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Haplodiploidy and paternal genome elimination (HD/PGE) are common in invertebrates, having evolved at least two dozen times, all from male heterogamety (i.e., systems with X chromosomes). However, why X chromosomes are important for the evolution of HD/PGE remains debated. The Haploid Viability Hypothesis posits that X-linked genes promote the evolution of male haploidy by facilitating purging recessive deleterious mutations. The Intragenomic Conflict Hypothesis holds that conflict between genes drives genetic system turnover; under this model, X-linked genes could promote the evolution of male haploidy due to conflicts with autosomes over sex ratios and genetic transmission. We studied lineages where we can distinguish these hypotheses: species with germline PGE that retain an XX/X0 sex determination system (gPGE+X). Because evolving PGE in these cases involves changes in transmission without increases in male hemizygosity, a high degree of X linkage in these systems is predicted by the Intragenomic Conflict Hypothesis but not the Haploid Viability Hypothesis. To quantify the degree of X linkage, we sequenced and compared 7 gPGE+X species’ genomes with 11 related species with typical XX/XY or XX/X0 genetic systems, representing three transitions to gPGE. We find highly increased X linkage in both modern and ancestral genomes of gPGE+X species compared to non-gPGE relatives, and recover a significant positive correlation between percent X linkage and the evolution of gPGE. These are among the first empirical results suggesting a role for intragenomic conflict in the evolution of novel genetic systems like HD/PGE.Significance StatementSex determination systems such as haplodiploidy, in which males’ gene transmission is haploid, are surprisingly common, however, the evolutionary paths to these systems are poorly understood. X chromosomes may play a particularly important role, either by increasing survival of males with only maternal genomes, or due to conflicts between X-chromosomal and autosomal genes. We studied X-chromosome gene richness in three arthropod lineages in which males are diploid as adults but only transmit their maternally-inherited haploid genome. We find that species with such atypical systems have far more X chromosomal genes than related diploid species. These results suggest that conflict between genetic elements within the genome drives the evolution of unusual sex determination systems.

Genome size in cellular organisms varies by six orders of magnitude, yet the cause of this large variation remains unexplained. The influential Drift-Barrier Hypothesis proposes that large genomes tend to evolve in small populations due to inefficient selection. However, to our knowledge no explicit tests of the Drift-Barrier Hypothesis have been reported. We performed the first explicit test, by comparing estimated census population size and genome size in mammals while incorporating potential covariates and the effect of shared evolutionary history. We found a lack of correlation between census population size and genome size among 199 species of mammals. These results suggest that population size is not the predominant factor influencing genome size and that the Drift-Barrier Hypothesis should be considered provisional.

Previous evolutionary reconstructions have concluded that early eukaryotic ancestors including both the last common ancestor of eukaryotes and of all fungi had intron-rich genomes. By contrast, some extant eukaryotes have few introns, underscoring the complex histories of intron–exon structures, and raising the question as to why these few introns are retained. Here, we have used recently available fungal genomes to address a variety of questions related to intron evolution. Evolutionary reconstruction of intron presence and absence using 263 diverse fungal species supports the idea that massive intron reduction through intron loss has occurred in multiple clades. The intron densities estimated in various fungal ancestors differ from zero to 7.6 introns per 1 kb of protein-coding sequence. Massive intron loss has occurred not only in microsporidian parasites and saccharomycetous yeasts, but also in diverse smuts and allies. To investigate the roles of the remaining introns in highly-reduced species, we have searched for their special characteristics in eight intron-poor fungi. Notably, the introns of ribosome-associated genes RPL7 and NOG2 have conserved positions; both intron-containing genes encoding snoRNAs. Furthermore, both the proteins and snoRNAs are involved in ribosome biogenesis, suggesting that the expression of the protein-coding genes and noncoding snoRNAs may be functionally coordinated. Indeed, these introns are also conserved in three-quarters of fungi species. Our study shows that fungal introns have a complex evolutionary history and underappreciated roles in gene expression.

Sex determination mechanisms vary widely across animals, but show remarkable degrees of recurrent evolution. Recurrent features of sex determination have largely been attributed to recurrent cooption of shared ancestral regulatory circuits. However, a new study on sex determination in Daphnia magna reveals both recurrent evolution of specific regulatory logic and apparently recurrent recruitment of a regulator, suggesting a role for optimization in recurrent patterns of sex determination mechanisms.

While much excitement has attended the discovery and study of circular RNAs, a new study in Cell Reports suggests that most mammalian circRNAs are not only functionless, but in fact costly. Comparison across three species is also consistent with the influential but rarely tested Drift-Barrier Hypothesis of molecular complexity. According to this hypothesis, nonessential genomic elements are slightly deleterious elements that fix by genetic drift and, thus, are generally more abundant in species with small effective population sizes. I discuss the implications of these new results for the Drift-Barrier hypothesis. In particular, I note the distinction between two classes of genomic elements, based on whether they are created by ‘standard’ small-scale mutations (basepair substitutions, indels, etc.) or larger, more idiosyncratic mutations (segmental duplications, transposable element propagation, etc.) I suggest that the Drift-Barrier Hypothesis is likely to apply to the former class, but perhaps not the latter class.

The sex-determining gene SRY has undergone rapid evolution in rodents. Curiously, a new study by Miyawaki et al. reveals that a recently evolved SRY gene sequence antagonizes SRY protein stability, necessitating splicing of a novel intron. Other data suggest that this troublesome gene region has noncoding RNA functions, possibly related to conflict between sex chromosomes.

Several bioinformatic tools have been developed for genome-wide identification of orthologous and paralogous genes. However, no corresponding tool allows the detection of exon homology relationships. Here, we present ExOrthist, a fully reproducible Nextflow-based software enabling inference of exon homologs and orthogroups, visualization of evolution of exon-intron structures, and assessment of conservation of alternative splicing patterns. ExOrthist evaluates exon sequence conservation and considers the surrounding exon-intron context to derive genome-wide multi-species exon homologies at any evolutionary distance. We demonstrate its use in different evolutionary scenarios: whole genome duplication in frogs and convergence of Nova-regulated splicing networks ( https://github.com/biocorecrg/ExOrthist). The research has been funded by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (ERC-StG-LS2-637591 to MI), the Spanish Ministerio de Ciencia (BFU2017-89201-P to MI, including and FPI PhD fellowship to FM), the “Centro de Excelencia Severo Ochoa 2013-2017” (SEV-2012-0208), EMBO Long Term postdoctoral fellowship (ALTF 1505-2015 to YM), and Marie Skłodowska-Curie actions (MSCA) grant (705938 to YM). We acknowledge the support of the CERCA Programme/Generalitat de Catalunya and of the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) to the EMBL partnership

During nuclear maturation of most eukaryotic pre-messenger RNAs and long non-coding RNAs, introns are removed through the process of RNA splicing. Different classes of introns are excised by the U2-type or the U12-type spliceosomes, large complexes of small nuclear ribonucleoprotein particles and associated proteins. We created intronIC, a program for assigning intron class to all introns in a given genome, and used it on 24 eukaryotic genomes to create the Intron Annotation and Orthology Database (IAOD). We then used the data in the IAOD to revisit several hypotheses concerning the evolution of the two classes of spliceosomal introns, finding support for the class conversion model explaining the low abundance of U12-type introns in modern genomes.

The discovery of introns four decades ago was one of the most unexpected findings in molecular biology. Introns are sequences interrupting genes that must be removed as part of messenger RNA production. Genome sequencing projects have shown that most eukaryotic genes contain at least one intron, and frequently many. Comparison of these genomes reveals a history of long evolutionary periods during which few introns were gained, punctuated by episodes of rapid, extensive gain. However, although several detailed mechanisms for such episodic intron generation have been proposed, none has been empirically supported on a genomic scale. Here we show how short, non-autonomous DNA transposons independently generated hundreds to thousands of introns in the prasinophyte Micromonas pusilla and the pelagophyte Aureococcus anophagefferens. Each transposon carries one splice site. The other splice site is co-opted from the gene sequence that is duplicated upon transposon insertion, allowing perfect splicing out of the RNA. The distributions of sequences that can be co-opted are biased with respect to codons, and phasing of transposon-generated introns is similarly biased. These transposons insert between pre-existing nucleosomes, so that multiple nearby insertions generate nucleosome-sized intervening segments. Thus, transposon insertion and sequence co-option may explain the intron phase biases and prevalence of nucleosome-sized exons observed in eukaryotes. Overall, the two independent examples of proliferating elements illustrate a general DNA transposon mechanism that can plausibly account for episodes of rapid, extensive intron gain during eukaryotic evolution.

Genomes show remarkable variation in architecture and complexity across organisms, with large differences in genome size and in numbers of genes, gene duplicates, introns and transposable elements. These differences have important implications for transcriptome and regulatory complexity and ultimately for organismal complexity. Numbers of spliceosomal introns show particularly striking differences, ranging across organisms from zero to hundreds of thousands of introns per genome. The causes of these differences remain poorly understood. According to one influential perspective, differences across species reflect the differential ability of selection in different populations to eliminate allegedly deleterious intron-containing alleles. Direct tests of this theory have been elusive. Here, I study evolution of intron-exon structures in genomic regions of recombination suppression (RRSs), which experience drastically reduced selective efficiency due to hitchhiking and background selection. I studied intron creation in eight independently evolved RRSs, spanning substantial diversity phylogenetically (plants, animals, fungi and brown algae) and biologically (sex chromosomes, mating type chromosomes, genomic regions flanking self-incompatibility loci, and the Drosophila "dot" chromosome). To identify newly created introns in RRSs, I compared intron positions in RRS genes with those in homologous genes. I found very few intron gains: no intron gains were observed in 7/8 studied data sets, and only three intron gains were observed overall (on the Drosophila dot chromosome). These results suggest that efficiency of selection may not be a major cause of differences in intron-exon structures across organisms. Instead, rates of spontaneous intron-creating and intron-deleting mutations may play the central role in shaping intron-exon structures.

The evolutionary history of the spliceosomal introns that interrupt nuclear genes in eukaryotes has been debated for four decades. Positions of introns show a high degree of coincidence between various eukaryotes, implying either than many modern introns are very old and/or that intron creation is highly biased toward certain sites, leading to rampant parallel intron gain. A series of articles in this and other journals reported evidence for a strikingly high degree of parallel insertion of introns in different alleles of the water flea Daphnia pulex Here, I report several new analyses of these data. Among the 23 loci reported to be undergoing parallel intron gain, I find that in five cases introns reported to be unrelated show extended sequence similarity strongly suggesting that the introns are in fact homologous. Five additional cases show extended conserved motifs between allegedly unrelated introns. For nearly all loci including the 13 remaining loci, at least one intron shows hallmarks of rapid sequence evolution, thwarting confident inference about homology. In addition, I reanalyze gene trees reconstructed from flanking exonic sequences, claimed by the original authors as additional evidence for parallel gain. I show that these phylogenetic trees frequently fail to recover expected relationships, and in any case show relationships not consistent with parallel intron gains. In total, I conclude that the data do not support widespread parallel intron gain in D. pulex These findings strengthen the notion that shared intron positions generally reflect ancestral introns, and thus the notion of complex genes in early eukaryotes.

In the deadly human malaria parasite Plasmodium falciparum, several major merozoite surface proteins (MSPs) show a striking pattern of allelic diversity called allelic dimorphism (AD). In AD, the vast majority of observed alleles fall into two highly divergent allelic classes, with recombinant alleles being rare or not observed, presumably due to repression by natural selection (recombination suppression, or RS). The three AD loci, merozoite surface proteins (MSPs) 1, 2, and 6, along with MSP3, which also exhibits RS among four allelic classes, can be collectively called AD/RS. The causes of AD/RS and the evolutionary history of allelic diversity at these loci remain mysterious. The few available sequences from a single closely related chimpanzee parasite, P. reichenowi, have suggested that for 3/4 loci, AD/RS is an ancient state that has been retained in P. falciparum since well before the P. falciparum-P. reichenowi ancestor. On the other hand, based on comparative sequence analysis, we recently suggested that (i) AD/RS P. falciparum loci have undergone interallelic recombination over longer evolutionary times (on the timescale of recent speciation events), and thus (ii) AD/RS may be a recent phenomenon. The recent publication of genomic sequencing efforts for P. gaboni, an outgroup to P. falciparum and P. reichenowi, allows for improved reconstruction of the evolutionary history of these loci. In this work, I report genic sequence for P. gaboni for all four AD/RS P. falciparum loci (MSP1, 2, 3, and 6). Comparison of these sequences with available P. falciparum and P. reichenowi data strengthens the evidence for interallelic recombination over the evolutionary history of these species and also strengthens the case that AD/RS at these loci is ancient. Combined with previous results, these data provide evidence that AD/RS at different loci has evolved at several different times in the evolutionary history of P. falciparum: (i) before the P. gaboni-P. falciparum divergence, for much of MSP1 and MSP3; (ii) between the P. gaboni-P. falciparum and P. reichenowi-P. falciparum divergences, for the 5' end of the AD region of MSP6 and block 3 of MSP1; (iii) near the P. reichenowi-P. falciparum divergence, for the 3' end of the AD region of MSP6; and (iv) after the P. reichenowi-P. falciparum divergence, for MSP2. Based on these results, I suggest a new hypothesis for long-term evolutionary maintenance of AD/RS by recombination within allelic groups.

The emergence of jawed vertebrates (gnathostomes) from jawless vertebrates was accompanied by major morphological and physiological innovations, such as hinged jaws, paired fins and immunoglobulin-based adaptive immunity. Gnathostomes subsequently diverged into two groups, the cartilaginous fishes and the bony vertebrates. Here we report the whole-genome analysis of a cartilaginous fish, the elephant shark (Callorhinchus milii). We find that the C. milii genome is the slowest evolving of all known vertebrates, including the 'living fossil' coelacanth, and features extensive synteny conservation with tetrapod genomes, making it a good model for comparative analyses of gnathostome genomes. Our functional studies suggest that the lack of genes encoding secreted calcium-binding phosphoproteins in cartilaginous fishes explains the absence of bone in their endoskeleton. Furthermore, the adaptive immune system of cartilaginous fishes is unusual: it lacks the canonical CD4 co-receptor and most transcription factors, cytokines and cytokine receptors related to the CD4 lineage, despite the presence of polymorphic major histocompatibility complex class II molecules. It thus presents a new model for understanding the origin of adaptive immunity.

The order of genes along metazoan chromosomes has generally been thought to be largely random, with few implications for organismal function. However, two recent studies, reporting hundreds of pairs of genes that have remained linked in diverse metazoan species over hundreds of millions of years of evolution, suggest widespread functional implications for gene order. These associations appear to largely reflect cis -regulatory constraints, with either (i) multiple genes sharing transcriptional regulatory elements, or (ii) regulatory elements for a developmental gene being found within a neighboring ‘bystander' gene (known as a genomic regulatory block). We discuss implications, questions raised, and new research directions arising from these studies, as well as evidence for similar phenomena in other eukaryotic groups.

Ever since Luria and Delbruck, the notion that mutation is random with respect to fitness has been foundational to modern biology. However, various studies have claimed striking exceptions to this rule. One influential case involves toxin-encoding genes in snails of the genus Conus, termed conotoxins, a large gene family that undergoes rapid diversification of their protein-coding sequences by positive selection. Previous reconstructions of the sequence evolution of conotoxin genes claimed striking patterns: (1) elevated synonymous change, interpreted as being due to targeted "hypermutation" in this region; (2) elevated transversion-to-transition ratios, interpreted as reflective of the particular mechanism of hypermutation; and (3) much lower rates of synonymous change in the codons encoding several highly conserved cysteine residues, interpreted as strong position-specific codon bias. This work has spawned a variety of studies on the potential mechanisms of hypermutation and on causes for cysteine codon bias, and has inspired hypermutation hypotheses for various other fast-evolving genes. Here, I show that all three findings are likely to be artifacts of statistical reconstruction. First, by simulating nonsynonymous change I show that high rates of dN can lead to overestimation of dS. Second, I show that there is no evidence for any of these three patterns in comparisons of closely related conotoxin sequences, suggesting that the reported findings are due to breakdown of statistical methods at high levels of sequence divergence. The current findings suggest that mutation and codon bias in conotoxin genes may not be atypical, and that random mutation and selection can explain the evolution of even these exceptional loci.

The recent explosion of genome sequences from all major phylogenetic groups has unveiled an unexpected wealth of cases of recurrent evolution of strikingly similar genomic features in different lineages. Here, we review the diverse known types of recurrent evolution in eukaryotic genomes, with a special focus on metazoans, ranging from reductive genome evolution to origins of splice-leader trans-splicing, from tandem exon duplications to gene family expansions. We first propose a general classification scheme for evolutionary recurrence at the genomic level, based on the type of driving force-mutation or selection-and the environmental and genomic circumstances underlying these forces. We then discuss various cases of recurrent genomic evolution under this scheme. Finally, we provide a broader context for repeated genomic evolution, including the unique relationship of genomic recurrence with the genotype-phenotype map, and the ways in which the study of recurrent genomic evolution can be used to understand fundamental evolutionary processes.

The structural complexity of the vertebrate brain is mirrored by its unparalleled transcriptome complexity. In particular, two post-transcriptional processes, alternative splicing and RNA editing, greatly diversify brain transcriptomes. Here we report a close connection between these two processes: we show A-to-I RNA editing in Nova1, a key brain-specific regulator of alternative splicing. Nova1 editing levels increase during embryonic development in mouse and chicken brains and show significant variation across postnatal brain regions. Evolutionary conservation of both editing and editing-associated RNA secondary structure of the Nova1 mRNA for 300 million years attests to the functional importance of Nova1 editing. Using a combination of different assays in human HEK293T cell lines, we report a novel post-translational role for this RNA editing. Whereas functional assays showed no effect of RNA editing on the regulatory splicing activity of the encoded proteins, we found evidence that edited forms exhibit reduced proteasome targeting and increased protein half-life. In addition, we found evidence for similar regulation of protein half-life by an evolutionarily conserved alternative splicing event in Nova1. These results open new venues of research on the multi-level integration of gene expression by: (1) revealing the novel role of RNA editing in regulating protein stability, and (2) establishing protein stability as a new target of multifaceted regulation.

Spliceosomal introns are hallmarks of eukaryotic genomes, dividing coding regions into separate exons, which are joined during mRNA intron removal catalyzed by the spliceosome. With few known exceptions, spliceosomal introns are cis-spliced, that is, removed from one contiguous pre-mRNA transcript. The protistan intestinal parasite Giardia lamblia exhibits one of the most reduced eukaryotic genomes known, with short intergenic regions and only four known spliceosomal introns. Our genome-wide search for additional introns revealed four unusual cases of spliceosomal intron fragmentation, with consecutive exons of conserved protein-coding genes being dispersed to distant genomic sites. Independent transcripts are trans-spliced to yield contiguous mature mRNAs. Most strikingly, a dynein heavy chain subunit is both interrupted by two fragmented introns and also predicted to be assembled as two separately translated polypeptides, a remarkably complex expression pathway for a nuclear-encoded sequence. For each case, we observe extensive base-pairing potential between intron halves. This base pairing provides both a rationale for the in vivo association of independently transcribed mRNAs transcripts and the apparent specificity of splicing. Similar base-pairing potential in two cis-spliced G. lamblia introns suggests an evolutionary pathway whereby intron fragmentation of cis-spliced introns is permissible and a preliminary evolutionary step to complete gene fission. These results reveal remarkably complex genome dynamics in a severely genomically reduced parasite.

Novel organismal structures in metazoans are often undergirded by complex gene regulatory networks; as such, understanding the emergence of new structures through evolution requires reconstructing the series of evolutionary steps leading to these underlying networks. Here, we reconstruct the step-by-step assembly of the vertebrate splicing network regulated by Nova , a splicing factor that modulates alternative splicing in the vertebrate central nervous system by binding to clusters of YCAY motifs on pre-RNA transcripts. Transfection of human HEK293T cells with Nova orthologs indicated vertebrate-like splicing regulatory activity in bilaterian invertebrates, thus Nova acquired the ability to bind YCAY clusters and perform vertebrate-like splicing modulation at least before the last common ancestor of bilaterians. In situ hybridization studies in several species showed that Nova expression became restricted to CNS later on, during chordate evolution. Finally, comparative genomics studies revealed a diverse history for Nova -regulated exons, with target exons arising through both de novo exon creation and acquisition of YCAY motifs by preexisting exons throughout chordate and vertebrate history. In addition, we find that tissue-specific Nova expression patterns emerged independently in other lineages, suggesting independent assembly of tissue-specific regulatory networks.

Organisms show striking differences in genome structure; however, the functional implications and fundamental forces that govern these differences remain obscure. The intron–exon organization of nuclear genes is involved in a particularly large variety of structures and functional roles. We performed a 22-species study of Meis/hth genes, intron-rich homeodomain-containing transcription factors involved in a wide range of developmental processes. Our study revealed three surprising results that suggest important and very different functions for Meis intron–exon structures. First, we find unexpected conservation across species of intron positions and lengths along most of the Meis locus. This contrasts with the high degree of structural divergence found in genome-wide studies and may attest to conserved regulatory elements residing within these conserved introns. Second, we find very different evolutionary histories for the 5′ and 3′ regions of the gene. The 5′-most 10 exons, which encode the highly conserved Meis domain and homeodomain, show striking conservation. By contrast, the 3′ of the gene, which encodes several domains implicated in transcriptional activation and response to cell signaling, shows a remarkably active evolutionary history, with diverse isoforms and frequent creation and loss of new exons and splice sites. This region-specific diversity suggests evolutionary “tinkering,” with alternative splicing allowing for more subtle regulation of protein function. Third, we find a large number of cases of convergent evolution in the 3′ region, including 1) parallel losses of ancestral coding sequence, 2) parallel gains of external and internal splice sites, and 3) recurrent truncation of C-terminal coding regions. These results attest to the importance of locus-specific splicing functions in differences in structural evolution across genes, as well as to commonalities of forces shaping the evolution of individual genes along different lineages.

The 2.9-Mbp genome of the microsporidian Encephalitozoon cuniculi is severely reduced and compacted, possessing only 16 known tiny spliceosomal introns. Based on motif and expression data, intron profiles were constructed to screen the genome. Twenty additional introns were predicted and verified, doubling the previous estimate. We further predict that accurate 3' splice site (3'SS) selection is accomplished via a scanning mechanism with specificity achieved by maintaining a constrained variable length between the branch point motif and 3'SS. Only introns in ribosomal protein genes exhibit positional bias, and we hypothesize that splicing could be regulating expression of these genes. The large set of new introns in non-ribosomal protein genes suggests that current models of intron loss are unlikely sufficient to explain the distribution of introns. Together, these results extend our understanding of the role of intron loss in genome evolution and contribute to a novel model for splice site selection.

In contrast to the typically streamlined genomes of prokaryotes, many eukaryotic genomes are riddled with long intergenic regions, spliceosomal introns, and repetitive elements. What explains the persistence of these and other seemingly suboptimal structures? There are three general hypotheses: (1) the structures in question are not actually suboptimal but optimal, being favored by selection, for unknown reasons; (2) the structures are not suboptimal, but of (essentially) equal fitness to “optimal” ones; or (3) the structures are truly suboptimal, but selection is too weak to systematically eliminate them. The 5′ splice sites of introns offer a rare opportunity to directly test these hypotheses. Intron-poor species show a clear consensus splice site; most introns begin with the same six nucleotide sequence (typically GTAAGT or GTATGT), indicating efficient selection for this consensus sequence. In contrast, intron-rich species have much less pronounced boundary consensus sequences, and only small minorities of introns in intron-rich species share the same boundary sequence. We studied rates of evolutionary change of 5′ splice sites in three groups of closely related intron-rich species—three primates, five Drosophila species, and four Cryptococcus fungi. Surprisingly, the results indicate that changes from consensus-to-variant nucleotides are generally disfavored by selection, but that changes from variant to consensus are neither favored nor disfavored. This evolutionary pattern is consistent with selective differences across introns, for instance, due to compensatory changes at other sites within the gene, which compensate for the otherwise suboptimal consensus-to-variant changes in splice boundaries.

Gene and intron prediction are essential for accurate inferences about genome evolution. Recently, two genome-wide studies searched for recent intron gains in humans, reaching very different conclusions: either of a complete absence of intron gain since early mammalian evolution, or of creation of numerous introns by genomic duplication in repetitive regions. We discuss one possible explanation: the underappreciated phenomenon of “template switching”, by which reverse transcriptase may create artifactual splicing-like events in the preparation of cDNA/EST libraries, may cause complications in searches for newly gained introns in repetitive regions. We report large numbers of apparent template switching in transcript sequences from the intron-poor protists Trichomonas vaginalis and Giardia lamblia. Supplementary material for this article can be found on the BioEssays website (http://www.interscience.wiley.com/jpages/0265-9247/suppmat/index.html). BioEssays 30:601–605, 2008. © 2008 Wiley Periodicals, Inc.

Over the past 5 years, the availability of dozens of whole genomic sequences from a wide variety of eukaryotic lineages has revealed a very large amount of information about the dynamics of intron loss and gain through eukaryotic history, as well as the evolution of intron sequences. Implicit in these advances is a great deal of information about the structure and evolution of surrounding sequences. Here, we review the wealth of ways in which structures of spliceosomal introns as well as their conservation and change through evolution may be harnessed for evolutionary and genomic analysis. First, we discuss uses of intron length distributions and positions in sequence assembly and annotation, and for improving alignment of homologous regions. Second, we review uses of introns in evolutionary studies, including the utility of introns as indicators of rates of sequence evolution, for inferences about molecular evolution, as signatures of orthology and paralogy, and for estimating rates of nucleotide substitution. We conclude with a discussion of phylogenetic methods utilizing intron sequences and positions.

Despite substantial work, the phylogeny of malaria parasites remains debated. The matter is complicated by concerns about patterns of evolution in potentially strongly selected genes as well as the extreme AT bias of some Plasmodium genomes. Particularly contentious has been the position of the most virulent human parasite Plasmodium falciparum, whether grouped with avian parasites or within a larger clade of mammalian parasites. Here, we study 3 classes of rare genomic changes, as well as the sequences of mitochondrial ribosomal RNA (rRNA) genes. We report 3 lines of support for a clade of mammalian parasites: 1) we find no instances of spliceosomal intron loss in a hypothetical ancestor of P. falciparum and the avian parasite Plasmodium gallinaceum, suggesting against a close relationship between those species; 2) we find 4 genomic mitochondrial indels supporting a mammalian clade, but none grouping P. falciparum with avian parasites; and 3) slowly evolving mitochondrial rRNA sequences support a mammalian parasite clade with 100% posterior probability. We further report a large deletion in the mitochondrial large subunit rRNA gene, which suggests a subclade including both African and Asian parasites within the clade of closely related primate malarias. This contrasts with previous studies that provided strong support for separate Asian and African clades, and reduces certainty about the historical and geographic origins of Plasmodium vivax. Finally, we find a lack of synapomorphic gene losses, suggesting a low rate of ancestral gene loss in Plasmodium.

Alternative splicing (AS) contributes to increased transcriptome and proteome diversity in various eukaryotic lineages. Previous studies showed low levels of conservation of alternatively spliced (cassette) exons within mammals and within dipterans. We report a strikingly different pattern in Caenorhabditis nematodes-more than 92% of cassette exons from Caenorhabditis elegans are conserved in Caenorhabditis briggsae and/or Caenorhabditis remanei. High levels of conservation extend to minor-form exons (present in a minority of transcripts) and are particularly pronounced for exons showing complex patterns of splicing. The functionality of the vast majority of cassette exons is underscored by various other features. We suggest that differences in conservation between lineages reflect differences in levels of functionality and further suggest that these differences are due to differences in intron length and the strength of consensus boundaries across lineages. Finally, we demonstrate an inverse relationship between AS and gene duplication, suggesting that the latter may be primarily responsible for the emergence of new functional transcripts in nematodes.

Chromosome rearrangements occur in a variety of eukaryotic life cycles, including during the development of the somatic macronuclear genome in ciliates. Previous work on the phyllopharyngean ciliate Chilodonella uncinata revealed that macronuclear β-tubulin and protein kinase gene families share alternatively processed germ line segments nested within divergent regions. To study genome evolution in this ciliate further, we characterized two additional alternatively processed gene families from two cryptic species of the ciliate morphospecies C. uncinata: those encoding histidine acid phosphatase protein (Hap) and leishmanolysin family protein (Lei). Analyses of the macronuclear Hap and Lei sequences reveal that each gene family consists of three members in the macronucleus that are marked by identical regions nested among highly divergent regions. Investigation of the micronuclear Hap sequences revealed a complex pattern in which the three macronuclear sequences are derived either from a single micronuclear region or from a combination of this shared region recombined with additional duplicate micronuclear copies of Hap. We propose a model whereby gene scrambling evolves by gene duplication followed by partial and reciprocal degradation of the duplicate sequences. In this model, alternative processing represents an intermediate step in the evolution of scrambled genes. Finally, we speculate on the possible role of genome architecture in speciation in ciliates by describing what might happen if changes in alternatively processed loci occur in subdivided populations., IMPORTANCE Genome rearrangements occur in a variety of eukaryotic cells and serve as an important mechanism for generating genomic diversity. The unusual genome architecture of ciliates with separate germline and somatic nuclei in each cell, provides an ideal system to study further principles of genome evolution. Previous analyses revealed complex forms of chromosome rearrangements, including gene scrambling and alternative processing of germ line chromosomes. Here we describe more complex rearrangements between germ line and somatic chromosomes than previously seen in alternatively processed gene families. Drawing on the present and previous findings, we propose a model in which alternative processing of duplicated micronuclear regions represents an intermediate stage in the evolution of scrambled genes. Under this model, alternative processing may provide insights into a mechanism for speciation in ciliates. Our data on gene scrambling and alternative processing also enhance views on the dynamic nature of genomes across the eukaryotic tree of life.

The age of modern introns and the evolutionary forces controlling intron loss and gain remain matters of much debate. In the case of the apicomplexan malaria parasite Plasmodium falciparum, previous studies have shown that while the positions of two thirds of P. falciparum introns are not shared with surveyed non-apicomplexans (leaving open the possibility that they were relatively recently gained), 99.1% are shared with Plasmodium yoelii, which diverged from P. falciparum at least 100 Mya. We show here that 60.6% of P. falciparum intron positions in conserved regions are shared with the distantly related apicomplexan Theileria parva, whereas only 18.2% of introns in the more intron-rich T. parva are shared with P. falciparum. Comparison of 3305 pairs of orthologous genes between T. parva and Theileria annulata showed that 7089/7111 (99.7%) introns in conserved regions are shared between species. These levels of conservation imply significant differences in rates of intron loss and gain through apicomplexan history. Because transposable elements (TEs) and/or (often TE-encoded) reverse transcriptase are implicated in models of intron loss and gain, the observed low rates of intron loss and gain in recent Plasmodium and Theileria evolution are consistent with the lack of known TE in those groups. We suggest that intron loss/gain in some eukaryotic lineages may be concentrated in relatively short episodes coincident with occasional TE invasions.

We compared intron positions in conserved regions of 3479 orthologous gene pairs from Plasmodium falciparum and Plasmodium yoelii, which likely diverged ≥100 million years ago (Mya). Only 27 out of 2212 positions were specific to one of the two species. Intron presence in related species shows that at least 19 and possibly 26 of the changes are due to intron loss, depending on phylogeny. The implied intron loss and gain rates are much lower than previously estimated for nematodes, arthropods, fungi, and plants, and are comparable only with the rates in vertebrates. That all observed changes were exact, occurring without loss or gain of flanking coding sequence, suggests intron loss via an mRNA intermediate, as does a nonsignificant trend toward loss of introns at adjacent positions. Many of the intron changes occurred in genes encoding proteins involved in nucleic acid-related processes, as previously found for intron gains in nematodes. Two changes occurred in the chloroquine resistance transporter, suggesting a role for positive selection in intron loss in Plasmodium. The dearth of intron loss and gain could be explained by the lack of known transposable elements in Plasmodium, since transposable elements and/or reverse transcriptase are thought to be necessary for both processes. The observed pattern suggests that the availability of stochastic intron loss and gain mutations can be a major determinant of changes in intron number.

The origins and importance of spliceosomal introns comprise one of the longest-abiding mysteries of molecular evolution. Considerable debate remains over several aspects of the evolution of spliceosomal introns, including the timing of intron origin and proliferation, the mechanisms by which introns are lost and gained, and the forces that have shaped intron evolution. Recent important progress has been made in each of these areas. Patterns of intron-position correspondence between widely diverged eukaryotic species have provided insights into the origins of the vast differences in intron number between eukaryotic species, and studies of specific cases of intron loss and gain have led to progress in understanding the underlying molecular mechanisms and the forces that control intron evolution.

The relationship between three biologically important groups, arthropods, nematodes, and deuterostomes, remains unresolved. It is unknown whether arthropods are more closely related to nematodes (consistent with the “ecdysozoa” hypothesis) or to deuterostomes (consistent with “coelomata”). We present a method in which we use the pattern of spliceosomal intron conservation to develop a series of inequalities that characterize each possible relationship. We find that only the ecdysozoa grouping satisfies these predictions, with P < 10 –6 . Simulations show that our method, unlike some previous methods, is largely insensitive to rate variation between branches.

We use the pattern of intron conservation in 684 groups of orthologs from seven fully sequenced eukaryotic genomes to provide maximum likelihood estimates of the number of introns present in the same orthologs in various eukaryotic ancestors. We find: ( i ) intron density in the plant–animal ancestor was high, perhaps two-thirds that of humans and three times that of Drosophila ; and ( ii ) intron density in the ancestral bilateran was also high, equaling that of humans and four times that of Drosophila . We further find that modern introns are generally very old, with two-thirds of modern bilateran introns dating to the ancestral bilateran and two-fifths of modern plant, animal, and fungus introns dating to the plant–animal ancestor. Intron losses outnumber gains over a large range of eukaryotic lineages. These results show that early eukaryotic gene structures were very complex, and that simplification, not embellishment, has dominated subsequent evolution.

We studied intron loss in 684 groups of orthologous genes from seven fully sequenced eukaryotic genomes. We found that introns closer to the 3′ ends of genes are preferentially lost, as predicted if introns are lost through gene conversion with a reverse transcriptase product of a spliced mRNA. Adjacent introns tend to be lost in concert, as expected if such events span multiple intron positions. Directly contrary to the expectations of some, introns that do not interrupt codons (phase zero) are more, not less, likely to be lost, an intriguing and previously unappreciated result. Adjacent introns with matching phases are not more likely to be retained, as would be expected if they enjoyed a relative selective advantage. The findings of 3′ and phase zero intron loss biases are in direct contradiction to an extremely recent study of fungi intron evolution. All patterns are less pronounced in the lineage leading to Caenorhabditis elegans , suggesting that the process of intron loss may be qualitatively different in nematodes. Our results support a reverse transcriptase-mediated model of intron loss.

We compared intron–exon structures in 1,560 human–mouse orthologs and 360 mouse–rat orthologs. The origin of differences in intron positions between species was inferred by comparison with an outgroup, Fugu for human–mouse and human for mouse–rat. Among 10,020 intron positions in the human–mouse comparison, we found unequivocal evidence for five independent intron losses in the mouse lineage but no evidence for intron loss in humans or for intron gain in either lineage. Among 1,459 positions in rat–mouse comparisons, we found evidence for one loss in rat but neither loss in mouse nor gain in either lineage. In each case, the intron losses were exact, without change in the surrounding coding sequence, and involved introns that are extremely short, with an average of 200 bp, an order of magnitude shorter than the mammalian average. These results favor a model whereby introns are lost through gene conversion with intronless copies of the gene. In addition, the finding of widespread conservation of intron–exon structure, even over large evolutionary distances, suggests that comparative methods employing information about gene structures should be very successful in correctly predicting exon boundaries in genomic sequences.

For nearly 15 years, it has been widely believed that many introns were recently acquired by the genes of multicellular organisms. However, the mechanism of acquisition has yet to be described for a single animal intron. Here, we report a large-scale computational analysis of the human, Drosophila melanogaster, Caenorhabditis elegans, and Arabidopsis thaliana genomes. We divided 147,796 human intron sequences into batches of similar lengths and aligned them with each other. Different types of homologies between introns were found, but none showed evidence of simple intron transposition. Also, 106,902 plant, 39,624 Drosophila, and 6021 C. elegans introns were examined. No single case of homologous introns in nonhomologous genes was detected. Thus, we found no example of transposition of introns in the last 50 million years in humans, in 3 million years in Drosophila and C. elegans, or in 5 million years in Arabidopsis. Either new introns do not arise via transposition of other introns or intron transposition must have occurred so early in evolution that all traces of homology have been lost.

In ancient genes whose products have known 3-dimensional structures, an excess of phase zero introns (those that lie between the codons) appear in the boundaries of modules, compact regions of the polypeptide chain. These excesses are highly significant and could support the hypothesis that ancient genes were assembled by exon shuffling involving compact modules. (Phase one and two introns, and many phase zero introns, appear to arise later.) However, as more genes, with larger numbers of homologs and intron positions, were examined, the effects became smaller, dropping from a 40% excess to an 8% excess as the number of intron positions increased from 570 to 3,328, even though the statistical significance remained strong. An interpretation of this behavior is that novel inserted positions appearing in homologs washed out the signal from a finite number of ancient positions. Here we show that this is likely to be the case. Analyses of intron positions restricted to those in genes for which relatively few intron positions from homologs are known, or to those in genes with a small number of known homologous gene structures, show a significant correlation of phase zero intron positions with the module structure, which weakens as the density of attributed intron positions or the number of homologs increases. These effects do not appear for phase one and phase two introns. This finding matches the expectation of the mixed model of intron origin, in which a fraction of phase zero introns are left from the assembly of the first genes, while other introns have been added in the course of evolution.

The intron-exon structures of eukaryotic nuclear genomes exhibit tremendous diversity across different species. The availability of many genomes from diverse eukaryotic species now allows for the reconstruction of the evolutionary history of this diversity. Consideration of spliceosomal systems in comparative context reveals a surprising and very complex portrait: in contrast to many expectations, gene structures in early eukaryotic ancestors were highly complex and "animal or plant-like" in many of their spliceosomal structures has occurred; pronounced simplification of gene structures, splicing signals, and spliceosomal machinery occurring independently in many lineages. In addition, next-generation sequencing of transcripts has revealed that alternative splicing is more common across eukaryotes than previously thought. However, much alternative splicing in diverse eukaryotes appears to play a regulatory role: alternative splicing fulfilling the most famous role for alternative splicing-production of multiple different proteins from a single gene-appears to be much more common in animal species than in nearly any other lineage.

Do introns delineate elements of protein tertiary structure? This issue is crucial to the debate about the role and origin of introns. We present an analysis of the full set of proteins with known three-dimensional structures that have homologs with intron positions recorded in GenBank. A computer program was generated that maps on a reference sequence the positions of all introns in homologous genes. We have applied this program to a set of 665 nonredundant protein sequences with defined three-dimensional structures in the Protein Data Bank (PDB), which yielded 8,217 introns in 407 proteins. For the subset of proteins corresponding to ancient conserved regions (ACR), we find that there is a correlation of phase-zero introns with the boundary regions of modules and no correlation for the phase-one and phase-two positions. However, for a subset of proteins without prokaryotic counterparts (131 non-ACR proteins), a set of presumably modern proteins (or proteins that have diverged extremely far from any ancestral form), we do not find any correlation of phase-zero intron positions with three-dimensional structure. Furthermore, we find an anticorrelation of phase-one intron positions with module boundaries: they actually have a preference for the interior of modules. This finding is explicable as a preference for phase-one introns to lie in glycines, between G|G sequences, the preference for glycines being anticorrelated with the three-dimensional modules. We interpret this anticorrelation as a sign that a number of phase-one introns, and hence many modern introns, have been inserted into G|G “protosplice” sequences.

Another expected legacy of ancient phase-zero introns is that putatively ancient introns (those widely phylogenetically distributed) should have a stronger phase-zero bias. We tested this notion by examining widely distributed C. elegans intron positions: those present in two or more kingdoms. We used a database of ancient genes with known corresponding protein structures (culled from the Protein Data Bank) and the introns from their homologues, compiled by Fedorov and collaborators (unpublished). Of 6568 total intron positions, C. elegans introns are found at 1539 positions within 280 gene families. At 233 of these C. elegans positions there are also introns in the copies of the gene from non-animal organisms. Such coincident positions are candidates for the positions of ancient introns, based on this very wide phylogenetic distribution. (These coincident intron positions do, in fact, show a stronger correlation with module boundaries 6xCentripetal modules and ancient introns. Roy, S.W. et al. Gene. 1999; 238: 85–91Crossref | PubMed | Scopus (40)See all References6.) If these positions are indeed ancient, and if the phase-zero bias of introns is due to the presence of ancient introns, this ancient subset of C. elegans introns should have a higher phase-zero bias than should the set of animal-unique C. elegans introns.Table 2Table 2 shows that this ancient subset does have a significantly stronger phase-zero bias (62.2% versus 50.8%), with a χ2 of 10.4 (P = 0.0013). This is a striking verification of the expectation that part of the phase-zero bias is due to the presence of truly ancient introns. Table 3Table 3 shows further that this is a general phenomenon. There is a highly-significant difference between the phase bias of animal intron positions at which introns are found in two or more kingdoms and those positions that are exclusively animal. The same is true for invertebrates and vertebrates separately, for plants, and for fungi, and there is a significant difference for protists. This trend is seen even further in positions where introns are found in three or more kingdoms. This result suggests that there were in fact ancient intron positions and that the observed excess of phase-zero positions is, at least in part, due to their presence in modern eukaryotes.Table 2Phase patterns of introns in Caenorhabditis elegans genesPhase zeroχ2Phase onePhase twoTotal intronsExclusively animal C. elegans positions663 50.8%306 23.4%337 25.8%1306Widely distributed C. elegans positions145 62.2%10.446 19.7%42 18.0%233Widely distributed signifies that an intron is also found at that position in a non-animal copy of the gene. The introns are from the database of 6568 intron positions in the set of ancient genes that have three-dimensional structures for the corresponding proteins compiled by Fedorov and collaborators (unpublished). The χ2 is calculated comparing phase zero with (phase one + phase two).Table 3Phase patterns of introns in different kingdomsaPhase zeroχ2Phase onePhase twoTotal intronsAnimalExclusively animal1607 50.2%860 26.9%735 23.0%3202Animal and other kingdom(s)329 59.8%17.4127 23.1%94 17.1%550Exclusively vertebrate504 48.2%324 31.0%217 20.8%1045Vertebrate and other kingdom(s)194 61.8%17.869 22.0%51 16.2%314Exclusively invertebrate906 51.6%423 24.1%428 24.4%1757Invertebrate and other kingdom(s)246 58.9%7.296 23.0%76 18.2%418 PlantExclusively plant1166 59.9%398 20.5%381 19.6%1945Plant and other kingdom(s)323 66.1%6.199 20.2%67 13.7%489 FungiExclusively fungi303 42.7%236 33.3%170 24.0%709Fungi and other kingdom(s)118 54.1%8.756 25.7%44 20.2%218 ProtistExclusively protist34 38.6%28 31.8%26 29.5%88Protists and other kingdom(s)41 56.9%5.316 22.2%15 20.8%72 AllOnly one kingdom3110 52.3%1522 25.6%1312 22.1%5944Two or more kingdoms371 60.4%14.7142 23.1%101 16.4%614Three or more kingdoms51 64.6%13 16.5%15 19.0%79The introns are from the database of 6568 intron positions in the set of ancient genes that have three-dimensional structures for the corresponding proteins compiled by Fedorov and collaborators (unpublished). The χ2 is calculated comparing phase zero with (phase one + phase two).These twin findings – first, that the intron phases in ancient genes, defined by BLAST scores, differ from those of recently transferred genes by having a stronger phase-zero bias, and second, that phylogenetically ancient intron positions also show a stronger phase-zero bias – support the theory that introns, mostly in phase zero, were present in the LUCA.Furthermore, as phase-zero, widely phylogenetically-distributed intron positions are highly correlated with the boundaries of modules 6xCentripetal modules and ancient introns. Roy, S.W. et al. Gene. 1999; 238: 85–91Crossref | PubMed | Scopus (40)See all References6, the subset of introns possessing one characteristic expected of ancient intron positions – wide phylogenetic distribution – also possesses two other traits expected of ancient positions. This is a result easily explained on a mixed intron-origin model, but not easily explained on strictly insertional ones.

Understanding gene duplication and gene structure evolution are fundamental goals of molecular evolutionary biology. A previous study by Babenko et al. (2004. Prevalence of intron gain over intron loss in the evolution of paralogous gene families. Nucleic Acids Res. 32:3724-3733) employed Dollo parsimony to infer spliceosomal intron losses and gains in paralogous gene families and concluded that there was a general excess of gains over losses. This result contrasts with patterns in orthologous genes, in which most lineages show an excess of intron losses over gains, suggesting the possibility of fundamentally different modes of intron evolution between orthologous and paralogous genes. We further studied the data and found a low level of intron position conservation with outgroups, and this led to problems with using Dollo parsimony to analyze the data. Statistical reanalysis of the data suggests, instead, that intron losses have outnumbered intron gains in paralogous gene families.

Cryptophyte and chlorarachniophyte algae are transitional forms in the widespread secondary endosymbiotic acquisition of photosynthesis by engulfment of eukaryotic algae. Unlike most secondary plastid-bearing algae, miniaturized versions of the endosymbiont nuclei (nucleomorphs) persist in cryptophytes and chlorarachniophytes. To determine why, and to address other fundamental questions about eukaryote–eukaryote endosymbiosis, we sequenced the nuclear genomes of the cryptophyte Guillardia theta and the chlorarachniophyte Bigelowiella natans. Both genomes have

Background The Amoebozoa constitute one of the primary divisio ns of eukaryotes encompassing taxa of both biomedic al and evolutionary importance, yet its genomic divers ity remains largely unsampled. Here we present an analysis of a whole genome assembly of Acanthamoeba castellanii ( Ac ) the first representative from a solitary free-living amoebozoan. Results Ac encodes 15,455 compact intron rich genes a signifi cant number of which are predicted to have arisen through interkingdom lateral gene transfer (LGT). A majority of the LGT candidates have undergone a substantial degree of intronization and Ac appears to have incorporated them into established transcriptional programs. Ac manifests a complex signaling and cell communicati on repertoire including a complete tyrosine kinase signaling toolkit and a comparable diversity of predicted extracellular receptors to that found in the facultatively multicellular dictyostelids. An impor tant environmental host of a diverse range of bacte ria and viruses, Ac utilizes a diverse repertoire of predicted pattern recognition receptors many with predicted orthologous functions in the innate immune systems of higher organisms. Conclusions Our analysis highlights the important role of LGT i n the biology of Ac and in the diversification of microbial eukaryotes. The early evolution of a key signaling facility implicated in the evolution of metazoan multicellularity strongly argues for its emergence early in the Unikont lineage. Overall the availabil ity of an Ac genome should aid in deciphering the biology of th e Amoebozoa and facilitate functional genomic studi es in this important model organism and environmental host.

This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date; after six months, it is available under a Creative Commons License.-- et al., The order of genes in eukaryotic genomes has generally been assumed to be neutral, since gene order is largely scrambled over evolutionary time. Only a handful of exceptional examples are known, typically involving deeply conserved clusters of tandemly duplicated genes (e.g., Hox genes and histones). Here we report the first systematic survey of microsynteny conservation across metazoans, utilizing 17 genome sequences. We identified nearly 600 pairs of unrelated genes that have remained tightly physically linked in diverse lineages across over 600 million years of evolution. Integrating sequence conservation, gene expression data, gene function, epigenetic marks, and other genomic features, we provide extensive evidence that many conserved ancient linkages involve (1) the coordinated transcription of neighboring genes, or (2) genomic regulatory blocks (GRBs) in which transcriptional enhancers controlling developmental genes are contained within nearby bystander genes. In addition, we generated ChIP-seq data for key histone modifications in zebrafish embryos, which provided further evidence of putative GRBs in embryonic development. Finally, using chromosome conformation capture (3C) assays and stable transgenic experiments, we demonstrate that enhancers within bystander genes drive the expression of genes such as Otx and Islet, critical regulators of central nervous system development across bilaterians. These results suggest that ancient genomic functional associations are far more common than previously thought—involving ∼12% of the ancestral bilaterian genome—and that cis-regulatory constraints are crucial in determining metazoan genome architecture., M.I., M.S.A., S.W.R., and H.B.F. were funded by NIH grant 1R21HG005240-01A1. H.B.F. is an Alfred P. Sloan Fellow and Pew Scholar in the Biomedical Sciences. J.J.T., A.F-M., O.B., E.C-M., and J.L.G-S. were funded by grants BFU2010-14839, CSD2007-00008, and Proyecto de Excelencia CVI-3488.

The scallop is an economically important sea food prized for its large and delicious adductor muscle. Studying the molecular basis of scallop muscle growth is important for both scallop breeding and our understanding of muscle mass regulation in bivalve. Myostatin (MSTN) is a conserved negative regulator of muscle growth and development. Here we report the MSTN gene from Zhikong scallop (Chlamys farreri Jones et Preston 1904). The C. farreri MSTN consists of 11651 nucleotides encoding 457 amino acids. The gene has a 3-exon/2-intron structure that is conserved with vertebrate homologs. The exons are 586, 380 and 408 bp in length, respectively, and separated by introns of 5086 and 1518 bp. The protein sequence contains characteristic conserved residues including a cleavage motif of proteolysis (RXXR) and nine cysteines. Three transcription initiation sites were found at 62, 146, and 296 bp upstream of the translation start codon ATG. In silico analysis of the promoter region identified a TATA-box and several muscle-specific regulatory elements including COMP, MEF2s, MTBFs and E-boxes. Minisatellite DNA was found in intron 1. By fluorescence in situ hybridization (FISH), the gene was mapped to the long arm of a pair of middle subtelocentric chromosome. Quantitative analysis of MSTN transcripts in embryos/larvae indicated high expression level in gastrulae and limited expression at other stages. In adult scallops, MSTN is predominantly expressed in striated muscle, with different expression levels in other tissues. Our data provide valuable genomic and expression information which will aid the further study on scallop MSTN function and MSTN evolution.

Whether a trade-off exists between robustness and evolvability is an important issue for protein evolution. Although traditional viewpoints have assumed that existing functions must be compromised by the evolution of novel activities, recent research has suggested that existing phenotypes can be robust to the evolution of novel protein functions. Enzymes that are targets of antibiotics that are competitive inhibitors must evolve decreased drug affinity while maintaining their function and sustaining growth. Utilizing a transgenic Saccharomyces cerevisiae model expressing the dihydrofolate reductase (DHFR) enzyme from the malarial parasite Plasmodium falciparum, we examine the robustness of growth rate to drug-resistance mutations. We assay the growth rate and resistance of all 48 combinations of 6 DHFR point mutations associated with increased drug resistance in field isolates of the parasite. We observe no consistent relationship between growth rate and resistance phenotypes among the DHFR alleles. The three evolutionary pathways that dominate DHFR evolution show that mutations with increased resistance can compensate for initial declines in growth rate from previously acquired mutations. In other words, resistance mutations that occur later in evolutionary trajectories can compensate for the fitness consequences of earlier mutations. Our results suggest that high levels of resistance may be selected for without necessarily jeopardizing overall fitness.

Nature contains a tremendous diversity of forms both at the organismal and genomic levels. This diversity motivates the twin central questions of molecular evolution: what are the molecular mechanisms of adaptation, and what are the functional consequences of genomic diversity. We report a 22-species comparative analysis of tropomyosin (PPM) genes, which exist in a variety of forms and are implicated in the emergence of a wealth of cellular functions, including the novel muscle functions integral to the functional diversification of bilateral animals. TPM genes encode either or both of long-form [284 amino acid (aa)] and short-form (approximately 248 aa) proteins. Consistent with a role of TPM diversification in the origins and radiation of bilaterians, we find evidence that the muscle-specific long-form protein arose in proximal bilaterian ancestors (the bilaterian 'stem'). Duplication of the 5' end of the gene led to alternative promoters encoding long- and short-form transcripts with distinct functions. This dual-function gene then underwent strikingly parallel evolution in different bilaterian lineages. In each case, recurrent tandem exon duplication and mutually exclusive alternative splicing of the duplicates, with further association between these alternatively spliced exons along the gene, led to long- and short-form-specific exons, allowing for gradual emergence of alternative "internal paralogs" within the same gene. We term these Mutually exclusively Alternatively spliced Tandemly duplicated Exon sets "MATEs". This emergence of internal paralogs in various bilaterians has employed every single TPM exon in at least one lineage and reaches striking levels of divergence with up to 77% of long- and short-form transcripts being transcribed from different genomic regions. Interestingly, in some lineages, these internal alternatively spliced paralogs have subsequently been "externalized" by full gene duplication and reciprocal retention/loss of the two transcript isoforms, a particularly clear case of evolution by subfunctionalization. This parallel evolution of TPM genes in diverse metazoans attests to common selective forces driving divergence of different gene transcripts and represents a striking case of emergence of evolutionary novelty by alternative splicing.

Immune evasion by Plasmodium falciparum is favored by extensive allelic diversity of surface antigens. Some of them, most notably the vaccine-candidate merozoite surface protein (MSP)-1, exhibit a poorly understood pattern of allelic dimorphism, in which all observed alleles group into two highly diverged allelic families with few or no inter-family recombinants. Here we describe contrasting levels and patterns of sequence diversity in genes encoding three MSP-1-associated surface antigens of P. falciparum, ranging from an ancient allelic dimorphism in the Msp-6 gene to a near lack of allelic divergence in Msp-9 to a more classical multi-allele polymorphism in Msp-7. Other members of the Msp-7 gene family exhibit very little polymorphism in non-repetitive regions. A comparison of P. falciparum Msp-6 sequences to an orthologous sequence from P. reichenowi provided evidence for distinct evolutionary histories of the 5' and 3' segments of the dimorphic region in PfMsp-6, consistent with one dimorphic lineage having arisen from recombination between now-extinct ancestral alleles. In addition, we uncovered two surprising patterns of evolution in repetitive sequence. First, in Msp-6, large deletions are associated with (nearly) identical sequence motifs at their borders. Second, a comparison of PfMsp-9 with the P. reichenowi ortholog indicated retention of a significant inter-unit diversity within an 18-base pair repeat within the coding region of P. falciparum, but homogenization in P. reichenowi.

Alternative splicing (AS) is a widespread mechanism with an important role in increasing transcriptome and proteome diversity by generating multiple different products from the same gene. Evolutionary studies of AS have focused primarily on the conservation of alternatively spliced sequences or of the AS pattern of those sequences itself. Less is known about the evolution of the regulation of AS, but several studies, working from different perspectives, have recently made significant progress. Here, we categorize the different levels of AS evolution, and summarize the studies on evolution of AS regulation, which point to a high level of evolutionary conservation of the regulation of AS events conserved between related species. This suggests that the quantitative regulation of AS is an intrinsic part of AS function. We discuss the potential role of changes in developmental regulation of AS as an additional layer in complex gene regulatory networks and in the emergence of genetic novelties.

Comparative genomics has begun to unravel the evolutionary history of transcript splicing in eukaryotes. The last common ancestor of modern eukaryotes is now known to have had at least moderately intron-dense genes and two complex spliceosomes. For other splicing-related phenomena the evolutionary history is less clear. We suggest that frequent mis-splicing is likely to be ancestral to eukaryotes, whereas trans-splicing and operon splicing are likely to be more recent. The origins of regulated splicing, alternative splicing and splicing of untranslated transcript regions are less certain. The data discussed underscore the significant genomic complexity of early eukaryotes, and should help to frame future questions about the origins of eukaryotic genome structure.

Despite their ubiquity, the mechanisms and evolutionary forces responsible for the origins of spliceosomal introns remain mysterious. Recent molecular evidence supports the idea that intronic RNAs can reverse splice into RNA transcripts, a crucial step for an influential model of intron gain. However, a paradox attends this model because the rate of intron gain is expected to be orders of magnitude lower than the rate of intron loss in general, in contrast to findings from several lineages. We suggest two possible resolutions to this paradox, based on steric considerations and on the possibility of co-option by specific introns of retroelement transposition pathways, respectively. In addition, we introduce two potential mechanisms for intron creation, based on hybrid RNA-DNA reverse splicing and on template switching errors by reverse transcriptase.