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Next-generation sequencing (NGS) based HLA genotyping can generate PCR artifacts corresponding to IMGT/HLA Database alleles, for which multiple examples have been observed, including sequence corresponding to the HLA-DRB1(∗)03:42 allele. Repeat genotyping of 131 samples, previously genotyped as DRB1(∗)03:01 homozygotes using probe-based methods, resulted in the heterozygous call DRB1(∗)03:01+DRB1(∗)03:42. The apparent rare DRB1(∗)03:42 allele is hypothesized to be a "hybrid amplicon" generated by PCR crossover, a process in which a partial PCR product denatures from its template, anneals to a different allele template, and extends to completion. Unlike most PCR crossover products, "hybrid amplicons" always corresponds to an IMGT/HLA Database allele, necessitating a case-by-case analysis of whether its occurrence reflects the actual allele or is simply the result of PCR crossover. The Hybrid Amplicon/PCR Crossover Artifact Detector (HAPCAD) program mimics jumping PCR in silico and flags allele sequences that may also be generated as hybrid amplicon.

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.

In his landmark 1987 study of the merozoite surface protein-1 locus in Plasmodium falciparum, Kazuyuki Tanabe and coauthors introduced the phenomenon of allelic dimorphism, in which antigenic diversity is arranged into two maximally diverged haplotypes. Further work has extended this finding to other loci in P. falciparum. Each of the loci at which allelic dimorphism is observed encodes major surface antigens of blood-stage malaria parasites, and is consequently a major vaccine target, thus understanding the origins and implications of allelic dimorphism is of crucial importance. Here we examine the essential features of allelic dimorphism in dimorphic malarial surface antigens. From sequence analysis, we conclude that the ancestral population may have been recombining/multimorphic rather than dimorphic. We hypothesize a pathway to allelic dimorphism in which an ancestral allele-rich recombining population could have undergone a severe population bottleneck, putatively caused by the lateral transfer of P. falciparum from apes to humans. This bottleneck produced a reduction in allelic diversity, favoring the survival of the most divergent alleles, which in turn led to recombination suppression by strong natural selection against recombinants.

Many efforts to improve science teaching in higher education focus on a few faculty members at an institution at a time, with limited published evidence on attempts to engage faculty across entire departments. We created a long-term, department-wide collaborative professional development program, Biology Faculty Explorations in Scientific Teaching (Biology FEST). Across 3 years of Biology FEST, 89% of the department’s faculty completed a weeklong scientific teaching institute, and 83% of eligible instructors participated in additional semester-long follow-up programs. A semester after institute completion, the majority of Biology FEST alumni reported adding active learning to their courses. These instructor self-reports were corroborated by audio analysis of classroom noise and surveys of students in biology courses on the frequency of active-learning techniques used in classes taught by Biology FEST alumni and nonalumni. Three years after Biology FEST launched, faculty participants overwhelmingly reported that their teaching was positively affected. Unexpectedly, most respondents also believed that they had improved relationships with departmental colleagues and felt a greater sense of belonging to the department. Overall, our results indicate that biology department–wide collaborative efforts to develop scientific teaching skills can indeed attract large numbers of faculty, spark widespread change in teaching practices, and improve departmental relations.

Active-learning pedagogies have been repeatedly demonstrated to produce superior learning gains with large effect sizes compared with lecture-based pedagogies. Shifting large numbers of college science, technology, engineering, and mathematics (STEM) faculty to include any active learning in their teaching may retain and more effectively educate far more students than having a few faculty completely transform their teaching, but the extent to which STEM faculty are changing their teaching methods is unclear. Here, we describe the development and application of the machine-learning-derived algorithm Decibel Analysis for Research in Teaching (DART), which can analyze thousands of hours of STEM course audio recordings quickly, with minimal costs, and without need for human observers. DART analyzes the volume and variance of classroom recordings to predict the quantity of time spent on single voice (e.g., lecture), multiple voice (e.g., pair discussion), and no voice (e.g., clicker question thinking) activities. Applying DART to 1,486 recordings of class sessions from 67 courses, a total of 1,720 h of audio, revealed varied patterns of lecture (single voice) and nonlecture activity (multiple and no voice) use. We also found that there was significantly more use of multiple and no voice strategies in courses for STEM majors compared with courses for non-STEM majors, indicating that DART can be used to compare teaching strategies in different types of courses. Therefore, DART has the potential to systematically inventory the presence of active learning with ∼90% accuracy across thousands of courses in diverse settings with minimal effort.

The giant, single-celled organism Stentor coeruleus has a long history as a model system for studying pattern formation and regeneration in single cells. Stentor (Figure 1A,B [1,2]) is a heterotrichous ciliate distantly related to familiar ciliate models such as Tetrahymena or Paramecium. The primary distinguishing feature of Stentor is its incredible size: a single cell is 1 millimeter long. Early developmental biologists, including T.H. Morgan[3], were attracted to the system because of its regenerative abilities -- if large portions of a cell are surgically removed, the remnant reorganizes into a normal-looking but smaller cell with correct proportionality [2,3]. These biologists were also drawn to Stentor because it exhibits a rich repertoire of behaviors, including light avoidance, mechanosensitive contraction, food selection, and even the ability to habituate to touch, a simple form of learning usually seen in higher organisms [4]. While early microsurgical approaches demonstrated a startling array of regenerative and morphogenetic processes in this single-celled organism, Stentor was never developed as a molecular model system. We report the sequencing of the Stentor coeruleus macronuclear genome and reveal key features of the genome: First, we find that Stentor uses the standard genetic code, suggesting that ciliate specific genetic codes arose after Stentor branched from other ciliates. We also discover that ploidy correlates with Stentor’s cell size. Finally, in the Stentor genome, we discover the smallest spliceosomal introns reported for any species. The sequenced genome opens the door to molecular analysis of single-cell regeneration in Stentor.

Instructor Talk—noncontent language used by instructors in classrooms—is a recently defined and promising variable for better understanding classroom dynamics. Having previously characterized the Instructor Talk framework within the context of a single course, we present here our results surrounding the applicability of the Instructor Talk framework to noncontent language used by instructors in novel course contexts. We analyzed Instructor Talk in eight additional biology courses in their entirety and in 61 biology courses using an emergent sampling strategy. We observed widespread use of Instructor Talk with variation in the amount and category type used. The vast majority of Instructor Talk could be characterized using the originally published Instructor Talk framework, suggesting the robustness of this framework. Additionally, a new form of Instructor Talk—Negatively Phrased Instructor Talk, language that may discourage students or distract from the learning process—was detected in these novel course contexts. Finally, the emergent sampling strategy described here may allow investigation of Instructor Talk in even larger numbers of courses across institutions and disciplines. Given its widespread use, potential influence on students in learning environments, and ability to be sampled, Instructor Talk may be a key variable to consider in future research on teaching and learning in higher education.

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.

Background The mechanisms by which DNA sequences are expressed is the central preoccupation of molecular genetics. Recently, ourselves and others reported that in the diplomonad protist Giardia lamblia, the coding regions of several mRNAs are produced by ligation of independent RNA species expressed from distinct genomic loci. Such trans-splicing of introns was found to affect nearly as many genes in this organism as does classical cis-splicing of introns. These findings raised questions about the incidence of intron trans-splicing both across the G. lambliatranscriptome and across diplomonad diversity in general, however a dearth of transcriptomic data at the time prohibited systematic study of these questions. Methods I leverage newly available transcriptomic data from G. lamblia and the related diplomonad Spironucleus salmonicidato search for trans-spliced introns. My computational pipeline recovers all four previously reported trans-spliced introns in G. lamblia, suggesting good sensitivity. Results Scrutiny of thousands of potential cases revealed only a single additional trans-spliced intron in G. lamblia, in the p68 helicase gene, and no cases in S. salmonicida. The p68 intron differs from the previously reported trans-spliced introns in its high degree of streamlining: the core features of G. lamblia trans-spliced introns are closely packed together, revealing striking economy in the implementation of a seemingly inherently uneconomical molecular mechanism. Discussion These results serve to circumscribe the role of trans-splicing in diplomonads both in terms of the number of genes effected and taxonomically. Future work should focus on the molecular mechanisms, evolutionary origins and phenotypic implications of this intriguing phenomenon.

Available genomic sequences from diverse eukaryotes attest to creation of millions of spliceosomal introns throughout the course of evolution, however the question of how introns are created remains unresolved. Resolution of this question has been thwarted by the fact that many modern introns appear to be hundreds of millions of years old, obscuring the mechanisms by which they were initially created. As such, analysis of lineages undergoing rapid intron creation is crucial. Recently, Hoy et al. reported the genome of the predatory mite Metaseiulus occidentalis, revealing generally rapid molecular evolution including wholesale loss of ancestral introns and gain of new ones. I sought to test several potential mechanisms of intron creation. BLAST searches did not reveal patterns of similarity between intronic sequences from different sites or between intron sequences and non-intronic sequences, which would be predicted if introns are created by propagation of pre-existing intronic sequences or by transposable element insertion. To test for evidence that introns are created by any of multiple mechanisms that are expected to lead to duplication of sequences at the two splice boundaries of an intron, I compared introns likely to have been gained in the lineage leading to M. occidentalis and likely ancestral introns. These comparisons did initially reveal greater similarity between boundaries in M. occidentalis-specific introns, however this excess appeared to be largely or completely due to greater adherence of newer introns to the so-called protosplice site, and therefore may not provide strong evidence for particular intron gain mechanisms. The failure to find evidence for particular intron creation mechanisms could reflect the relatively old age of even these introns, intron creation by variants of tested mechanisms that do not leave a clear sequence signature, or by intron creation by unimagined mechanisms.

Broad transcriptomic sequencing of eukaryotes has revealed the ubiquity of splicing of nuclear genes. While the vast majority of splicing events join segments of the same RNA transcript, various studies have found a few intriguing cases of trans-splicing of introns, in which splicing events within protein coding regions join segments of different RNA transcripts. The most structurally intricate case known involves the bursicon gene in mosquitos, in which an internal exon is encoded at a distinct locus, requiring multiple trans-splicing events form the mature mRNA. This arrangement is known to be ancestral to mosquitos, however the exact timing of the origin of trans-splicing and the history of the bursicon gene within mosquitos is unknown. Taking advantage of the recent availability of genomes from various Anopheles mosquitos and from relatives of mosquitos, I determined trans versus cis encoding of bursicon across Culicomorpha. I conclude that trans-splicing emerged in the last common ancestor of mosquitos, and that trans-splicing has been retained in all 19 studied Anopheles species. The retention of trans-splicing could indicate functional importance of this arrangement, or could alternatively reflect the rarity of mutations giving rise to viable allelic alternatives.

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.

Recently there has been a lively debate about a new class of rare genomic characters, RGC_CAMs, and their implications for deep bilaterian phylogeny. Most recently, nine bilaterian species were analyzed along with subsets of six outgroups (Rogozin et al. 2007b), and support for a coelomate clade reported. The authors suggested that our previously reported support for an ecdysozoan clade (Irimia et al. 2007) reflected (i) one outgroup, Nematostella vectensis, being too closely related to bilaterians and (ii) lack of "rigorous statistical analysis." Here, we report further studies of these characters. First, we discuss general issues of outgroup choice. Second, we point out that an argument used by Rogozin et al. against backmutation is not statistically significant. Third, we point out that the statistical method of Rogozin et al. fails to incorporate backmutations, leading to systematic placement of the long-branch taxon as the outgroup. A simple modification of the method yields very different results: 51 of 63 outgroup combinations favor Ecdysozoa, including 51 of 52 with at least eight phylogenetically informative characters, and all 19 with statistically significant signal. These results indicate that the Coelomata signal is a long-branch artifact.

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.

Accurate gene prediction in eukaryotes is a difficult and subtle problem. Here we point out a useful feature of expected distributions of spliceosomal intron lengths. Since introns are removed from transcripts prior to translation, intron lengths are not expected to respect coding frame, thus the number of genomic introns that are a multiple of three bases (‘3n introns’) should be similar to the number that are a multiple of three plus one bases (or plus two bases). Skewed predicted intron length distributions thus suggest systematic errors in intron prediction. For instance, a genome-wide excess of 3n introns suggests that many internal exonic sequences have been incorrectly called introns, whereas a deficit of 3n introns suggests that many 3n introns that lack stop codons have been mistaken for exonic sequence. A survey of genomic annotations for 29 diverse eukaryotic species showed that skew in intron length distributions is a common problem. We discuss several examples of skews in genome-wide intron length distributions that indicate systematic problems with gene prediction. We suggest that evaluation of length distributions of predicted introns is a fast and simple method for detecting a variety of possible systematic biases in gene prediction or even problems with genome assemblies, and discuss ways in which these insights could be incorporated into genome annotation protocols.

Although spliceosomal introns are present in all characterized eukaryotes, intron numbers vary dramatically, from only a handful in the entire genomes of some species to nearly 10 introns per gene on average in vertebrates. For all previously studied intron-rich species, significant fractions of intron positions are shared with other widely diverged eukaryotes, indicating that 1) large numbers of the introns date to much earlier stages of eukaryotic evolution and 2) these lineages have not passed through a very intron-poor stage since early eukaryotic evolution. By the same token, among species that have lost nearly all of their ancestral introns, no species is known to harbor large numbers of more recently gained introns. These observations are consistent with the notion that intron-dense genomes have arisen only once over the course of eukaryotic evolution. Here, we report an exception to this pattern, in the intron-rich diatom Thalassiosira pseudonana. Only 8.1% of studied T. pseudonana intron positions are conserved with any of a variety of divergent eukaryotic species. This implies that T. pseudonana has both 1) lost nearly all of the numerous introns present in the diatom-apicomplexan ancestor and 2) gained a large number of new introns since that time. In addition, that so few apparently inserted T. pseudonana introns match the positions of introns in other species implies that insertion of multiple introns into homologous genic sites in eukaryotic evolution is less common than previously estimated. These results suggest the possibility that intron-rich genomes may have arisen multiple times in evolution. These results also provide evidence that multiple intron insertion into the same site is rare, further supporting the notion that early eukaryotic ancestors were very intron rich.

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.

Numerous previous studies have elucidated 2 surprising patterns of spliceosomal intron evolution in diverse eukaryotes over the past roughly 100 Myr. First, rates of recent intron gain in a wide variety of eukaryotic lineages have been surprisingly low, far too low to explain modern intron densities. Second, intron losses have outnumbered intron gains over a variety of lineages. For several reasons, land plants might be expected to have comparatively high rates of intron gain and thus to represent a possible exception to this pattern. However, we report several studies that indicate low rates of intron gain and an excess of intron losses over intron gains in a variety of plant lineages. We estimate that intron losses have outnumbered intron gains in recent evolution in Arabidopsis thaliana (roughly 12.6 times more losses than gains), Oryza sativa (9.8 times), the green alga Chlamydomonas reinhardtii (5.1 times), and the Bigelowiella natans nucleomorph, an enslaved green algal nucleus (2.8 times). We estimate that during recent evolution, A. thaliana and O. sativa have experienced very low rates of intron gain of around one gain per gene per 2.6-8.0 billion years. In addition, we compared 8,258 pairs of putatively orthologous A. thaliana-O. sativa genes. We found that 5.3% of introns in conserved coding regions are species-specific. Observed species-specific A. thaliana and O. sativa introns tend to be exact and to lie adjacent to each other along the gene, in a pattern suggesting mRNA-mediated intron loss. Our results underscore that low intron gain rates and intron number reduction are common features of recent eukaryotic evolution. This pattern implies that rates of intron creation were higher during earlier periods of evolution and further focuses attention on the causes of initial intron proliferation.

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.

In the present study we have utilized the Allium cepa root tip meristem model to evaluate the cytotoxic and anti-mitotic activities of latex of Calotropis procera (DL) and podophyllotoxin. Standard cytotoxic drug cyclophosphamide and non-cytotoxic drugs cyprohcptadine and aspirin served as controls. Like cyclophosphamide, both DL and podophyllotoxin significantly inhibited the growth of roots and mitotic activity in a dose-dependent manner. However, podophyllotoxin was more potent in this regard and produced root decay. Cyproheptadine and aspirin, on the other hand, showed a marginal effect on the root growth and mitotic activity at much higher concentrations.

In the present study, dry latex (DL) of Calotropis procera possessing potent anti-inflammatory activity was evaluated for its antioxidant and anti-hyperglycemic effects against alloxan-induced diabetes in rats. Daily oral administration of DL at 100 and 400 mg/kg doses produced a dose-dependent decrease in the blood glucose and increase in the hepatic glycogen content. DL also prevented the loss of body weight in diabetic rats and brought down the daily water consumption to values comparable to normal rats. DL also produced an increase in the hepatic levels of the endogenous antioxidants, namely superoxide dismutase (SOD), catalase and glutathione, while it brought down the levels of thiobarbituric acid-reactive substances (TBARS) in alloxan-induced diabetic rats. The efficacy of DL as an antioxidant and as an anti-diabetic agent was comparable to the standard anti-diabetic drug, glibenclamide.

We study the intron–exon structures of 684 groups of orthologs from seven diverse eukaryotic genomes and provide maximum likelihood estimates for rates and numbers of intron losses and gains in these same genes for a variety of lineages. Rates of intron loss vary from ≈2 × 10 –9 to 2 × 10 –10 per year. Rates of gain vary from 6 × 10 –13 to 4 × 10 –12 per possible intron insertion site per year. There is an inverse correspondence between rates of intron loss and gain, leading to a 20-fold variation among lineages in the ratio of the rates of the two processes. The observed rates of intron gain are insufficient to explain the large number of introns estimated to have been present in the plant–animal ancestor, suggesting that introns present in early eukaryotes may have been created by a fundamentally different process than more recently gained introns.

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.

Background Plasmodium erythrocyte invasion genes play a key role in malaria parasite transmission, host-specificity and immuno-evasion. However, the evolution of the genes responsible remains understudied. Investigating these genes in avian malaria parasites, where diversity is particularly high, offers new insights into the processes that confer malaria pathogenesis. These parasites can pose a significant threat to birds and since birds play crucial ecological roles they serve as important models for disease dynamics. Comprehensive knowledge of the genetic factors involved in avian malaria parasite invasion is lacking and has been hampered by difficulties in obtaining nuclear data from avian malaria parasites. Thus the first Illumina-based de novo transcriptome sequencing and analysis of the chicken parasite Plasmodium gallinaceum was performed to assess the evolution of essential Plasmodium genes. Methods White leghorn chickens were inoculated intravenously with erythrocytes containing P. gallinaceum. cDNA libraries were prepared from RNA extracts collected from infected chick blood and sequencing was run on the HiSeq2000 platform. Orthologues identified by transcriptome sequencing were characterized using phylogenetic, ab initio protein modelling and comparative and population-based methods. Results Analysis of the transcriptome identified several orthologues required for intra-erythrocytic survival and erythrocyte invasion, including the rhoptry neck protein 2 (RON2) and the apical membrane antigen-1 (AMA-1). Ama-1 of avian malaria parasites exhibits high levels of genetic diversity and evolves under positive diversifying selection, ostensibly due to protective host immune responses. Conclusion Erythrocyte invasion by Plasmodium parasites require AMA-1 and RON2 interactions. AMA-1 and RON2 of P. gallinaceum are evolutionarily and structurally conserved, suggesting that these proteins may play essential roles for avian malaria parasites to invade host erythrocytes. In addition, host-driven selection presumably results in the high levels of genetic variation found in ama-1 of avian Plasmodium species. These findings have implications for investigating avian malaria epidemiology and population dynamics. Moreover, this work highlights the P. gallinaceum transcriptome as an important public resource for investigating the diversity and evolution of essential Plasmodium genes. Electronic supplementary material The online version of this article (doi:10.1186/1475-2875-13-382) contains supplementary material, which is available to authorized users.

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.

Venkatesh; Byrappa et al.-- This work is licensed under a Creative Commons Attribution- NonCommercial-Share Alike 3.0 Unported licence., 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. © 2014 Macmillan Publishers Limited.

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.

We test the hypothesis that introns were used to construct the first genes from small exons, whose protein products represent compact elements of structure. For any three-dimensional structure, a computer program analyzes the structure into a set of modules, segments of the polypeptide chain bounded in space by a maximum diameter, separated by a set of ‘boundary regions’. The ‘boundary regions’ are such that if the gene were divided by an intron in each ‘boundary region’, the protein would be divided into modules less than the specified diameter. Using a set of 32 ancient proteins, which have no introns in prokaryotes, we examine the intron positions in their eukaryotic homologs and show that the introns are correlated with modules of diameter 21, 28 and 33 A, with P values below 0.001.

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