selection and reflect adaptation to altitudinal variation among the study sites. Genes with footprints of selection were significantly enriched in binding and metabolic processes. Several genes potentially related to high altitude adaptation were identified, although the identity and functional significance of most genomic targets of selection remain unknown. In general, the results provide genomic support for results of earlier common garden and low coverage genetic studies that have uncovered substantial adaptive differentiation along altitudinal and latitudinal gradients in amphibians.

Population genomic evidence for adaptive differentiation in Baltic Sea three-spined sticklebacks.

BACKGROUND: The degree of genetic differentiation among populations experiencing high levels of gene flow is expected to be low for neutral genomic sites, but substantial divergence can occur in sites subject to directional selection. Studies of highly mobile marine fish populations provide an opportunity to investigate this kind of heterogeneous genomic differentiation, but most studies to this effect have focused on a relatively low number of genetic markers and/or few populations. Hence, the patterns and extent of genomic divergence in high-gene flow marine fish populations remain poorly understood. RESULTS: We here investigated genome-wide patterns of genetic variability and differentiation in 10 marine populations of three-spined stickleback (Gasterosteus aculeatus) distributed across a steep salinity and temperature gradient in the Baltic Sea, by utilizing > 30,000 single nucleotide polymorphisms obtained with a pooled RAD-seq approach. We found that genetic diversity and differentiation varied widely across the genome, and identified numerous fairly narrow genomic regions exhibiting signatures of both divergent and balancing selection. Evidence was uncovered for substantial genetic differentiation associated with both salinity and temperature gradients, and many candidate genes associated with local adaptation in the Baltic Sea were identified. CONCLUSIONS: The patterns of genetic diversity and differentiation, as well as candidate genes associated with adaptation in Baltic Sea sticklebacks were similar to those observed in earlier comparisons between marine and freshwater populations, suggesting that similar processes may be driving adaptation to brackish and freshwater environments. Taken together, our results provide strong evidence for heterogenic genomic divergence driven by local adaptation in the face of gene flow along an environmental gradient in the post-glacially formed Baltic Sea.

Genomic signatures associated with recurrent scale loss in cyprinid fish.

Scale morphology represents a fundamental feature of fish and a key evolutionary trait underlying fish diversification. Despite frequent and recurrent scale loss throughout fish diversification, comprehensive genome-wide analyses of the genomic signatures associated with scale loss in divergent fish lineages remain scarce. In the current study, we investigated genome-wide signatures, specifically convergent protein-coding gene loss, amino acid substitutions, and cis-regulatory sequence changes, associated with recurrent scale loss in two divergent Cypriniformes lineages based on large-scale genomic, transcriptomic, and epigenetic data. Results demonstrated convergent changes in many genes related to scale formation in divergent scaleless fish lineages, including loss of P/Q-rich scpp genes (e.g. scpp6 and scpp7), accelerated evolution of non-coding elements adjacent to the fgf and fgfr genes, and convergent amino acid changes in genes (e.g. snap29) under relaxed selection. Collectively, these findings highlight the existence of a shared genetic architecture underlying recurrent scale loss in divergent fish lineages, suggesting that evolutionary outcomes may be genetically repeatable and predictable in the convergence of scale loss in fish.

Cryptic diversity begets challenges and opportunities in biodiversity research.

How many species of life are there on Earth? This is a question that we want to know but cannot yet answer. Some scholars speculate that the number of species may reach 2.2 billion when considering cryptic diversity and that each morphology-based insect species may contain an average of 3.1 cryptic species. With nearly two million described species, such high estimates of cryptic diversity would suggest that cryptic species are widespread. The development of molecular species delimitation has led to the discovery of a large number of cryptic species, and cryptic biodiversity has gradually entered our field of vision and attracted more attention. This paper introduces the concept of cryptic species, how they evolve, and methods by which they may be discovered and confirmed, and provides theoretical and methodological guidance for the study of hidden species. A workflow of how to confirm cryptic species is provided. In addition, the importance and reliability of multi-evidence-based integrated taxonomy are reaffirmed as a way to better standardize decision-making processes. Special focus on cryptic diversity and increased funding for taxonomy is needed to ensure that cryptic species in hyperdiverse groups are discoverable and described. An increased focus on cryptic species in the future will naturally arise as more difficult groups are studied, and thereby, we may finally better understand the rules governing the evolution and maintenance of cryptic biodiversity.

Genomic divergence between nine- and three-spined sticklebacks.

BACKGROUND: Comparative genomics approaches help to shed light on evolutionary processes that shape differentiation between lineages. The nine-spined stickleback (Pungitius pungitius) is a closely related species of the ecological 'supermodel' three-spined stickleback (Gasterosteus aculeatus). It is an emerging model system for evolutionary biology research but has garnered less attention and lacks extensive genomic resources. To expand on these resources and aid the study of sticklebacks in a phylogenetic framework, we characterized nine-spined stickleback transcriptomes from brain and liver using deep sequencing. RESULTS: We obtained nearly eight thousand assembled transcripts, of which 3,091 were assigned as putative one-to-one orthologs to genes found in the three-spined stickleback. These sequences were used for evaluating overall differentiation and substitution rates between nine- and three-spined sticklebacks, and to identify genes that are putatively evolving under positive selection. The synonymous substitution rate was estimated to be 7.1 × 10(-9) per site per year between the two species, and a total of 165 genes showed patterns of adaptive evolution in one or both species. A few nine-spined stickleback contigs lacked an obvious ortholog in three-spined sticklebacks but were found to match genes in other fish species, suggesting several gene losses within 13 million years since the divergence of the two stickleback species. We identified 47 SNPs in 25 different genes that differentiate pond and marine ecotypes. We also identified 468 microsatellites that could be further developed as genetic markers in nine-spined sticklebacks. CONCLUSION: With deep sequencing of nine-spined stickleback cDNA libraries, our study provides a significant increase in the number of gene sequences and microsatellite markers for this species, and identifies a number of genes showing patterns of adaptive evolution between nine- and three-spined sticklebacks. We also report several candidate genes that might be involved in differential adaptation between marine and freshwater nine-spined sticklebacks. This study provides a valuable resource for future studies aiming to identify candidate genes underlying ecological adaptation in this and other stickleback species.

Pervasive indels and their evolutionary dynamics after the fish-specific genome duplication.

Insertions and deletions (indels) in protein-coding genes are important sources of genetic variation. Their role in creating new proteins may be especially important after gene duplication. However, little is known about how indels affect the divergence of duplicate genes. We here study thousands of duplicate genes in five fish (teleost) species with completely sequenced genomes. The ancestor of these species has been subject to a fish-specific genome duplication (FSGD) event that occurred approximately 350 Ma. We find that duplicate genes contain at least 25% more indels than single-copy genes. These indels accumulated preferentially in the first 40 my after the FSGD. A lack of widespread asymmetric indel accumulation indicates that both members of a duplicate gene pair typically experience relaxed selection. Strikingly, we observe a 30-80% excess of deletions over insertions that is consistent for indels of various lengths and across the five genomes. We also find that indels preferentially accumulate inside loop regions of protein secondary structure and in regions where amino acids are exposed to solvent. We show that duplicate genes with high indel density also show high DNA sequence divergence. Indel density, but not amino acid divergence, can explain a large proportion of the tertiary structure divergence between proteins encoded by duplicate genes. Our observations are consistent across all five fish species. Taken together, they suggest a general pattern of duplicate gene evolution in which indels are important driving forces of evolutionary change.

Enlarged fins of Tibetan catfish provide new evidence of adaptation to high plateau.

The uplift of the Tibetan Plateau significantly altered the geomorphology and climate of the Euroasia by creating large mountains and rivers. Fishes are more likely to be affected relative to other organisms, as they are largely restricted to river systems. Faced with the rapidly flowing water in the Tibetan Plateau, a group of catfish has evolved greatly enlarged pectoral fins with more numbers of fin-rays to form an adhesive apparatus. However, the genetic basis of these adaptations in Tibetan catfishes remains elusive. In this study, we performed comparative genomic analyses based on the chromosome-level genome of Glyptosternum maculatum in family Sisoridae and detected some proteins with conspicuously high evolutionary rates in particular in genes involved in skeleton development, energy metabolism, and hypoxia response. We found that the hoxd12a gene evolved faster and a loss-of-function assay of hoxd12a supports a potential role for this gene in shaping the enlarged fins of these Tibetan catfishes. Other genes with amino acid replacements and signatures of positive selection included proteins involved in low temperature (TRMU) and hypoxia (VHL) responses. Functional assays reveal that the G. maculatumTRMU allele generates more mitochondrial ATP than the ancestral allele found in low-altitude fishes. Functional assays of VHL alleles suggest that the G. maculatum allele has lower transactivation activity than the low-altitude forms. These findings provide a window into the genomic underpinnings of physiological adaptations that permit G. maculatum to survive in the harsh environment of the Tibetan Himalayas that mirror those that are convergently found in other vertebrates such as humans.

Functional Innovation through Gene Duplication Followed by Frameshift Mutation.

In his influential book "Evolution by Gene Duplication", Ohno postulated that frameshift mutation could lead to a new function after duplication, but frameshift mutation is generally thought to be deleterious, and thus drew little attention in functional innovation in duplicate evolution. To this end, we here report an exhaustive survey of the genomes of human, mouse, zebrafish, and fruit fly. We identified 80 duplicate genes that involved frameshift mutations after duplication. The frameshift mutation preferentially located close to the C-terminus in most cases (55/88), which indicated that a frameshift mutation that changed the reading frame in a small part at the end of a duplicate may likely have contributed to adaptive evolution (e.g., human genes NOTCH2NL and ARHGAP11B) otherwise too deleterious to survive. A few cases (11/80) involved multiple frameshift mutations, exhibiting various patterns of modifications of the reading frame. Functionality of duplicate genes involving frameshift mutations was confirmed by sequence characteristics and expression profile, suggesting a potential role of frameshift mutation in creating functional novelty. We thus showed that genomes have non-negligible numbers of genes that have experienced frameshift mutations following gene duplication. Our results demonstrated the potential importance of frameshift mutations in molecular evolution, as Ohno verbally argued 50 years ago.

Contrasting population differentiation in two sympatric Triplophysa loaches on the Qinghai-Tibet Plateau.

Genetic differentiation in aquatic organisms is usually shaped by drainage connectivity. Sympatric aquatic species are thus expected to show similar population differentiation patterns and similar genetic responses to their habitats. Water bodies on the Qinghai-Tibet Plateau (QTP) have recently experienced dramatic physicochemical changes, threatening the biodiversity of aquatic organisms on the "roof of the world." To uncover ecological genetics in Tibetan loaches (Triplophysa)-the largest component of the QTP ichthyofauna-we characterized population differentiation patterns and adaptive mechanisms to salinity change in two sympatric and phylogenetically closely related Tibetan loaches, T. stewarti and T. stenura, by integrating population genomic, transcriptomic, and electron probe microanalysis approaches. Based on millions of genome-wide SNPs, the two Tibetan loach species show contrasting population differentiation patterns, with highly geographically structured and clear genetic differentiation among T. stewarti populations, whereas there is no such observation in T. stenura, which is also supported by otolith microchemistry mapping. While limited genetic signals of parallel adaption to salinity changes between the two species are found from either genetic or gene expression variation perspective, a catalog of genes involved in ion transport, energy metabolism, structural reorganization, immune response, detoxification, and signal transduction is identified to be related to adaptation to salinity change in Triplophysa loaches. Together, our findings broaden our understanding of the population characteristics and adaptive mechanisms in sympatric Tibetan loach species and would contribute to biodiversity conservation and management of aquatic organisms on the QTP.