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Hundreds of cell types form the vertebrate brain but it is largely unknown how similar cellular repertoires are between or within species or how cell-type diversity evolves. To examine cell-type diversity across and within species, we performed single-cell RNA sequencing of ~130,000 hypothalamic cells from zebrafish (Danio rerio) and surface and cave morphs of Mexican tetra (Astyanax mexicanus). We found that over 75% of cell types were shared between zebrafish and Mexican tetra, which diverged from a common ancestor over 150 million years ago. Shared cell types displayed shifts in paralogue expression that were generated by subfunctionalization after genome duplication. Expression of terminal effector genes, such as neuropeptides, was more conserved than the expression of their associated transcriptional regulators. Species-specific cell types were enriched for the expression of species-specific genes and characterized by the neofunctionalization of expression patterns of members of recently expanded or contracted gene families. Comparisons between surface and cave morphs revealed differences in immune repertoires and transcriptional changes in neuropeptidergic cell types associated with genomic differences. The single-cell atlases presented here are a powerful resource to explore hypothalamic cell types and reveal how gene family evolution and shifts in paralogue expression contribute to cellular diversity. Single-cell transcriptomics of hypothalamic cells from zebrafish and surface and cave morphs of Mexican tetra shows conservation of cell types and that cellular novelty is associated with genetic novelty and the species-specific expression of paralogous genes.

Summary The critical role of cytokinin in early nodulation in legumes is well known. In our study, exogenous cytokinin application to roots of the important crop legume, chickpea (Cicer arietinum L.), led to the formation of pseudo‐nodules even in the absence of rhizobia. Hence, a genome‐wide analysis of the cytokinin signalling, two‐component system (TCS) genes, was conducted in chickpea, Medicago and Cajanus cajan. The integrated phylogenetic, evolutionary and expression analysis of the TCS genes was carried out, which revealed that histidine kinases (HKs) were highly conserved, whereas there was diversification leading to neofunctionalization at the level of response regulators (RRs) especially the TypeB RRs. Further, the functional role of the CaHKs in nodulation was established by complementation of the sln1Δ mutant of yeast and cre1 mutants of (Medicago) which led to restoration of the nodule‐deficient phenotype. Additionally, the highest expressing TypeB RR of chickpea, CaRR13, was functionally characterized. Its localization in the nucleus and its Y1H assay‐based interaction with the promoter of the early nodulation gene CaNSP2 indicated its role as a transcription factor regulating early nodulation. Overexpression, RNAi lines and complementation of cre1 mutants with CaRR13 revealed its critical involvement as an important signalling molecule regulating early events of nodule organogenesis in chickpea.

Ascorbate-glutathione (AsA-GSH) cycle plays an important role in tuning beneficial ROS accumulation for intracellular signals and imparts plant tolerance to oxidative stress by detoxifying excess of ROS. Here, we present genome-wide identification of AsA-GSH cycle genes (APX, MDHAR, DHAR, and GR) in several leguminous species and expression analyses in G. max during stress, germination and tissue development. Our data revealed 24 genes in Glycine genus against the maximum of 15 in other leguminous species, which was due to 9 pars of duplicated genes mostly originated from sub/neofunctionalization. Cytosolic APX and MDHAR genes were highly expressed in different tissues and physiological conditions. Germination induced genes encoding AsA-GSH proteins from different cell compartments, whereas vegetative phase (leaves) stimulated predominantly genes related to chloroplast/mitochondria proteins. Moreover, cytosolic APX-1, 2, MDHAR-1a, 1b and GR genes were the primary genes linked to senescence and biotic stresses, while stAPX-a, b and GR (from organelles) were the most abiotic stress related genes. Biotic and abiotic stress tolerant genotypes generally showed increased MDHAR, DHAR and/or GR mRNA levels compared to susceptible genotypes. Overall, these data clarified evolutionary events in leguminous plants and point to the functional specificity of duplicated genes of the AsA-GSH cycle in G. max.

Group A PP2C (PP2CA) genes form a gene subfamily whose members play an important role in regulating many biological processes by dephosphorylation of target proteins. In this study we examined the effects of evolutionary changes responsible for functional divergence of BnaABI1 paralogs in Brassica napus against the background of the conserved PP2CA gene subfamily in Brassicaceae. We performed comprehensive phylogenetic analyses of 192 PP2CA genes in 15 species in combination with protein structure homology modeling. Fundamentally, the number of PP2CA genes remained relatively constant in these taxa, except in the Brassica genus and Camelina sativa. The expansion of this gene subfamily in these species has resulted from whole genome duplication. We demonstrated a high degree of structural conservation of the PP2CA genes, with a few minor variations between the different PP2CA groups. Furthermore, the pattern of conserved sequence motifs in the PP2CA proteins and their secondary and 3D structures revealed strong conservation of the key ion-binding sites. Syntenic analysis of triplicated regions including ABI1 paralogs revealed significant structural rearrangements of the Brassica genomes. The functional and syntenic data clearly show that triplication of BnaABI1 in B. napus has had an impact on its functions, as well as the positions of adjacent genes in the corresponding chromosomal regions. The expression profiling of BnaABI1 genes showed functional divergence, i.e. subfunctionalization, potentially leading to neofunctionalization. These differences in expression are likely due to changes in the promoters of the BnaABI1 paralogs. Our results highlight the complexity of PP2CA gene subfamily evolution in Brassicaceae.

Aedes aegypti can act as a vector for viral pathogens but the mechanism of viral resistance and evolving host-pathogen tolerance are poorly understood. Here the authors structurally characterise a duplicated pair of interacting Toll immunoreceptors and the cytokine ligand Spaetzle1C and show their dose-dependent function and mechanism of activation. Aedes aegypti has evolved to become an efficient vector for arboviruses but the mechanisms of host-pathogen tolerance are unknown. Immunoreceptor Toll and its ligand Spaetzle have undergone duplication which may allow neofunctionalization and adaptation. Here we present cryo-EM structures and biophysical characterisation of low affinity Toll5A complexes that display transient but specific interactions with Spaetzle1C, forming asymmetric complexes, with only one ligand clearly resolved. Loop structures of Spaetzle1C and Toll5A intercalate, temporarily bridging the receptor C-termini to promote signalling. By contrast unbound receptors form head-to-head homodimers that keep the juxtamembrane regions far apart in an inactive conformation. Interestingly the transcriptional signature of Spaetzle1C differs from other Spaetzle cytokines and controls genes involved in innate immunity, metabolism and tissue regeneration. Taken together our results explain how upregulation of Spaetzle1C in the midgut and Toll5A in the salivary gland shape the concomitant immune response.

SummaryThe hemiparasitic plant Phtheirospermum japonicum is a nutritional specialist that supplements its nutrient requirements by parasitizing other plants through haustoria. During parasitism, the Phtheirospermum haustorium transfers hypertrophy-inducing cytokinins (CKs) to the infected host root. The CK biosynthesis genes required for haustorium-derived CKs and the induction of hypertrophy are still unknown. We searched for haustorium-expressed isopentenyltransferases (IPTs) that catalyse the first step of CK biosynthesis, confirmed the specific expression by in vivo imaging of a promoter-reporter, and further analysed the subcellular localization, the enzymatic function, and contribution to inducing hypertrophy by studying CRISPR-Cas9 induced Phtheirospermum mutants. PjIPT1a was expressed in intrusive cells of the haustorium close to the host vasculature. PjIPT1a and its closest homolog PjIPT1b located to the cytosol and showed isopentenyltransferases activity in vitro with differences in substrate specificity. Mutating PjIPT1a abolished parasite-induced CK responses in the host. A homolog of PjIPT1a with shared characteristics was also identified in the related weed Striga hermonthica. We propose that PjIPT1a exemplifies how parasitism-related functions evolve through gene duplications and neofunctionalization.

Emerging evidence links genes within human-specific segmental duplications (HSDs) to traits and diseases unique to our species. Strikingly, despite being nearly identical by sequence (>98.5%), paralogous HSD genes are differentially expressed across human cell and tissue types, though the underlying mechanisms have not been examined. We compared cross-tissue mRNA levels of 75 HSD genes from 30 families between humans and chimpanzees and found expression patterns consistent with relaxed selection on or neofunctionalization of derived paralogs. In general, ancestral paralogs exhibited greatest expression conservation with chimpanzee orthologs, though exceptions suggest certain derived paralogs may retain or supplant ancestral functions. Concordantly, analysis of long-read isoform sequencing data sets from diverse human tissues and cell lines found that about half of derived paralogs exhibited globally lower expression. To understand mechanisms underlying these differences, we leveraged data from human lymphoblastoid cell lines (LCLs) and found no relationship between paralogous expression divergence and post-transcriptional regulation, sequence divergence, or copy-number variation. Considering cis-regulation, we reanalyzed ENCODE data and recovered hundreds of previously unidentified candidate CREs in HSDs. We also generated large-insert ChIP-sequencing data for active chromatin features in an LCL to better distinguish paralogous regions. Some duplicated CREs were sufficient to drive differential reporter activity, suggesting they may contribute to divergent cis-regulation of paralogous genes. This work provides evidence that cis-regulatory divergence contributes to novel expression patterns of recent gene duplicates in humans.

Horizontal gene transfer (HGT) plays an important role in evolutionary processes as organisms adapt to their environments, and now cases of gene duplication after HGT in eukaryotes are emerging at an increasing rate. However, the fate and roles of the duplicated genes over time in eukaryotes remain unclear. Here we conducted a comprehensive analysis of the evolution of cysteine synthase (CYS) in lepidopteran insects. Our results indicate that HGT-derived CYS genes are widespread and have undergone duplication following horizontal transfer in many lepidopteran insects. Moreover, lepidopteran CYS proteins not only have β-cyanoalanine synthase activity but also possess cysteine synthase activity that is involved in sulfur amino acid biosynthesis. Duplicated CYS genes show marked divergence in gene expression patterns and enzymatic properties, suggesting that they probably have undergone subfunctionalization and/or neofunctionalization in Lepidoptera. The gene transfer of CYS genes and subsequent duplication appears to have facilitated the adaptation of lepidopteran insects to different diets and promoted their ecological diversification. Overall, this study provides valuable insights into the ecological and evolutionary contributions of CYS in lepidopteran insects.

Key message Mapping combined with expression and variant analyses in switchgrass, a crop with complex genetics, identified a cluster of candidate genes for leaf wax in a fast-evolving region of chromosome 7K. Abstract Switchgrass (Panicum virgatum L.) is a promising warm-season candidate energy crop. It occurs in two ecotypes, upland and lowland, which vary in a number of phenotypic traits, including leaf glaucousness. To initiate trait mapping, two F2 mapping populations were developed by crossing two different F1 sibs derived from a cross between the tetraploid lowland genotype AP13 and the tetraploid upland genotype VS16, and high-density linkage maps were generated. Quantitative trait locus (QTL) analyses of visually scored leaf glaucousness and of hydrophobicity of the abaxial leaf surface measured using a drop shape analyzer identified highly significant colocalizing QTL on chromosome 7K (Chr07K). Using a multipronged approach, we identified a cluster of genes including Pavir.7KG077009, which encodes a Type III polyketide synthase-like protein, and Pavir.7KG013754 and Pavir.7KG030500, two highly similar genes that encode putative acyl-acyl carrier protein (ACP) thioesterases, as strong candidates underlying the QTL. The lack of homoeologs for any of the three genes on Chr07N, the relatively low level of identity with other switchgrass KCS proteins and thioesterases, as well as the organization of the surrounding region suggest that Pavir.7KG077009 and Pavir.7KG013754/Pavir.7KG030500 were duplicated into a fast-evolving chromosome region, which led to their neofunctionalization. Furthermore, sequence analyses showed all three genes to be absent in the two upland compared to the two lowland accessions analyzed. This study provides an example of and practical guide for trait mapping and candidate gene identification in a complex genetic system by combining QTL mapping, transcriptomics and variant analysis.

Gene duplication is an important driver of genomic diversity that can promote adaptive evolution. However, like most mutations, a newly duplicated gene is often deleterious and removed from the genome by drift or natural selection. The early molecular changes that occur soon after duplication therefore may influence the long-term survival of gene duplicates, but relatively little empirical data exist on the events near the onset of duplication before mutations have time to accumulate. In this study, we contrast gene expression and DNA methylation levels of duplicate genes in the threespine stickleback, Gasterosteus aculeatus, including recently emerged duplications that segregate as copy number variations (CNVs). We find that younger duplicate genes have higher levels of promoter methylation than older genes, and that gene CNVs have higher promoter methylation than non-CNVs. These results suggest preferential duplication of highly methylated genes or rapid methylation changes soon after duplication. We also find a negative association between methylation and expression, providing a putative role for methylation in suppressing transcription that compensates for increases in gene copy numbers and promoting paralog retention. We propose that methylation contributes to the longevity of young duplicate genes, extending the window of opportunity for functional divergence via mutation.

Variations in gene expression patterns represent a powerful source of evolutionary innovation. In a rodent living about 70 million years ago, a genomic accident led an immune formyl peptide receptor (FPR) gene to hijack a vomeronasal receptor regulatory sequence. This gene shuffling event forced an immune pathogen sensor to transition into an olfactory chemoreceptor, which thus moved from sensing the internal world to probing the outside world. We here discuss the evolution of the FPR gene family, the events that led to their neofunctionalization in the vomeronasal organ and the functions of immune and vomeronasal FPRs.

New genes (or lineage‐specific genes) can facilitate functional innovations. MALE STERILITY 5 (MS5) in Brassica napus is a fertility‐related new gene, which has two wild‐type alleles (BnMS5a and BnMS5c) and two mutant alleles (BnMS5b and BnMS5d) that could induce male sterility. Here, we studied the history and functional evolution of MS5 homologs in plants by phylogenetic analysis and molecular genetic experiments. We identified 727 MS5 homologs and found that they define a Brassicaceae‐specific gene family that has expanded partly via multiple tandem gene duplications and also probably transpositions. The MS5 in B. napus is inherited from a basic diploid ancestor of B. rapa. Molecular genetic experiments indicate that BnMS5a and BnMS5c are functionally distinct in B. napus and that BnMS5d can inhibit BnMS5a in B. napus in a dosage‐dependent manner. The BnMS5a protein can move in coordination with meiotic telomeres and interact with the nuclear envelope protein SUN1, with a possible crucial role in meiotic chromosome behavior. In summary, BnMS5 belongs to a Brassicaceae‐specific new gene family, and has gained a novel function that is essential for male fertility in B. napus through neofunctionalization that has likely occurred since the origin of B. rapa.

The regulation of floral organ identity was investigated using a forward genetic approach in five floral homeotic mutants of Thalictrum, a noncore eudicot. We hypothesized that these mutants carry defects in the floral patterning genes. Mutant characterization comprised comparative floral morphology and organ identity gene expression at early and late developmental stages, followed by sequence analysis of coding and intronic regions to identify transcription factor binding sites and protein-protein interaction (PPI) motifs. Mutants exhibited altered expression of floral MADS-box genes, which further informed the function of paralogs arising from gene duplications not found in reference model systems. The ensuing modified BCE models for the mutants supported instances of neofunctionalization (e.g., B-class genes expressed ectopically in sepals), partial redundancy (E-class), or subfunctionalization (C-class) of paralogs. A lack of deleterious mutations in the coding regions of candidate floral MADS-box genes suggested that cis-regulatory or trans-acting mutations are at play. Consistent with this hypothesis, double-flower mutants had transposon insertions or showed signs of transposon activity in the regulatory intron of AGAMOUS (AG) orthologs. Single amino acid substitutions were also found, yet they did not fall on any of the identified DNA binding or PPI motifs. In conclusion, we present evidence suggesting that transposon activity and regulatory mutations in floral homeotic genes likely underlie the striking phenotypes of these Thalictrum floral homeotic mutants.

Although SQUAMOSA promoter-binding-like (SPL) transcription factors are important regulators of development in rice (Oryza sativa), prior assessments of the SPL family have been limited to single genes. A functional comparison across the full gene family in standardized genetic backgrounds has not been reported previously. Here, we demonstrate that the SPL gene family in rice is enriched due to the most recent whole genome duplication (WGD). Notably, 10 of 19 rice SPL genes (52%) cluster in four units that have persisted for at least 50 million years. We show that SPL gene grouping and retention following WGD is widespread in angiosperms, suggesting the conservatism and importance of this gene arrangement. We used Cas9 editing to generate transformation lines for all 19 SPL genes in a common set of backgrounds, and found that knockouts of 14 SPL genes exhibited defects in plant height, 10 exhibited defects in panicle size, and nine had altered grain lengths. We observed subfunctionalization of genes in the paleoduplicated pairs, but little evidence of neofunctionalization. Expression of OsSPL3 was negatively correlated with that of its closest neighbor in its synteny group, OsSPL4, and its sister paired gene, OsSPL12, in the opposing group. Nucleotide diversity was lower in eight of the nine singleton genes in domesticated rice, relative to wild rice, whereas the reverse was true for the paired genes. Together, these results provide functional information on eight previously unexamined OsSPL family members and suggest that paleoduplicate pair redundancy benefits plant survival and innovation.

Learning about the roles that duplicate genes play in the origins of novel phenotypes requires an understanding of how their functions evolve. To date, only one method—CDROM—has been developed with this goal in mind. In particular, CDROM employs gene expression distances as proxies for functional divergence, and then classifies the evolutionary mechanisms retaining duplicate genes from comparisons of these distances in a decision tree framework. However,CDROMdoes not account for stochastic shifts in gene expression or leverage advances in contemporary statistical learning for performing classification, nor is it capable of predicting the underlying parameters of duplicate gene evolution. Thus, here we developCLOUD, a multi-layer neural network built upon a model of gene expression evolution that can both classify duplicate gene retention mechanisms and predict their underlying evolutionary parameters. We show that not only is theCLOUDclassifier substantially more powerful and accurate thanCDROM, but that it also yields accurate parameter predictions, enabling a better understanding of the specific forces driving the evolution and long-term retention of duplicate genes. Further, application of theCLOUDclassifier and predictor to empirical data fromDrosophilarecapitulates many previous findings about gene duplication in this lineage, showing that new functions often emerge rapidly and asymmetrically in younger duplicate gene copies, and that functional divergence is driven by strong natural selection. Hence,CLOUDrepresents the best available method for classifying retention mechanisms and predicting evolutionary parameters of duplicate genes, thereby also highlighting the utility of incorporating sophisticated statistical learning techniques to address long-standing questions about evolution after gene duplication.

Whole genome duplication (WGD) is a common phenomenon in plants, inducing species diversity, promoting speciation, and playing an important role in providing new genetic material in plant evolution. So far, numerous new gene functions such as pseudogenization, neofunctionalization, and subfunctionalization have evolved through WGD. WGD has occurred several times during the evolution of angiosperms over the past 200 million years. Poaceae, one of the largest plant families in the angiosperms, is considered one of the most economically important and ecologically successful plants, with approximately 600 genera and 10,000 species. Many genetic studies have been conducted on these plants. However, study of the most active phylogeny in the Poaceae family study has not yet been completed. Through comparative studies of major Poaceae groups, this review focuses on the effects of genome duplication in the Poaceae family on its evolutionary history and the changes in chromosome numbers.

Vision represents an excellent model for studying adaptation, given the genotype-to-phenotype-map that has been characterized in a number of taxa. Fish possess a diverse range of visual sensitivities and adaptations to underwater light making them an excellent group to study visual system evolution. In particular, some speciose but understudied lineages can provide a unique opportunity to better understand aspects of visual system evolution such as opsin gene duplication and neofunctionalization. In this study, we characterized the visual system of Neotropical Characiformes, which is the result of several spectral tuning mechanisms acting in concert including gene duplications and losses, gene conversion, opsin amino acid sequence and expression variation, and A1/A2-chromophore shifts. The Characiforms we studied utilize three cone opsin classes (SWS2, RH2, LWS) and a rod opsin (RH1). However, the characiform’s entire opsin gene repertoire is a product of dynamic evolution by opsin gene loss (SWS1, RH2) and duplication (LWS, RH1). The LWS- and RH1-duplicates originated from a teleost specific whole-genome duplication as well as characiform-specific duplication events. Both LWS-opsins exhibit gene conversion and, through substitutions in key tuning sites, one of the LWS-paralogs has acquired spectral sensitivity to green light. These sequence changes suggest reversion and parallel evolution of key tuning sites. In addition, characiforms exhibited species-specific differences in opsin expression. Finally, we found interspecific and intraspecific variation in the use of A1/A2-chromophores correlating with the light environment. These multiple mechanisms may be a result of the highly diverse visual environments where Characiformes have evolved.

Auxin controls numerous growth processes in land plants through a gene expression system that modulates ARF transcription factor activity1–3. Gene duplications in families encoding auxin response components have generated tremendous complexity in most land plants, and neofunctionalization enabled various unique response outputs during development1,3,4. However, it is unclear what fundamental biochemical principles underlie this complex response system. By studying the minimal system in Marchantia polymorpha, we derive an intuitive and simple model where a single auxin-dependent A-ARF activates gene expression. It is antagonized by an auxin-independent B-ARF that represses common target genes. The expression patterns of both ARF proteins define developmental zones where auxin response is permitted, quantitatively tuned or prevented. This fundamental design probably represents the ancestral system and formed the basis for inflated, complex systems. The minimal regulatory logic and the evolutionary history of the auxin signalling pathway are explored by studying the much simpler auxin network in Marchantia with a combination of genetic, domain shuffling and biochemical strategies.

AT-hook motif nuclear localized (AHL) genes have diverse but poorly understood biological functions. We identified and analyzed 37 AHL genes in maize. We also discovered four and one additional AHLs in rice and sorghum, respectively, besides those reported earlier. The maize AHLs were classified into two clades (A and B) and three distinct types (I, II, and III) as also reported in Arabidopsis. Phylogenetic and ortholog analyses showed that, while the evolutionary classification was conserved in plants, expansion of the AHL gene family in maize was accompanied with new biological functions. Gene structure analysis showed that, while all but one Type-I AHLs lacked an intron, origin of Type-II and Type-III AHLs was associated with the gain of introns suggesting evolutionarily distinct temporal and spatial expression patterns and, likely, neofunctionalization. Gene duplication analysis revealed that AHLs in maize expanded via dispersive duplication further supporting their functional diversity. To discern these functions, we analyzed 71 transcriptomes from diverse tissues and developmental stages of maize and classified AHLs into eight groups with distinct temporal/spatial expression profiles. Coexpression analysis implicated 5 AHLs and 33 novel genes in networks specific to endosperm, seed, root, leaf, and reproductive tissues indicating their role in the development of these organs. Major processes coregulated by AHLs include pollen development, drought response, senescence, and wound response. We also identified interactions of AHL proteins in coregulating important processes including stress response. These novel insights into the role of AHLs in plant development provide a platform for functional analyses in maize and related grasses.

UDP- glucose: glycoprotein glucosyltransferase (UGGT) is a protein that operates as the gatekeeper for the endoplasmic reticulum (ER) quality control mechanism of glycoprotein folding. It is known that vertebrates and Caenorhabditis genomes harbor two uggt gene copies that exhibit differences in their properties.Bayesian phylogenetic inference based on 195 UGGT and UGGT-like protein sequences of an ample spectrum of eukaryotic species showed that uggt genes went through independent duplications in Caenorhabditis and vertebrates. In both lineages, the catalytic domain of the duplicated genes was subjected to a strong purifying selective pressure, while the recognition domain was subjected to episodic positive diversifying selection. Selective relaxation in the recognition domain was more pronounced in Caenorhabditis uggt-b than in vertebrates uggt-2. Structural bioinformatics analysis revealed that Caenorhabditis UGGT-b protein lacks essential sequences proposed to be involved in the recognition of unfolded proteins. When we assayed glucosyltrasferase activity of a chimeric protein composed by Caenorhabditis uggt-b recognition domain fused to S. pombe catalytic domain expressed in yeast, no activity was detected.The present results support the conservation of the UGGT activity in the catalytic domain and a putative divergent function of the recognition domain for the UGGT2 protein in vertebrates, which would have gone through a specialization process. In Caenorhabditis, uggt-b evolved under different constraints compared to uggt-a which, by means of a putative neofunctionalization process, resulted in a non-redundant paralog. The non-canonical function of uggt-b in the worm lineage highlights the need to take precautions before generalizing gene functions in model organisms.

The conservation of orthologs of most subunits of the origin recognition complex (ORC) has served to propose that the whole complex is common to all eukaryotes. However, various uncertainties have arisen concerning ORC subunit composition in a variety of lineages. Also, it is unclear whether the ancestral diversification of ORC in eukaryotes was accompanied by the neofunctionalization of some subunits, for example, role of ORC1 in centriole homeostasis. We have addressed these questions by reconstructing the distribution and evolutionary history of ORC1-5/CDC6 in a taxon-rich eukaryotic data set. First, we identified ORC subunits previously undetected in divergent lineages, which allowed us to propose a series of parsimonious scenarios for the origin of this multiprotein complex. Contrary to previous expectations, we found a global tendency in eukaryotes to increase or decrease the number of subunits as a consequence of genome duplications or streamlining, respectively. Interestingly, parasites show significantly lower number of subunits than free-living eukaryotes, especially those with the lowest genome size and gene content metrics. We also investigated the evolutionary origin of the ORC1 role in centriole homeostasis mediated by the PACT region in human cells. In particular, we tested the consequences of reducing ORC1 levels in the centriole-containing green alga Chlamydomonas reinhardtii. We found that the proportion of centrioles to flagella and nuclei was not dramatically affected. This, together with the PACT region not being significantly more conserved in centriole-bearing eukaryotes, supports the notion that this neofunctionalization of ORC1 would be a recent acquisition rather than an ancestral eukaryotic feature., E.O.-P. was supported by a predoctoral FPI grant from Ministerio de Economía y Empresa (MINECO). Research in our laboratories was supported by grants BIO2017-92329-EXP, RTI2018-094793-B-I00 (MINECO, Ministerio de Ciencia e Innovación, Fondo Europeo de Desarrollo Regional [FEDER]), and ERC-2018-AdG_833617 (European Union [EU]) to C.G., grant BFU2017-90114-P (MINECO, FEDER) to I.R.-T., and grants BFU2015-70649-P (MINECO) and U.E.INTERREG VA POCTEP-055_ALGARED_PLUS5_E (EU) to E.F., and by institutional grants from Banco de Santander and Fundacion Ramon Areces to the CBMSO.

Abstract Earlier we suggested a new hypothesis of the possible evolutionary role of hereditary tumors (Kozlov, Evolution by tumor Neofunctionalization, 2014), and described a new class of genes – tumor specifically expressed, evolutionarily novel (TSEEN) genes - that are predicted by this hypothesis (Kozlov, Infect Agents Cancer 11:34, 2016). In this paper we studied evolutionarily novel genes expressed in fish tumors after regression, as a model of evolving organs. As evolutionarily novel genes may not yet have organismal functions, we studied the acquisition of new gene functions by comparing fish evolutionarily novel genes with their human orthologs. We found that many genes involved in development of progressive traits in humans (lung, mammary gland, placenta, ventricular septum, etc.) originated in fish and are expressed in fish tumors and tumors after regression. These findings support a possible evolutionary role of hereditary tumors, and in particular the hypothesis of evolution by tumor neofunctionalization. Research highlights Earlier we described a new class of genes that are tumor-specifically expressed and evolutionarily novel (TSEEN). As the functions of TSEEN genes are often uncertain, we decided to study TSEEN genes of fishes so that we could trace the appearance of their new functions in higher vertebrates. We found that many human genes which are involved in development of progressive traits (placenta development, mammary gland and lung development etc.,) originated in fishes and are expressed in fish tumors.

Germ-soma differentiation evolved independently in many eukaryotic lineages and contributed to complex multicellular organizations. However, the molecular genetic bases of such convergent evolution remain unresolved. Two multicellular volvocine green algae, Volvox and Astrephomene, exhibit convergent evolution of germ-soma differentiation. The complete genome sequence is now available for Volvox, while genome information is scarce for Astrephomene. Here, we generated the de novo whole genome sequence of Astrephomene gubernaculifera and conducted RNA-seq analysis of isolated somatic and reproductive cells. In Volvox, tandem duplication and neofunctionalization of the ancestral transcription factor gene (RLS1/rlsD) might have led to the evolution of regA, the master regulator for Volvox germ-soma differentiation. However, our genome data demonstrated that Astrephomene has not undergone tandem duplication of the RLS1/rlsD homolog or acquisition of a regA-like gene. Our RNA-seq analysis revealed the downregulation of photosynthetic and anabolic gene expression in Astrephomene somatic cells, as in Volvox. Among genes with high expression in somatic cells of Astrephomene, we identified three genes encoding putative transcription factors, which may regulate somatic cell differentiation. Thus, the convergent evolution of germ-soma differentiation in the volvocine algae may have occurred by the acquisition of different regulatory circuits that generate a similar division of labor.

Gene duplications generate new genes that can contribute to expression changes and the evolution of new functions. Genomes often consist of gene families that undergo expansions, some of which occur in specific lineages that reflect recent adaptive diversification. In this study, lineage-specific genes and gene family expansions were studied across five dictyostelid species to determine when and how they are expressed during multicellular development. Lineage-specific genes were found to be enriched among genes with biased expression (predominant expression in one developmental stage) in each species and at most developmental time points, suggesting independent functional innovations of new genes throughout the phylogeny. Biased duplicate genes had greater expression divergence than their orthologs and paralogs, consistent with subfunctionalization or neofunctionalization. Lineage-specific expansions in particular had biased genes with both molecular signals of positive selection and high expression, suggesting adaptive genetic and transcriptional diversification following duplication. Our results present insights into the potential contributions of lineage-specific genes and families in generating species-specific phenotypes during multicellular development in dictyostelids.

Reproductive proteins mediating fertilization commonly exhibit rapid sequence diversification driven by positive selection. This pattern has been observed among nearly all taxonomic groups, including mammals, invertebrates, and plants, and is remarkable given the essential nature of the molecular interactions mediating fertilization. Gene duplication is another iimportant mechanism that facilitates the generation of molecular novelty. Following duplication, paralogs may parse ancestral gene function (subfunctionalization) or acquire new roles (neofunctionalization). However, the contributions of duplication followed by sequence diversification to the molecular diversity of gamete recognition genes has been understudied in many models of fertilization. The marine gastropod mollusk abalone is a classic model for fertilization. Its two acrosomal proteins (lysin and sp18) are ancient gene duplicates with unique gamete recognition functions. Through detailed genomic and bioinformatic analyses we show how duplication events followed by sequence diversification has played an ongoing role in the evolution of abalone acrosomal proteins. The common ancestor of abalone had four members of its acrosomal protein family in a tandem gene array that repeatedly experienced positive selection. We find that both sp18 paralogs contain positively selected sites located in different regions of the paralogs, consistent with a subfunctionalization model where selection acted upon distinct binding interfaces in each paralog. Further, a more recent species-specific duplication of both lysin and sp18 in the European abalone H. tuberculata is described. Despite clade-specific acrosomal protein paralogs, there are no concomitant duplications of egg coat proteins in H. tuberculata, indicating that duplication of egg proteins per se is not responsible for retention of duplicated acrosomal proteins. We hypothesize that, in a manner analogous to host/pathogen evolution, sperm proteins are selected for increased diversity through extensive sequence divergence and recurrent duplication driven by conflict mechanisms.

How and to what degree gene duplication events create regulatory innovation, redundancy, or neofunctionalization remain important questions in animal evolution and comparative genetics. Ankfn1 genes are single copy in most invertebrates, partially duplicated in jawed vertebrates, and only the derived copy retained in most mammals. Null mutations in the single mouse homolog have vestibular and neurological abnormalities. Null mutation of the single Drosophila homolog is typically lethal with severe sensorimotor deficits in rare survivors. The functions and potential redundancy of paralogs in species with two copies is not known. Here we define a vestibular role for Ankfn1 homologs in zebrafish based on simultaneous disruption of each locus. Zebrafish with both paralogs disrupted showed vestibular defects and early lethality from swim bladder inflation failure. One intact copy at either locus was sufficient to prevent major phenotypes. Our results show that vertebrate Ankfn1 genes are required for vestibular-related functions, with at least partial redundancy between ancestral and derived paralogs.

Phytoplasmas are insect-transmitted plant pathogens that cause substantial losses in agriculture. In addition to economic impact, phytoplasmas induce distinct disease symptoms in infected plants, thus attracting attention for research on molecular plant-microbe interactions and plant developmental processes. Due to the difficulty of establishing an axenic culture of these bacteria, culture-independent genome characterization is a crucial tool for phytoplasma research. However, phytoplasma genomes have strong nucleotide composition biases and are repetitive, which make it challenging to produce complete assemblies. In this study, we utilized Illumina and Oxford Nanopore sequencing technologies to obtain the complete genome sequence of ‘Candidatus Phytoplasma luffae’ strain NCHU2019 that is associated with witches’ broom disease of loofah (Luffa aegyptiaca) in Taiwan. The fully assembled circular chromosome is 769 kb in size and is the first representative genome sequence of group 16SrVIII phytoplasmas. Comparative analysis with other phytoplasmas revealed that NCHU2019 has an exceptionally repetitive genome, possessing a pair of 75 kb repeats and at least 13 potential mobile units (PMUs) that account for ~25% of its chromosome. This level of genome repetitiveness is exceptional for bacteria, particularly among obligate pathogens with reduced genomes. Our genus-level analysis of PMUs demonstrated that these phytoplasma-specific mobile genetic elements can be classified into three major types that differ in gene organization and phylogenetic distribution. Notably, PMU abundance explains nearly 80% of the variance in phytoplasma genome sizes, a finding that provides a quantitative estimate for the importance of PMUs in phytoplasma genome variability. Finally, our investigation found that in addition to horizontal gene transfer, PMUs also contribute to intra-genomic duplications of effector genes, which may provide redundancy for neofunctionalization or subfunctionalization. Taken together, this work improves the taxon sampling for phytoplasma genome research and provides novel information regarding the roles of mobile genetic elements in phytoplasma evolution.

Bread wheat is an essential crop with the second-highest global production after maize. Currently, wheat diseases are a serious threat to wheat production. Therefore, efficient breeding for disease resistance is extremely urgent in modern wheat. Here, we identified 2012 NLR genes from hexaploid wheat, and Ks values of paired syntenic NLRs showed a significant peak at 3.1–6.3 MYA, which exactly coincided with the first hybridization event between A and B genome lineages at ~5.5 MYA. We provided a landscape of dynamic diversity of NLRs from Triticum and Aegilops and found that NLR genes have higher diversity in wild progenitors and relatives. Further, most NLRs had opposite diversity patterns between genic and 2 Kb-promoter regions, which might respectively link sub/neofunctionalization and loss of duplicated NLR genes. Additionally, we identified an alien introgression of chromosome 4A in tetraploid emmer wheat, which was similar to that in hexaploid wheat. Transcriptome data from four experiments of wheat disease resistance helped to profile the expression pattern of NLR genes and identified promising NLRs involved in broad-spectrum disease resistance. Our study provided insights into the diversity evolution of NLR genes and identified beneficial NLRs to deploy into modern wheat in future wheat disease-resistance breeding.

Plants are a remarkable source of high-value specialized metabolites having significant physiological and ecological functions. Genes responsible for synthesizing specialized metabolites are often clustered together for a coordinated expression, which is commonly observed in bacteria and filamentous fungi. Similar to prokaryotic gene clustering, plants do have gene clusters encoding enzymes involved in the biosynthesis of specialized metabolites. More than 20 gene clusters involved in the biosynthesis of diverse metabolites have been identified across the plant kingdom. Recent studies demonstrate that gene clusters are evolved through gene duplications and neofunctionalization of primary metabolic pathway genes. Often, these clusters are tightly regulated at nucleosome level. The prevalence of gene clusters related to specialized metabolites offers an attractive possibility of an untapped source of highly useful biomolecules. Accordingly, the identification and functional characterization of novel biosynthetic pathways in plants need to be worked out. In this review, we summarize insights into the evolution of gene clusters and discuss the organization and importance of specific gene clusters in the biosynthesis of specialized metabolites. Regulatory mechanisms which operate in some of the important gene clusters have also been briefly described. Finally, we highlight the importance of gene clusters to develop future metabolic engineering or synthetic biology strategies for the heterologous production of novel metabolites.

The superfamily of cytochrome P450 (CYP) enzymes plays key roles in plant evolution and metabolic diversification. This review provides a status on the CYP landscape within green algae and land plants. The 11 conserved CYP clans known from vascular plants are all present in green algae and several green algae-specific clans are recognized. Clan 71, 72, and 85 remain the largest CYP clans and include many taxa-specific CYP (sub)families reflecting emergence of linage-specific pathways. Molecular features and dynamics of CYP plasticity and evolution are discussed and exemplified by selected biosynthetic pathways. High substrate promiscuity is commonly observed for CYPs from large families, favoring retention of gene duplicates and neofunctionalization, thus seeding acquisition of new functions. Elucidation of biosynthetic pathways producing metabolites with sporadic distribution across plant phylogeny reveals multiple examples of convergent evolution where CYPs have been independently recruited from the same or different CYP families, to adapt to similar environmental challenges or ecological niches. Sometimes only a single or a few mutations are required for functional interconversion. A compilation of functionally characterized plant CYPs is provided online through the Plant P450 Database (erda.dk/public/vgrid/PlantP450/).

The Sox family of transcription factors regulate many different processes during metazoan development, including stem cell maintenance, nervous system specification and germline development. In addition, it has recently become apparent that SoxB genes are involved in embryonic segmentation in several arthropod species. Segmentation in arthropods occurs in two main ways: long germ animals form all segments at once, best exemplified in the well-studied Drosophila melanogaster system, and short germ animals form anterior segments simultaneously, with posterior segments added sequentially from a segment addition zone. In both D. melanogaster and the short germ beetle Tribolium castaneum, the SoxB gene Dichaete is required for correct segmentation and, more recently, we showed that a close relative of Dichaete, Sox21b-1, is required for the simultaneous formation of prosomal segments and sequential addition of opisthosomal segments in the spider Parasteatoda tepidariorum. Here we further analysed the function and expression of Sox21b-1 in P. tepidariorum. We found that while this gene regulates the generation of both prosomal and opisthosomal segments, it plays different roles in the formation of these tagma reflecting their contrasting modes of segmentation and deployment of gene regulatory networks with different architectures. To further investigate the evolution of Sox genes and their roles we characterised the repertoire of the gene family across several arachnid species with and without an ancestral whole genome duplication, and compared Sox expression between P. tepidariorum and the harvestman Phalangium opilio. The results suggest that Sox21b-1 was likely involved in segmentation ancestrally in arachnids, but that other Sox genes could also regulate this process in these animals. We also found that most Sox families have been retained as duplicates or ohnologs after WGD and evidence for potential subfunctionalisation and/or neofunctionalization events.

Gene duplication is a prevalent phenomenon across the tree of life. The processes that lead to the retention of duplicated genes are not well understood. Functional genomics approaches in model organisms, such as yeast, provide useful tools to test the mechanisms underlying retention with functional redundancy and divergence of duplicated genes, including fates associated with neofunctionalization, subfunctionalization, back-up compensation, and dosage amplification. Duplicated genes may also be retained as a consequence of structural and functional entanglement. Advances in human gene editing have enabled the interrogation of duplicated genes in the human genome, providing new tools to evaluate the relative contributions of each of these factors to duplicate gene retention and the evolution of genome structure.

SummaryTermites are model social organisms characterized by a polyphenic caste system. Subterranean termites (Rhinotermitidae) are ecologically and economically important species, including acting as destructive pests. Rhinotermitidae occupies an important evolutionary position within the clade representing an intermediate taxon between the higher (Termitidae) and lower (other families) termites. Here, we report the genome, transcriptome and methylome of the Japanese subterranean termite Reticulitermes speratus. The analyses highlight the significance of gene duplication in social evolution in this termite. Gene duplication associated with caste-biased gene expression is prevalent in the R. speratus genome. Such duplicated genes encompass diverse categories related to social functions, including lipocalins (chemical communication), cellulases (wood digestion and social interaction), lysozymes (social immunity), geranylgeranyl diphosphate synthase (social defense) and a novel class of termite lineage-specific genes with unknown functions. Paralogous genes were often observed in tandem in the genome, but the expression patterns were highly variable, exhibiting caste biases. Some duplicated genes assayed were expressed in caste-specific organs, such as the accessory glands of the queen ovary and frontal glands in soldier heads. We propose that gene duplication facilitates social evolution through regulatory diversification leading to caste-biased expression and subfunctionalization and/or neofunctionalization that confers caste-specialized functions.Significance StatementTermites are model social organisms characterized by a sophisticated caste system, where distinct castes arise from the same genome. Our genomics data of Japanese subterranean termite provides insights into the evolution of the social system, highlighting the significance of gene duplication. Gene duplication associated with caste-biased gene expression is prevalent in the termite genome. Many of the duplicated genes were related to social functions, such as chemical communication, social immunity and defense, and they often expressed in caste-specific organs. We propose that gene duplication facilitates social evolution through regulatory diversification leading to caste-biased expression and functional specialization. In addition, since subterranean termites are ecologically and economically important species including destructive pests in the world, our genomics data serves as a foundation for these studies.

In polyploids, whole genome duplication (WGD) played a significant role in genome expansion, evolution and diversification. Many gene families are expanded following polyploidization, with the duplicated genes functionally diversified by neofunctionalization or subfunctionalization. These mechanisms may support adaptation and have likely contributed plant survival during evolution. Flowering time is an important trait in plants, which affects critical features, such as crop yields. The flowering-time gene family is one of the largest expanded gene families in plants, with its members playing various roles in plant development. Here, we performed genome-wide identification and comparative analysis of flowering-time genes in three palnt families i.e., Malvaceae, Brassicaceae, and Solanaceae, which indicate these genes were expanded following the event/s of polyploidization. Duplicated genes have been retained during evolution, although genome reorganization occurred in their flanking regions. Further investigation of sequence conservation and similarity network analyses provide evidence for functional diversification of duplicated genes during evolution. These functionally diversified genes play important roles in plant development and provide advantages to plants for adaptation and survival in response to environmental changes encountered during evolution. Collectively, we show that flowering-time genes were expanded following polyploidization and retained as large gene family by providing advantages from functional diversification during evolution.

Reprogramming biosynthetic assembly-lines is a topic of intense interest. This is unsurprising as the scaffolds of most antibiotics in current clinical use are produced by such pathways. The modular nature of assembly-lines provides a direct relationship between the sequence of enzymatic domains and the chemical structure of the product, but rational reprogramming efforts have been met with limited success. To gain greater insight into the design process, we wanted to examine how Nature creates assembly-lines and searched for biosynthetic pathways that might represent evolutionary transitions. By examining the biosynthesis of the anti-tubercular wollamides, we show how whole gene duplication and neofunctionalization can result in pathway bifurcation. Importantly, we show that neofunctionalization occurs primarily through intragenomic recombination. This pathway bifurcation leads to redundancy, providing the genetic robustness required to enable large structural changes during the evolution of antibiotic structures. Should the new product be none-functional, gene loss can restore the original genotype. However, if the new product confers an advantage, depreciation and eventual loss of the original gene creates a new linear pathway. This provides the blind watchmaker equivalent to the ‘design, build, test’ cycle of synthetic biology.

A single insulin receptor (InR) gene has been identified and extensively studied in model species ranging from nematodes to mice. However, most insects possess additional copies of InR, yet the functional significance, if any, of alternate InRs is unknown. Here, we used the wing-dimorphic brown planthopper (BPH) as a model system to query the role of a second InR copy in insects. NlInR2 resembled the BPH InR homologue (NlInR1) in terms of nymph development and reproduction, but revealed distinct regulatory roles in fuel metabolism, lifespan, and starvation tolerance. Unlike a lethal phenotype derived from NlInR1 null, homozygous NlInR2 null mutants were viable and accelerated DNA replication and cell proliferation in wing cells, thus redirecting short-winged–destined BPHs to develop into long-winged morphs. Additionally, the proper expression of NlInR2 was needed to maintain symmetric vein patterning in wings. Our findings provide the first direct evidence for the regulatory complexity of the two InR paralogues in insects, implying the functionally independent evolution of multiple InRs in invertebrates., Author summary The highly conserved insulin/insulin-like growth factor signaling pathway plays a pivotal role in growth, development, and various physiological processes across a wide phylogeny of organisms. Unlike a single InR in the model species such as the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans, most insect lineages have two or even three InR copies. However, the function of the alternative InRs remains elusive. Here, we created a homozygous mutation for a second insulin receptor (InR2) in the wing-dimorphic brown planthopper (BPH), Nilaparvata lugens, using the clustered regularly interspaced palindromic repeats/CRISPR-associated (CRISPR/Cas9) system. Our findings revealed that InR2 possesses functions distinct from the BPH InR homologue (NlInR1), indicating that multiple InR paralogues may have evolved independently and may have functionally diversified in ways more complex than previously expected in invertebrates.

The constitution and regulation of effector repertoires shape host-microbe interactions. Ustilago maydis and Sporisorium reilianum are two closely related smut fungi, which both infect maize but cause distinct disease symptoms. Understanding how effector orthologs are regulated in these two pathogens can therefore provide insights into the evolution of different infection strategies. We tracked the infection progress of U. maydis and S. reilianum in maize leaves and used two distinct infection stages for cross-species RNA-sequencing analyses. We identified 207 of 335 one-to-one effector orthologs as differentially regulated during host colonization, which might reflect the distinct disease development strategies. Using CRISPR-Cas9-mediated gene conversion, we identified two differentially expressed effector orthologs with conserved function between two pathogens. Thus, differential expression of functionally conserved genes might contribute to species-specific adaptation and symptom development. Interestingly, another differentially expressed orthogroup (UMAG_05318/Sr10075) showed divergent protein function, providing a possible case for neofunctionalization. Collectively, we demonstrated that the diversification of effector genes in related pathogens can be caused both by alteration on the transcriptional level and through functional diversification of the encoded effector proteins.

Queen discrimination behavior in the fire ant Solenopsis invicta maintains its two types of societies: colonies with one (monogyne) or many (polygyne) queens, yet the underlying genetic mechanism is poorly understood. This behavior is controlled by two supergene alleles, SB and Sb, with ~600 genes. Polygyne workers, having either the SB/SB or SB/Sb genotype, accept additional SB/Sb queens into their colonies but kill SB/SB queens. In contrast, monogyne workers, all SB/SB, reject all additional queens regardless of genotype. Because the SB and Sb alleles have suppressed recombination, determining which genes within the supergene mediate this differential worker behavior is difficult. We hypothesized that the alternate worker genotypes sense queens differently because of the evolution of differential expression of key genes in their main sensory organ, the antennae. To identify such genes, we sequenced RNA from four replicates of pooled antennae from three classes of workers: monogyne SB/SB, polygyne SB/SB, and polygyne SB/Sb. We identified 81 differentially expressed protein‐coding genes with 13 encoding potential chemical metabolism or perception proteins. We focused on the two odorant perception genes: an odorant receptor SiOR463 and an odorant‐binding protein SiOBP12. We found that SiOR463 has been lost in the Sb genome. In contrast, SiOBP12 has an Sb‐specific duplication, SiOBP12b′, which is expressed in the SB/Sb worker antennae, while both paralogs are expressed in the body. Comparisons with another fire ant species revealed that SiOBP12b′ antennal expression is specific to S. invicta and suggests that queen discrimination may have evolved, in part, through expression neofunctionalization., Queen discrimination behavior in the fire ant Solenopsis invicta maintains its two types of societies: colonies with one (monogyne) or many (polygyne) queens, yet the underlying genetic mechanism is poorly understood. We hypothesized that the alternate worker genotypes sense queens differently because of the evolution of differential expression of key genes in their main sensory organ, the antennae. We identified 81 differentially expressed genes using RNA sequencing, and follow‐up experiments revealed that antennal expression of an odorant binding protein, SiOBP12b', is specific to S. invicta and suggests that queen discrimination may have evolved, in part, through expression neofunctionalization.

Fungal genomes encode highly organized gene clusters that underlie the production of specialized (or secondary) metabolites. Gene clusters encode key functions to exploit plant hosts or environmental niches. Promiscuous exchange among species and frequent reconfigurations make gene clusters some of the most dynamic elements of fungal genomes. Despite evidence for high diversity in gene cluster content among closely related strains, the microevolutionary processes driving gene cluster gain, loss, and neofunctionalization are largely unknown. We analyzed the Fusarium graminearum species complex (FGSC) composed of plant pathogens producing potent mycotoxins and causing Fusarium head blight on cereals. We de novo assembled genomes of previously uncharacterized FGSC members (two strains of F. austroamericanum, F. cortaderiae, and F. meridionale). Our analyses of 8 species of the FGSC in addition to 15 other Fusarium species identified a pangenome of 54 gene clusters within FGSC. We found that multiple independent losses were a key factor generating extant cluster diversity within the FGSC and the Fusarium genus. We identified a modular gene cluster conserved among distantly related fungi, which was likely reconfigured to encode different functions. We also found strong evidence that a rare cluster in FGSC was gained through an ancient horizontal transfer between bacteria and fungi. Chromosomal rearrangements underlying cluster loss were often complex and were likely facilitated by an enrichment in specific transposable elements. Our findings identify important transitory stages in the birth and death process of specialized metabolism gene clusters among very closely related species.

Macrophage migration inhibitory factor (MIF) dynamically connects innate and adaptive immune systems in vertebrate animals, allowing highly orchestrated systemic responses to various insults. The occurrence of MIF-like genes in non-vertebrate organisms suggests its origin from an ancestral metazoan gene, whose function is still a matter of debate. In the present work, by analyzing available genomic and transcriptomic data from bivalve mollusks, we identified 137 MIF-like sequences, which were classified into three types, based on phylogeny and conservation of key residues: MIF, D-DT, and the lineage-specific type MDL. Comparative genomics revealed syntenic conservation of homologous genes at the family level, the loss of D-DT in the Ostreidae family as well as the expansion of MIF-like genes in the Mytilidae family, possibly underpinning the neofunctionalization of duplicated gene copies. In M. galloprovincialis, MIF and one D-DT were mostly expressed in haemocytes and mantle rim of untreated animals, while D-DT paralogs often showed very limited expression, suggesting an accessory role or their persistence as relict genes.

Yersinia enterocolitica is a pectinolytic zoonotic foodborne pathogen, the genome of which contains pectin-binding proteins and several different classes of pectinases, including polysaccharide lyases (PLs) and an exopolygalacturonase. These proteins operate within a coordinated pathway to completely saccharify homogalacturonan (HG). Polysaccharide lyase family 2 (PL2) is divided into two major subfamilies that are broadly-associated with contrasting ‘endolytic’ (PL2A) or ‘exolytic’ (PL2B) activities on HG. In the Y. enterocolitica genome, the PL2A gene is adjacent to an independent carbohydrate binding module from family 32 (YeCBM32), which possesses a N-terminal secretion tag and is known to specifically bind HG. Independent CBMs are rare in nature and, most commonly, are fused to enzymes in order to potentiate catalysis. The unconventional gene architecture of YePL2A and YeCBM32, therefore, may represent an ancestral relic of a fission event that decoupled PL2A from its cognate CBM. To provide further insight into the evolution of this pectinolytic locus and the molecular basis of HG depolymerisation within Y. enterocolitica, we have resurrected a YePL2A-YeCBM32 chimera and demonstrated that the extant PL2A digests HG more efficiently. In addition, we have engineered a tryptophan from the active site of the exolytic YePL2B into YePL2A (YePL2A-K291W) and demonstrated, using X-ray crystallography of substrate complexes, that it is a structural determinant of exo-activity within the PL2 family. In this manner, surrogate structural platforms may assist in the study of phylogenetic relationships informed by extant and resurrected sequences, and can be used to overcome challenging structural problems within carbohydrate active enzyme families.

Cysteine-rich receptor-like kinases (CRK) constitute one of the largest subfamily of receptor-like kinases, which play crucial roles in plant development and stress response. In total, 43, 37, 36, 38 and 170 CRK genes including duplicated genes were identified in the genome of Brachypodium distachyon, Hordeum vulgare, Oryza sativa, Sorghum bicolor and Triticum aestivum, respectively. These CRK proteins were tightly clustered into four phylogenetic groups and exhibited close syntenic relationship among orthologous genes. Majority of CRK proteins contain a transmembrane domain for plasma membrane localization. The organization of exon/intron, domains and motifs were variably conserved. Tissue-specific expression suggested the involvement of certain CRK genes in plant development. Modulated expression revealed their specific stress-responsive functions. Co-expression and interaction analysis indicated their role in signaling. Ks value and divergence time analysis suggested duplication of TaCRK genes before the hybridization of T. aestivum sub-genomes. Expression comparison of duplicated TaCRK genes revealed functional retention, neofunctionalization or pseudo-functionalization. Recombinant expression of a stress-responsive gene TaCRK68-A in Escherichia coli and Saccharomyces cerevisiae displayed enhanced tolerance against heat, drought, cold and salinity stresses. The study suggested vital functions of CRKs during development and stresses, and provides the basis for functional characterization of each gene in future studies.

The plant O-methyltransferases are dependent on S-Adenosyl-l-methionine, which can catalyze a variety of secondary metabolites. Here we identified different number of OMT genes from the respective grass genomes. Phylogenetic analysis showed that this OMT gene family is a grass-specific gene family that is different from COMT. Most of genes were expanded by tandem and segment duplication after the species split from their progenitor. Furthermore, genes from Group I and two clusters from group II are only present in Panicoideae, which included Bx10 and Bx7 involved in the benzoxazinoids pathway, suggesting these genes could participate in insect resistance in Panicoideae. Gene expression profiles showed that OMT genes were preferentially expressed in vegetative stages, especially in roots. These results revealed that this grass-specific OMT gene family could affect the development of vegetative stages, and be involved in the benzoxazinoids pathway or suberin biosynthesis that was different from COMT.

Gibberellin (GA) functions as an essential natural regulator of growth and development in plants. For each step of the GA metabolic pathway, different copy numbers can be found in different species, as is the case with the 13 genes across four enzymatic steps in rice (Oryza sativa). A common view is that such gene duplication creates homologs that buffer organisms against loss-of-function (LOF) mutations. Therefore, knockouts of any single homolog might be expected to have little effect. To test this question, we generated clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) knockouts for these homologs and measured effects on growth and reproduction. Surprisingly, we report here that there is consistently one or more essential gene at each enzymatic step, for which LOF mutation induces death or sterility—suggesting that the GA pathway does not have a redundancy route and that each gene family is essential for GA metabolism. In most of these genes from the same gene family, we observed defects in plant height and infertility, suggesting that the duplicated members retain functions related to GA synthesis or degradation. We identified both subfunctionalization of the three recently diversified homologs OsKO1, OsKO2, and OsKO5 and neofunctionalization in OsKO3 and OsKO4. Thus, although the function of each step is conserved, the evolution of duplicates in that step is diversified. Interestingly, the CRISPR/Cas9 lines at the SD1 locus were typically sterile, whereas the natural sd1 mutants, related to the “Green Revolution” in rice, show normal setting rates. Collectively, our results identify candidates for control of GA production and provide insight into the evolution of four critical gene families in plants.

Background Gene duplication has played an important role in the evolution and domestication of flowering plants. Yet little is known about how plant duplicate genes evolve and are retained over long timescales, particularly those arising from small-scale duplication (SSD) rather than whole-genome duplication (WGD) events. Results We address this question in the Poaceae (grass) family by analyzing gene expression data from nine tissues of Brachypodium distachyon, Oryza sativa japonica (rice), and Sorghum bicolor (sorghum). Consistent with theoretical predictions, expression profiles of most grass genes are conserved after SSD, suggesting that functional conservation is the primary outcome of SSD in grasses. However, we also uncover support for widespread functional divergence, much of which occurs asymmetrically via the process of neofunctionalization. Moreover, neofunctionalization preferentially targets younger (child) duplicate gene copies, is associated with RNA-mediated duplication, and occurs quickly after duplication. Further analysis reveals that functional divergence of SSD-derived genes is positively correlated with both sequence divergence and tissue specificity in all three grass species, and particularly with anther expression in B. distachyon. Conclusions Our results suggest that SSD-derived grass genes often undergo rapid functional divergence that may be driven by natural selection on male-specific phenotypes. These observations are consistent with those in several animal species, suggesting that duplicate genes take similar evolutionary trajectories in plants and animals. Electronic supplementary material The online version of this article (10.1186/s12862-019-1415-2) contains supplementary material, which is available to authorized users.

Background RNA interference (RNAi) related pathways provide defense against viruses and transposable elements, and have been implicated in the suppression of meiotic drive elements. Genes in these pathways often exhibit high levels of adaptive substitution, and over longer timescales show gene duplication and loss—most likely as a consequence of their role in mediating conflict with these parasites. This is particularly striking for Argonaute 2 (Ago2), which is ancestrally the key effector of antiviral RNAi in insects, but has repeatedly formed new testis-specific duplicates in the recent history of the obscura species-group of Drosophila. Results Here we take advantage of publicly available genomic and transcriptomic data to identify six further RNAi-pathway genes that have duplicated in this clade of Drosophila, and examine their evolutionary history. As seen for Ago2, we observe high levels of adaptive amino-acid substitution and changes in sex-biased expression in many of the paralogs. However, our phylogenetic analysis suggests that co-duplications of the RNAi machinery were not synchronous, and our expression analysis fails to identify consistent male-specific expression. Conclusions These results confirm that RNAi genes, including genes of the antiviral and piRNA pathways, have undergone multiple independent duplications and that their history has been particularly labile within the obscura group. However, they also suggest that the selective pressures driving these changes have not been consistent, implying that more than one selective agent may be responsible., BMC Evolutionary Biology, 19, ISSN:1471-2148

Over several decades, glucosinolates have become a model system for the study of specialized metabolic diversity in plants. The near-complete identification of biosynthetic enzymes, regulators, and transporters has provided support for the role of gene duplication and subsequent changes in gene expression, protein function, and substrate specificity as the evolutionary bases of glucosinolate diversity. Here, we provide examples of how whole-genome duplications, gene rearrangements, and substrate promiscuity potentiated the evolution of glucosinolate biosynthetic enzymes, regulators, and transporters by natural selection. This in turn may have led to the repeated evolution of glucosinolate metabolism and diversity in higher plants.

Genome mergers between independently evolving lineages, via allopolyploidy, can potentially lead to instantaneous sympatric speciation. However, little is known about the consequences of allopolyploidy and the resultant "genome shock" on genome evolution and expression beyond the plant and fungal branches of the Tree of Life. The aim of this study was to compare substitution rates and gene expression patterns in two allopolyploid brown algae (Phaeophyceae and Heterokonta) and their progenitors in the genus Pelvetiopsis N. L. Gardner in the north-east Pacific, and to date their relationships. We used RNA-seq data, all potential orthologues, and putative single-copy loci for phylogenomic, divergence, and gene expression analyses. The multispecies coalescent placed the origin of allopolyploids in the late Pleistocene (0.35-0.05 Ma). Homoeologues displayed increased nonsynonymous divergence compared with parental orthologues, consistent with relaxed selective constraint following allopolyploidization, including for genes with no evidence of pseudogenization or neofunctionalization. Patterns of homoeologue-orthologue expression conservation and expression level dominance were largely shared with both natural plant and fungal allopolyploids. Our results provide further support for common cross-Kingdom patterns of allopolyploid genome evolution and transcriptional responses, here in the evolutionarily distinct marine heterokont brown algae.

The gene encoding the ubiquitous DNA repair protein, Ku70p, has undergone extensive copy number expansion during primate evolution. Gene duplications of KU70 have the hallmark of long interspersed element-1 mediated retrotransposition with evidence of target-site duplications, the poly-A tails, and the absence of introns. Evolutionary analysis of this expanded family of KU70-derived “NUKU” retrocopies reveals that these genes are both ancient and also actively being created in extant primate species. NUKU retrocopies show evidence of functional divergence away from KU70, as evinced by their altered pattern of tissue expression and possible tissue-specific translation. Molecular modeling predicted that amino acid changes in Nuku2p at the interaction interface with Ku80p would prevent the assembly of the Ku heterodimer. The lack of Nuku2p-Ku80p interaction was confirmed by yeast two-hybrid assay, which contrasts the robust interaction of Ku70p-Ku80p. While several NUKU retrocopies appear to have been degraded by mutation, NUKU2 shows evidence of positive natural selection, suggesting that this retrocopy is undergoing neofunctionalization. Although Nuku proteins do not appear to antagonize retrovirus transduction in cell culture, the observed expansion and rapid evolution of NUKUs could be being driven by alternative selective pressures related to infectious disease or an undefined role in primate physiology.

Gene duplication is increasingly recognized as an important mechanism for the origination of new genes, as revealed by comparative genomic analysis. However, the ways in which new duplicate genes contribute to phenotypic evolution remain largely unknown, especially in plants, owing to a lack of experimental and phenotypic data. In this study, we identified the new geneExov,derived from a partial gene region duplication of its parental geneExov-L, which is a member of an exonuclease family, into a different chromosome inArabidopsis thaliana. We experimentally investigated the phenotypic effects ofExovandExov-Lin an attempt to understand how the new gene diverged from the parental copy and contributes to phenotypic evolution. Evolutionary analysis demonstrated thatExovis a species-specific gene that originated within the last 3.5 million years and shows strong signals of positive selection. Unexpectedly, RNAseq analyses reveal that the new gene, despite its young age, has acquired a large number of novel direct and indirect interactions in which the parental gene does not engage. This is consistent with a high, selection-driven substitution rate in the protein sequence encoded byExovin contrast to the slowly evolvingExov-L, suggesting an important role forExovin phenotypic evolution. We analyzed phenotypic effects ofexovandexov-lsingle T-DNA-insertion mutants;doubleexov, exov-lT-DNA insertion mutants; and CRISPR/Cas9-mediatedexovcrpandexov-lcrpknockouts on seven morphological traits in both the new and parental genes. We detected significant segregation of morphological changes for all seven traits when assessed in terms of single mutants, as well as morphological changes for seven traits associated with segregation of doubleexov, exov-lmutants. Substantial divergence of phenotypic effects between new and parental genes was revealed by principal component analyses, suggesting neofunctionalization in the new gene. These results reveal a young gene that plays critical roles in biological processes that underlie morphological and developmental evolution inArabidopsis thaliana.