In the 25 years since they were first discovered, introns have puzzled molecular biologists because of their uncertain function and mysterious origin. Introns are non-coding DNA sequences that reside inside a gene, splitting it into discrete units called exons. The resulting disruption of coding sequence continuity would wreak havoc in protein assembly if eukaryotic cells did not dispose of introns in messenger RNAs—the intermediates in the decoding of gene sequences to produce protein chains—in a now well-described process known as splicing.At first glance, introns may seem like pesky parasites for which eukaryotes have cleverly evolved bypass mechanisms. But introns may also benefit their hosts. Evolutionary advantages of introns include the possibility to create new genes by cutting and pasting exons from existing genes or to diversify the protein output of a single gene by splicing the exons together in different ways. Thus, balancing intron gains and losses clearly has important evolutionary implications for a host.Saccharomyces cerevisiae averages less than one intron per gene, whereas mammalian genes routinely have 10 or more. Whether these differences reflect different propensities for gaining or losing introns is the subject of ongoing debates.Yet different organisms strike that balance differently. The budding yeast Organisms with low intron density display a bias for insertions at the beginning (5′ end) rather than the end (3′ end) of genes. A popular hypothesis is that in these organisms, genes lose their introns through a process that rewrites genomic DNA using as template the messenger RNAs purged of intron sequences. This process might preferentially remove 3′ introns because it relies on an enzyme called reverse transcriptase that can be primed to read RNAs starting at their 3′ end. The hypothesis has gained experimental support in yeast. It also presents the advantage of potentially explaining intron paucity and 5′ position bias in one stroke. In a new study, Cydney Nielsen and her colleagues present evidence that challenges this model.Neurospora crassa, Magnoporthe grisea, Fusarium gramineum, and Aspergillus nidulans) form an evolutionary tree with branching points estimated at 200, 230, and 330 million years ago. While they diverged from yeast some 500 million years ago, they share with yeast a low intron density (one to two introns per gene) and a 5′ position bias. The authors' approach is to tally intron gains and losses during the evolution of these four species and then plot their positions along the genes' length.They address intron dynamics with a genome-wide survey of intron distribution among four Ascomycete fungi with recently completed genome sequences. The four fungi (They identify 3,450 gene regions that are clearly conserved in all four species and harbor an intron in at least one of them. To distinguish intron gains from losses, they rely on a simple parsimony principle, which they refine with additional probability analyses. In brief, an intron present in only one species counts as a gain; an intron absent from one species but present in its closest relative and in a cousin counts as a loss.Nielsen and colleagues record between 150 and 350 intron losses in each lineage. Surprisingly, losses do not occur preferentially at the genes' 3′ end. The authors conclude that while a 3′ reverse transcriptase-based mechanism might be a factor, it cannot be the sole reason for the introns' 5′ bias. The other surprising result is that intron gains occur at almost the same rate as losses in all lineages. Intron gains therefore play an important role in the evolution of even intron-poor genomes. Clearly, intron distribution in fungi owes to forces more complex than simple 3′ intron elimination, forces that the authors propose may also shape evolution of other eukaryotic genomes.


Little is known about the patterns of intron gain and loss or the relative contributions of these two processes to gene evolution. To investigate the dynamics of intron evolution, we analyzed orthologous genes from four filamentous fungal genomes and determined the pattern of intron conservation. We developed a probabilistic model to estimate the most likely rates of intron gain and loss giving rise to these observed conservation patterns. Our data reveal the surprising importance of intron gain. Between about 150 and 250 gains and between 150 and 350 losses were inferred in each lineage. We discuss one gene in particular (encoding 1-phosphoribosyl-5-pyrophosphate synthetase) that displays an unusually high rate of intron gain in multiple lineages. It has been recognized that introns are biased towards the 5′ ends of genes in intron-poor genomes but are evenly distributed in intron-rich genomes. Current models attribute this bias to 3′ intron loss through a poly-adenosine-primed reverse transcription mechanism. Contrary to standard models, we find no increased frequency of intron loss toward the 3′ ends of genes. Thus, recent intron dynamics do not support a model whereby 5′ intron positional bias is generated solely by 3′-biased intron loss. A comparative study of four fungal genomes reveals the patterns of intron gain and loss over several hundred million years of evolution Saccharomyces cerevisiae. It has been suggested that both the paucity and positional bias of introns in yeast may be due to intron loss through a mechanism of homologous recombination of spliced messages reverse-transcribed from the 3′ poly-adenylated tail , to roughly seven introns per gene on average for some basidiomycetes . Finally, fungi display a strong 5′ bias in intron positions, enabling us to investigate the processes underlying this phenomenon.Fungal genomes are in many ways ideal for exploring questions of intron evolution. The fundamental aspects of intron biology are shared between fungi and other eukaryotes, making fungi appropriate model organisms for intron study. They are gene dense with relatively simple gene structures compared with plants and animals, making gene prediction more accurate. Fungi also display a wide diversity of gene structures, ranging from far less than one intron per gene for SRY. Evidence for the occurrence of multiple independent intron losses has also been reported in studies such as those by Caenorhabditis elegans.In principle, a 5′ intron bias could arise through various combinations of intron gain and loss, and a complete understanding of intron positional bias requires an assessment of the contributions of both of these processes. A number of studies demonstrate the occurrence of intron gain and loss in individual genes or gene families. Fugu as an outgroup) and between mouse and rat (with human as an outgroup), and observed a sparseness of intron loss and complete absence of intron gain in these closely related organisms. On the other hand, More recently, a number of genome-wide studies of intron dynamics have been conducted. Aspergillus nidulans, Fusarium graminearum, Magnaporthe grisea, and Neurospora crassa. Ortholog sets composed of one gene from each of the four genomes were identified as pairwise best bidirectional BLAST hits satisfying stringent overlap criteria. Orthologs in each set were subsequently aligned, and the locations of introns were marked. These intron positions  were subjected to rigorous alignment quality filtering to eliminate alignment and annotation errors  passed the quality filter. The complete set of aligned orthologs with passing and failing intron positions is provided in N. crassa (blue box), and the third is present only in A. nidulans, and, because of the ambiguity in inferring gain or loss in this case, is classified as “Other” (black-edged gray rectangle). Examining the region around the one raw gained intron in N. crassa at the nucleotide level  and M. grisea (M) but absent from F. graminearum (F) and N. crassa (N).where r is defined for a given organism as the fraction of introns in the parent that did not survive in any sibling. We define “sibling raw losses” for an organism as the number of introns that are present in the organism and at least one cousin but in no sibling. This quantity is then divided by the number of introns present in that organism and at least one cousin to give the sibling loss rate. For example, the sibling loss rate for F. graminearum is given byThe sibling loss rate, m, and subtract this quantity from the raw gains to obtain “inferred gains.” To calculate m we first count B0, the number of introns conserved in the organism and at least one sibling, but in no cousin. The quantities m and B0 are related through the variablen0, the number of introns present in an organism's parent but not in any cousin, by the equationsWe next correct the raw gains for each organism. Raw gains include some introns that were in fact lost in all but one lineage. We use the loss rates to calculate the expected number of these multiple losses, andThis follows from our assumption of independent gains and losses. Thus, we can calculate the expected number of multiple losses asn1, and the number not present in any cousin n0 (introduced above). To estimate the size of the set of parental introns present in at least one cousin, we first count the subset of these introns that are presently observable. An intron is in this set if it is present in at least one cousin and at least one sibling, or is present in at least one cousin and in the organism in question. We call this number of introns B1. By the assumption that gains and losses are independent, we haveWe use the loss rates to estimate the number of introns in each organism's parent. To do so, we estimate separately the number of parental introns present in at least one cousin Using this relation and the one in F. graminearum as an example, the true number of losses would also include some introns conserved in the patterns A, M, N, and MN. We calculate the number of inferred losses as ntotal q.Finally, we correct raw losses. Our definition of raw losses undercounts the true number by omitting those introns not conserved in at least one cousin and at least one sibling. Taking This method can be extended to any phylogenetic tree and to any organism with at least one cousin.M. grisea  than the average for other genes analyzed, resulting in unusually large numbers of introns in these genes  synthetase genes. These genes encode a widely conserved protein that catalyzes the production of PRPP, a precursor in the nucleotide biosynthesis pathway. In contrast to the majority of orthologs that displayed fewer than two gained introns, the set of PRPP synthetase genes displayed a total of 22 raw gains , we determined the frequency of phase 0, 1, and 2 introns in the set of all intron positions  based on parsimony. However, this does not accurately account for the possibility of multiple gain or loss events. We have developed a probabilistic model that allows for multiple events, providing a corrected estimate of the total number of gains and losses within the dataset. Our probabilistic method allows for a more accurate assessment of rates of gain and loss. In our dataset, allowing for multiple events results in only modest corrections to the rates estimated using parsimony.F. graminearum and M. grisea the number of introns gained was on par with the number lost and similar in magnitude to the number of introns gained in N. crassa.Our analysis demonstrates a significant role for intron gain over the past few hundred million years in the fungi analyzed. Previous analyses of specific gene families have provided evidence of specific instances of gained introns . HoweverThe mechanisms underlying intron gain are not known. We analyzed the set of predicted intron gains for possible signatures that might shed light on this process. No statistically significant bias was detected in the positions of gained introns along the coding sequence see , and no N. crassa and M. grisea suggests the possibility of independent increases in gain rate in the two species. Alternatively, the apparent high intron gain rate exhibited by this gene may have arisen just prior to the last common ancestor of N. crassa and M. grisea. Although it is premature to speculate about possible mechanisms, one possibility is that a factor or factors responsible for intron insertion evolved to associate with the PRPP synthetase gene locus, transcript, or message at this point, leading to a higher rate of intron insertion in this gene.Our data further indicate that intron gain can vary substantially between different gene families in a lineage-specific fashion. The PRPP synthetase gene is a particularly striking example, exhibiting significant increases in gained introns in two of the four lineages investigated. Moreover, the paucity of intron positions shared between Finally, our results do not support the mechanism commonly proposed to account for the 5′ positional bias of introns in intron-poor organisms . Contrarhttp://www.broad.mit.edu/annotation/fungi/fgi). The following datasets were used: A. nidulans , N. crassa , F. graminearum , and M. grisea .All sequences and annotations were taken from the Broad Institute Fungal Genome Initiative website . We repeated our analysis, requiring that each best bidirectional hit also be the only BLAST hit in each genome (spanning 60% the length of the longest protein). This protocol yielded only 1,178 ortholog sets, but gave qualitatively similar results for intron gains and losses .The proteins in each ortholog set were aligned using ClustalW 1.82 , and intRegions of low alignment quality were eliminated with a filter that required at least 30% identity and 50% similarity in a window of ten residues on each side of the intron position. These parameters were determined following manual classification of a set of 181 randomly selected intron positions as “clearly homologous,” “ambiguous/possibly homologous,” or “non-homologous” see B. Using To further exclude likely annotation and alignment errors, intron positions were also filtered by eliminating positions adjacent to gaps in the amino acid alignment and by eliminating positions with nearby introns but low evidence of homology in the intervening sequence. It is possible that some of these positions may in fact reflect intron gain or loss events that occurred simultaneously with coding sequence insertion or deletion. However, removing these positions did not significantly impact our results, as the number of positions adjacent to gaps was only about one-tenth of the number of positions that passed the quality filter, and the introns removed did not have an apparent positional bias see A. In the second case λ is given by the total number of raw gains observed in that organism multiplied by the length of that gene in amino acids and divided by the total number of amino acids in all genes in that organism . We reported the less significant of the two p-values in the results.We modeled the number of gains in a particular organism as a Poisson distribution under two different null hypotheses. One null hypothesis was that the gains were spread uniformly across all genes. The other was that the number of gains in each gene was proportional to the length of the gene. In the first case the Poisson parameter λ is given by the total number of raw gains observed in that organism divided by the total number of ortholog sets p-value for the significance of phase 0 bias was determined by the χ2 test with two degrees of freedom using equal expected phase frequencies. To detect sequence bias at intron insertion sites, we examined gained introns separately in F. graminearum,M. grisea, and N. crassa. For each gained intron, we extracted four bases upstream and downstream of orthologous sites in the other two sequences, consistent with For each of the three in-group lineages, the frequency of phase 0, 1, and 2 introns was determined for five different datasets: for each class of conservation , for all introns passing our filter, and for all introns in the ortholog set. The Figure S1Positional biases in intron gain, loss, and current distribution in three fungal genomes determined using orthologs predicted by a “bidirectional only hit” method. (A), (B), and (C) are roughly analogous to (D), (E), and (A), respectively, in (78 KB DOC).Click here for additional data file.Table S1http://genes.mit.edu/NielsenEtAl/.Also available at (4.3 MB ZIP).Click here for additional data file.


Analysis of over 3,000 co-linear paired genes in rice shows more intron loss than intron gain following segmental duplication. Introns are under less selection pressure than exons, and consequently, intronic sequences have a higher rate of gain and loss than exons. In a number of plant species, a large portion of the genome has been segmentally duplicated, giving rise to a large set of duplicated genes. The recent completion of the rice genome in which segmental duplication has been documented has allowed us to investigate intron evolution within rice, a diploid monocotyledonous species.Arabidopsis thaliana, we identified more intron loss (49 introns within 35 gene pairs) than intron gain (5 introns within 5 gene pairs) following segmental duplication. We were unable to demonstrate preferential intron loss at the 3' end of genes as previously reported in mammalian genomes. However, we did find that the four nucleotides of exons that flank lost introns had less frequently used 4-mers.Analysis of segmental duplication in rice revealed that 159 Mb of the 371 Mb genome and 21,570 of the 43,719 non-transposable element-related genes were contained within a duplicated region. In these duplicated regions, 3,101 collinear paired genes were present. Using this set of segmentally duplicated genes, we investigated intron evolution from full-length cDNA-supported non-transposable element-related gene models of rice. Using gene pairs that have an ortholog in the dicotyledonous model species We observed that intron evolution within rice following segmental duplication is largely dominated by intron loss. In two of the five cases of intron gain within segmentally duplicated genes, the gained sequences were similar to transposable elements. Plasmodium falciparum and other eukaryotes [Introns are under less selection pressure than exons, and consequently, their sequences diverge faster than exons. However, the position of the intron with respect to the protein sequence is relatively conserved and conservation of intron position has been observed between distinct eukaryotic lineages throughout about 1.5 billion years of evolution such as between animal and fungal genes [karyotes . With rekaryotes ,4, suggekaryotes  and intrkaryotes  in the 3karyotes .Introns can be classified into three categories based on location relative to the codon. Introns that do not interrupt the codons are termed phase 0, while phase 1 introns are located between the first and second bases of the codon and phase 2 introns are located between the second and third bases of the codon. It has been reported that eukaryotic genes have more phase 0 introns than phase 1 or phase 2 introns; on average a 5:3:2 ratio of phase 0: phase 1: phase 2 introns is observed, although the specific ratio of intron phase appears to be species specific -10. SeveArabidopsis thaliana, a model dicotyledonous plant [Arabidopsis and rice is approximately 130 to 200 million years ago (MYA) [Discovery of both intron loss and intron gain suggests that these two processes may be ongoing events in evolution. The rates of intron gain and loss seem to differ greatly among species ,7,14-16 us plant . With thus plant  and unifus plant , we havego (MYA) -25. Intego (MYA)  and occugo (MYA) , with thgo (MYA) .A. thaliana, a model dicotyledous plant with a near-complete genome sequence, as the outgroup to readily classify 'intron loss' and 'intron gain' events between the two duplicated rice genes.Segmental duplication in rice provides the opportunity to study intron gain and loss within a subset of genes that have recently diverged. In this study, we report on the evolution of introns within coding sequences (CDS) after segmental duplication in rice. Through our examination of segmentally duplicated genes, we anticipated that we would identify more intron gain or loss events than for non-duplicated genes due to the accelerated rate of intron loss or intron gain in duplicated versus orthologous genes, as reported previously in two malaria parasites . Other aPrevious analyses of segmental duplication in rice used sequence datasets that contained a substantial portion of unfinished genome sequence and lacked refined structural and functional annotation of the genes -29. ThusAn increase in genome coverage within the duplicated regions was observed if the maximum distance permitted between collinear gene pairs was expanded from 200 kb to 500 kb, 1 Mb, or 5 Mb, whereas a much smaller percentage of the genome was covered if the maximum distance was limited to 100 kb  that showed potential intron loss or intron gain were investigated further.Within the 43,719 non-transposable element-related gene models in rice, 140,827 introns within the CDS are present, with an average length of 385 base pairs  470) and an average GC content of 37.5%. Out of the 3,101 pairs of segmentally duplicated genes, 281 pairs had at least one intron that passed the manual review for full-length (fl)-cDNA support and single isoform. In total, 2,573 introns were present within these 281 gene pairs and had a similar length distribution (average 315 bp) and GC content (36.9% GC) to those found throughout the genome. We found that 197 of the 281 pairs (70%) had completely conserved exon-intron structure in the coding region , that is, the intron number, position, and phase were identical among the duplicated genes  , one gene pair with a single gained intron, and four gene pairs in which both intron loss and gain were observed (6 intron losses and 4 intron gains). An example of intron loss is shown in Figure Arabidopsis At4g29040.1. Alignments of all of the 36 gene pairs with their orthologs from Arabidopsis are displayed in Additional data file 3. The length of the lost introns  was shorter than the average intron length in the rice genome . The distribution of the length of the lost introns and gained introns and the frequency of the length of the 33,011 fl-cDNA supported (FLS) rice introns  are shown in Figure To determine whether the incongruence was due to intron gain or loss, we used the putative orthologous gene from Arabidopsis ortholog. However, within these 31 gene pairs, 34 introns in total were lost as for 3 gene pairs, both rice genes underwent separate intron loss events. In these 31 gene pairs, we observed no bias in intron loss position at the 3' ends of genes . Randomization tests showed that intron loss at phase 2 was unexpectedly high  and intron loss at phase 0 was unexpectedly low .To examine whether there was a bias in the phase of intron loss in segmentally duplicated genes in rice, we examined the 34 independently lost introns and excluded genes with multiple intron losses. The frequency of intron loss at phase 2 was higher, but not statistically significant, than intron loss at phase 0 and 1 , while that at the acceptor site of the lost introns was within the average range . The rank distributions of 4-mers flanking the acceptor splice site did not differ significantly between lost and retained introns (P approximately 0.069).The sum of the ranks (SoR) of the exonic 4-mers flanking the donor splice site of the lost introns  was very significantly lower than expected  [Two out of the five gained introns showed several matches to known rice transposon sequences. The intron of LOC_Os12g02840.1 had a significant hit to a putative Ty1-copia subclass retrotransposon protein (82% identity over the entire intron). A large portion of the other gained intron  was highly similar (92% identity) to Arabidopsis and poplar homologs. Thus, the most parsimonious explanation for the data is a single insertion into one of the rice duplicates prior to the divergence of rice, sorghum, and maize (data not shown).We examined these five cases of intron gain further by examining homologous genes from other plant species. With the exception of one case, the gained intron was clearly a straightforward insertion into one of the rice gene pairs . For LOC_Os3g16960.1, the gained intron was observed in the maize and sorghum homologs, but absent in the p to be the proportion of non-conserved introns between duplicated genes, we have p = 54/(137 + 958) = 0.0493, where 54 is the number of non-conserved introns, 137 is the total number of the aligned intron positions within the 36 gene pairs that have intron loss and gain, and 958 is the total number of aligned intron positions within the 197 conserved gene pairs. Given that intron loss and acquisition are rare events, the expected rate of intron loss and gain can be estimated under the simple Poisson model and calculated as:Intron loss and gain are two important processes in evolution. We observed more genes with intron loss than gain after segmental duplication in rice. We estimated the rates of intron loss and gain after the segmental genome duplication in rice. Allowing int = -ln (1 - p) = 0.0506DIf we estimate t = 70 MYA for the rice genome duplication ,27, we eint/2t = 0.0506/(2 × 70 × 106) = 3.61 × 10-10 per intron per yearμ = DAs a total of 49 lost introns and 5 gained introns were observed, we estimated the evolutionary rate of intron loss and intron gain after the genome duplication is:loss = 3.61 × 10-10 × 49/(49 + 5) = 3.28 × 10-10 per intron per yearμgain = 3.61 × 10-10 × 5(49 + 5) = 3.34 × 10-11 per intron per yearμArabidopsis of 2 to 3 × 10-10 per year and an intron gain rate of 2.2 to 2.9 × 10-12 per year [Arabidopsis [A previous study involving 684 groups of orthologous genes reported an intron loss rate in per year . Our stubidopsis .dS for the 233 gene pairs that had a single isoform, were supported by a fl-cDNA, and had been manually validated (197 gene pairs with congruent intron structure and 36 gene pairs with intron loss and/or intron gain). The dS values ranged from 0.03 to 24.86 with a clear peak between 0.6 to 1.4 (data not shown). Similar rates of intron loss (1.41 × 10-10 per intron per year) and intron gain (0.94 × 10-11 per intron per year) were obtained from the calculations performed using a subset of the 233 gene pairs in which the dS between duplicates was between 0.6 and 1.4 .It is possible that the rate of intron loss and gain differs within our set of segmentally duplicated genes as it has been previously reported that the segmental duplication between the top arms of chromosomes 11 and 12 is recent (within 5 MYA) in comparison to the bulk of the segmental duplication, estimated at 70 MYA ,27. ThusArabidopsis (data not shown) in which there is no 5' bias in intron location within single-intron genes [et al. [Caenorhabditis. However, according to the genomic deletion model, we would expect some instances of imprecise deletion of introns, which is not the case in our sample. Therefore, an unknown recognition signal may exist that allows the exact deletion of introns in rice.A reverse transcriptase-mediated model in which a segment of the genomic copy of a gene can be replaced by a reverse-transcribed copy via homologous recombination was previously proposed to explain the pattern of intron loss ,35,36 anon genes . The oth [et al. , who obsWe did not observe any statistically significant differences in the frequency of intron losses in different phases. Nor did examination of nucleotide compositional patterns in the exons surrounding the splicing site reveal an apparent pattern in the bi-nucleotide sequence of the exon at the boundary other than that shown by canonical splice site consensus sequence (AG|GT) in which '|' represents the intron position (data not shown). Yet conservation of the exon nucleotides adjacent to the exon-intron boundary has been reported to play an important role in correct splicing -40. WithWe were able to document intron loss and gain in segmentally duplicated rice genes with a rate of loss and gain similar to that observed within orthologous genes across a range of eukaryotes. While we did not observe preferential intron loss at the 3' end of genes, we did observe a nucleotide bias within the exonic sequence flanking the lost introns.A total of 43,719 non-transposable element-related rice protein sequences from release 3 of the TIGR Rice Genome Annotation  were useDuplicated genes with at least one intron were checked to ensure that they were supported by a fl-cDNA and that no alternative isoforms existed. Intron positions and phases were retrieved from the TIGR Osa1 genome annotation database . ClustalArabidopsis proteome  and intron positions and phases were inserted into the ClustalW alignment . Only loss or gain of introns after segmental duplication was examined further. An intron loss was defined if the intron was present at the same position in only a single rice gene and the putative Arabidopsis ortholog (referred to as a retained intron). An intron gain was defined if the intron was present in single rice gene but absent in the other rice paralog and the putative Arabidopsis ortholog.Simple phylogeny analysis was used to determine if the incongruent exon-intron structure was attributable to loss or gain of an intron. We identified putative orthologous genes by searching the predicted lease 5, ) with thA total of 233 pairs of duplicated genes, among which 197 pairs have completely conserved introns and 36 pairs show putative loss and gain of introns, were used in our randomization test. The total number of conserved intron alignment positions at each phase was counted . The total number of independently lost introns at each phase was counted . A total of 10,000 iterations were simulated. A total of 34 phases were randomly generated in each iteration based on the frequencies of the conserved aligned intron positions at each phase from the 233 gene pairs. The number of lost introns at each phase was then compared with those generated by simulation.4 = 256) precludes effective use of standard tests, such as the chi-square test, to compare the distributions. Instead, tests based on rank distributions were used as described below.To determine whether lost introns in duplicated rice genes tend to be flanked by rare nucleotide combinations, we compared the frequency distribution of the four nucleotides (4-mers) in the exonic sequence that flanked lost introns with the exonic 4-mers flanking the corresponding retained introns, as well as with the frequency distribution of the 4-mers flanking all introns in the genome. Comparisons were done independently for 4-mers flanking the donor and the acceptor ends of introns. The small number of lost introns, distributed over three intron phases  relative to the total number of 4-mer classes . In this way, three rank distributions, one for each intron phase 0, 1 and 2, and their attached frequency distributions, were generated for each the donor and the acceptor flanking regions.A total of 33,011 introns within the coding regions from 6,046 rice gene models that were supported with fl-cDNA, had no alternative splicing isoform, and had at least one intron within the CDS were used to determine the 4-mer distribution in exonic sequences that flank the introns. The four nucleotides that flank the donor and acceptor splice sites of each intron were extracted and their frequency calculated. For each intron phase, each 4-mer was given a rank between 1 and 256, to cover all of the 4P of obtaining this value by chance. P is approximately equal to the fraction of pseudo-replicated with a smaller or equal SoR value.We devised a statistic that we call 'sum of ranks', SoR, to determine if the 4-mers flanking lost introns are less common than expected by chance. This statistic SoR corresponds to the sum of the ranks of all introns in a sample, as determined by their nucleotide composition and phase. The test was conducted as follows: 10,000 pseudo-replicates were generated by randomly sampling the three rank distribution obtained for all introns, according to their frequency distribution . Each pseudo-replicate consisted of 34 sampled introns, 15, 7 and 12 of which were sampled from the rank distribution of phase 0, 1, and 2 introns, respectively, to preserve the characteristics of the observed distribution of lost introns. A SoR value was obtained for each pseudo-replicate to generate the distribution of expected 'sum of ranks'. The SoR for the 34 lost introns was compared against this distribution to determine the probability A rank was attributed to each lost intron, based on the composition of its 4-mer and its intron phase, according to the rank distributions obtained for all 33,011 introns (see above), to obtain a distribution of ranks for the set of lost introns. A distribution of ranks for the set of retained introns was obtained in a similar way. The two distributions were compared using a Wilcoxon's signed rank test. This procedure was done for both donor and acceptor flanking sequences.ab initio gene finder FGENESH [Arabidopsis, maize, sorghum and poplar) and inserted the intron positions/phases into the ClustalW alignment.Sequences of the five gained introns were searched against the NCBI non-redundant database and were further searched against all the 12 rice pseudomolecules . Signifi FGENESH  on the mdS) between each of the two rice duplicates was estimated by maximum likelihood, using the codon-based substitution model of Yang et al. [-9/site/year [The number of synonymous substitutions per synonymous site (g et al.  as impleg et al. ,52. Codeg et al. , and theite/year , rice paArabidopsis. Additional data file Arabidopsis, poplar, maize and sorghum.The following additional data are available with the online version of this paper. Additional data file The segmentally duplicated blocks within the rice genome.Click here for fileThe 3,101 pairs of segmentally duplicated genes along with their pairings and their sequence.Click here for fileArabidopsis.The ClustalW alignment of the two rice duplicated genes and their orthologous gene from Click here for fileThe occurrence of background exonic 4-mers at the donor splice sites of different intron phase.Click here for fileThe occurrence of background exonic 4-mer at the acceptor splice sites of different intron phase.Click here for fileArabidopsis, poplar, maize and sorghum.The ClustalW alignment of the two rice duplicated proteins with putative orthologous proteins from Click here for file


The timing of the origin of introns is of crucial importance for an understanding of early genome architecture. The Exon theory of genes proposed a role for introns in the formation of multi-exon proteins by exon shuffling and predicts the presence of conserved splice sites in ancient genes. In this study, large-scale analysis of potential conserved splice sites was performed using an intron-exon database (ExInt) derived from GenBank.A set of conserved intron positions was found by matching identical splice sites sequences from distantly-related eukaryotic kingdoms. Most amino acid sequences with conserved introns were homologous to consensus sequences of functional domains from conserved proteins including kinases, phosphatases, small GTPases, transporters and matrix proteins. These included ancient proteins that originated before the eukaryote-prokaryote split, for instance the catalytic domain of protein phosphatase 2A where a total of eleven conserved introns were found. Using an experimental setup in which the relation between a splice site and the ancientness of its surrounding sequence could be studied, it was found that the presence of an intron was positively correlated to the ancientness of its surrounding sequence. Intron phase conservation was linked to the conservation of the gene sequence and not to the splice site sequence itself. However, no apparent differences in phase distribution were found between introns in conserved versus non-conserved sequences.The data confirm an origin of introns deep in the eukaryotic branch and is in concordance with the presence of introns in the first functional protein modules in an 'Exon theory of genes' scenario. A model is proposed in which shuffling of primordial short exonic sequences led to the formation of the first functional protein modules, in line with hypotheses that see the formation of introns integral to the origins of genome evolution.This article was reviewed by Scott Roy (nominated by Anthony Poole), Sandro de Souza (nominated by Manyuan Long), and Gáspár Jékely. The question about the origin of introns is fundamental for an understanding of the evolution of the genome. Historically, there have been two opposite camps that try to explain the origin of introns see -4). The . The 4])In all introns-early scenarios, one would expect the presence of conserved introns between orthologous proteins that diverged before the eukaryotic lineage. There have been studies that used phylogenetic comparisons in an attempt to show that intron position is conserved and therefore ancient . The formation of multimodular proteins by exon shuffling ,29 has bSupport for a functional role of introns in exon shuffling has come from the distribution of intron phase and the excess of symmetrical internal exons, ,33 propoA large-scale search was performed that identified conserved introns and which allowed the gene sequence that harboured these introns to be studied. It was also found that the presence of an intron is positively correlated to the ancientness of its surrounding exon sequence, suggesting a role for ancient exons as the building blocks of the early protein modules.An experimental set-up was defined that allowed for a systematic search for potential conserved splice sites in a genomic intron-exon database derived from GenBank . A consBased on the method shown above, homologous sequences were identified with an identical intron position relative to the amino acids sequence. Figure A) and Rab related genes . The largest number of conserved splice sites was found in the consensus sequence of the catalytic domain of protein phosphatases 2, comprising a large family of serine/threonine phosphatases . Members of the Rab related genes are common throughout the eukaryotic tree, and members of the serine/threonine kinases and the protein phosphatase 2 domain are also present in Bacteria and Archaea, indicating their ancient origin. These results show that the mapping of these individual conserved splice sites to its ancestor protein can reveal the wide-spread occurrence of these sites in ancient proteins.The names of the genes and the observed conservation of the sequences already suggested a common function of the sequences involved cf. Fig. , as expees cd0015; B. The es cd0015; B. The From an introns-early perspective, the conserved introns that were found in ancient protein domains, such as those for phosphatase and kinase, could represent the remnants of the introns between the exons that could have been used for the exon concatenation or shuffling in early genome evolution. However, since the query used to look for ancient introns is based upon selecting conserved sequences, the introns found in ancient protein modules could also represent a sample of a larger number of randomly distributed introns and not be specific for ancient genes. This can be investigated by studying the relation between the ancientness of the selected sequences and the presence of an intron where a positive correlation would be expected according to the Exon theory. No relation could point to a bias in the results, where the putative ancient introns are just a sub-selection of conserved sequences that happen to contain a conserved intron.In order to quantify the relation between ancientness of the gene and the presence of an intron, an experimental setup was used in which the effect of the splice site on the ancientness of the sequence could be studied. A variable-length splice site region was defined in which the length of the identical exon sequence could be stepwise increased from 2 to 10 identical residues by adjusting the SQL query Fig. . A measuUsing the described set-up above Fig. , the lenA second, independent measure of the ancientness of the sequences that contain conserved introns can be made based on their presence in the main Life groups Bacteria, Archaea and Eukarya. Instead of counting the number of common residues as done in Fig. As expected when the introns were present before the protein sequences diverged, intron phase was found to be conserved in the set of putative conserved splice sites cf. Fig. . HoweverIn this study, a straightforward search method in a relational database was used to identify candidate conserved introns that could have delineated ancient exons. One of the advantages of this method was that the definition of a conserved intron can be easily translated in terms of the query language SQL. Also, the method allowed to precisely define and control the variables used, permitting quantitative analysis of the queries. A strict definition of a conserved splice site was used where not only the position, but also the sequence was conserved and where intron-sliding was not allowed. Other datasets of conserved introns have been published ,23 that a priori homology outside this region, allowing to find conserved splice sites in sequences from genes that show little overall homology, for instance in co-orthologous genes. The strict definition of a conserved splice site therefore limited the result set, but did select a well-defined subset of splice sites that could represent the ancient exon building blocks of protein modules. In summary, the present methodological concept can be seen as complementary to more conventional methods to study the conservation of introns.The stringency of the method used in the present article, a high similarity over a short sequence, was designed to select virtually no false-positive conserved introns. The set of conserved introns is therefore not exhaustive, but was intended to identify conserved introns that would satisfy the definition for ancient introns. Since the method involves only a short sequence for a potential match, it does not rely on any The results in this and other studies identified conserved introns that are shared between different eukaryotic kingdoms. The rationale behind this article was based on the assumption that exons originated at the origins of the genome itself  versus an insertion early in the eukaryotic tree (cf. introns-late). However, the emerging view of an ancestor eukaryote that already contained a complex, intron-rich genome -44, this, this44,within a variety of ancient protein modules, suggesting that the initial function of exons did not represent the boundaries of functional protein modules, but that the domains itself were assembled from exons. In such a model, individual exons do not have to coincide with functional modules but could represent structural modules or just connectors (like LEGO blocks) with no further intrinsic function, in line with the relative small size of some of the proposed ancient exons. Shuffling and recombination of these building blocks could then quickly generate a large variety of potential functional modules, on which natural selection can then act. It is the author's opinion that such active recombination mechanisms are a requirement for building a genome and may underlie an intrinsic evolvability in the genome. Also, such a scenario fits in a model in which a eukaryotic-like genome was the ancestor genome and prokaryotes were derived from this ancestor ..within aLarge-scale analysis of intron position and exon amino acid sequence was performed using a bioinformatics approach on publicly available genomic databases. The exon/intron database ExInt  was usedThe ExInt database contains a total of 120,573 gene sequence entries with 518,168 exons and 417,178 introns. Genomic databases like GenBank contain significant numbers of redundant sequences that have to be dealt with when performing large-scale comparisons. A large part of the redundancy in GenBank comes from non-identified sequences where a gene was identified by an algorithm that detects open reading frames and introns. The pre-selection on the presence of the word 'gene' in the sequence annotation selected only identified genes and greatly reduced redundancy of the result sets, effectively reducing the size of the database from 120,573 to 27,861 entries. The available species annotation of genes was used to link sequences to their taxonomic category. An additional taxon table was created by selecting representative species from the ExInt database and classifying these into the main eukaryotic kingdoms categories Protists, Fungi, Animals and Plants. The NCBI taxonomy browser was used for the classification of these species. The resulting taxon table contained 94 entries with at least 10 entries within each eukaryotic kingdom, covering 60% of the GenBank entries in the ExInt database and leaving 16,741 gene entries. Since there were more than one species per defined kingdom, result sets generally included homologous sequences when multiple species within a single kingdom gave a match to a single gene from another kingdom. In order to reduce redundancy, results were therefore cleaned by removing data with identical eukaryotic kingdom combinations (e.g. human vs. yeast and mouse vs. yeast was reduced to one entry).The conserved domain database (CDD) at NCBI was used All experiments were performed on a normal laptop PC  with Windows XP running a MySQL relational database (version 4). Querying was done using MySQL Control Center. The result of each query was visually inspected for redundancy and specificity and was further manually checked for consistency by using the found sequences to search the PubMed protein sequence. In general, result sets were imported in a new single-table database for further analysis. Analyses for graphical representations and statistics were done in Excel.The author(s) declare that they have no competing interests.AdR designed all the experiments, analyzed the data and wrote the manuscript.Scott Roy, Allan Wilson Centre for Molecular Ecology and Evolution, Massey University, Palmerston North, New Zealand. Nominated by Anthony Poole."Conserved intron positions in ancient protein modules" by de Roos. The paper takes an ingenious approach to the attempt to distinguish between the introns-late and introns-early perspectives. Much previous evidence for the introns-early theory has relied on the relationship of intron positions to the coding frame of the flanking exonic sequence, or to the three-dimensional structure of the corresponding protein, findings whose existence and relevance have not always been accepted by proponents of introns-late. Avoiding these mine strewn landscapes, de Roos investigates another prediction of the presence of introns in LUCA – if introns were (primarily) created in ancient times, they should preferentially fall within ancient sequences. However, I am very concerned about various methodological issues, and therefore suspect that the results do not inform the debate.In concordance with the comments of other reviewers, I have focused less on a discrimination between introns-late and introns-early, but more towards support of the Exon theory of genes. I also discussed the potential bias in the results and the way this was investigated.The author presents three related lines of evidence for pre-LUCA introns. First, conserved introns tend to be found in conserved protein domains. Second, among conserved sequences, those that contain introns are more conserved . Third, conserved introns are preferentially found in genes common to all three domains of life.An alternative explanation for the first finding, that introns tend to be found in conserved protein domains, is that there is a bias in the finding of 'conserved' introns. Here, conserved introns means those introns that share positions across eukaryotic groups in very highly conserved sequences (for instance 6 residues exactly conserved). Similarly, domains are currently generally defined by sequence conservation across long evolutionary distances. Thus, this finding reduces to: introns in short highly conserved sequences are preferentially found in longer highly conserved sequences. This is not particularly surprising. Without a subtle negative control, it is hard to discern a signal of ancientness above this potential bias.I am aware that the finding of conserved introns in conserved domains does not prove an ancient origin, since the search method could not discriminate directly between an insertion early in the eukaryotic tree. However, the Exon theory of genes expects conserved introns in ancient modules and the first part of the article set out to find those potential conserved introns. In this way it is a confirmation of the hypothesis, although it cannot be considered evidence. I designed the experiments in Figs 5 and 6 to address this point (see below). The nature of the bias and how this has been investigated is now stated explicitly.The second finding is curious, but I fear may also be explained by limitations in the methodology. Here, the central finding is that short highly-conserved sequences containing conserved introns are more likely to lie in long highly-conserved sequences than are those that do not contain introns. Given the enormity of the sequence database, it is likely that some highly-conserved sixmers will be false positives – i.e. they will not reflect true homology. The probability of a false positive is greatly reduced by the presence of a conserved intron . Thus, the conserved intron-containing set is likely to contain far fewer false-positives than the control set. The higher false positive rate among the control set predicts a lower extent of sequence conservation, just as seen. Thus it is again difficult to discern a signal of ancientness.In order to investigate a potential bias, the experiments in Fig. 5 were designed, were a control situation was created that used exactly the same methodology, but now without an intron. It was shown that sequences without an intron are less often found within a conserved domain or sequence. In other words, if a sample is taken from a genomic database that selects short, highly similar sequences, the gene it is in is more ancient if an intron is also shared between these sequences. This would not be expected when the sequences with an intron are just a subset of a larger set with conserved sequences regardless of an intron.The assumption that the probability of a false positive is greatly reduced by the presence of a conserved intron was actually tested in the experiments. Although not evidence for the ancient origins, this does strengthen the finding that introns at similar positions in conserved genes are homologous and would be in line with an Exon theory of genes.P = 0.07 by a Fisher Exact Test). Thus while the difference in fractions of ancient genes is suggestive, we cannot currently conclude anything from this finding. In addition, I am concerned that this test may also suffer from problems similar to those discussed above.The final finding, that intron-containing matching sequences are preferentially found across domains of life, is not statistically significant: the fraction of intron-containing sequences in 'ancient' genes (8/19) is not statistically different from the fraction of intronless sequences , but did not show any further substantial gene similarity. It is true that this value is not statistically different, and this is now shown (I found p = 0.053 using Student T test). However, there were more than one test that suggested the ancientness: a) overall sequence similarity, b) representatives of ancient protein modules, and c) representatives of protein modules that are shared between prokaryotes as well as eukaryotes. Although these tests cannot be considered truly independent, the set of controls does suggest that the introns are preferentially located in ancient sequences.In addition, the unorthodox methods used (complete sequence conservation over short sequence fragments) are hard to evaluate given the lack of a history for such methods. It is not clear why these methods have been chosen over more traditional tools, which are abundant. This makes it difficult to interpret the data.I have included a paragraph that discusses the method used, and added the result set of potential conserved introns.Assuming an 'Exon theory of genes'-scenario , I set out to look for conserved introns. I defined a potential conserved intron just by a shared intron in a short sequence, without any preselection of the type of genes. Since SQL is a powerful tool to extract specific information from large relational data sources, this quickly led to introns that could be considered conserved. Since sequence matching was done using a simple algorithm that compared sequence similarity, it was easy to control the variables and do the experiments with control sequences as in Fig. I believe this is one of the main advantages: the straightforward way of querying makes it possible to design predictive experiments by changing one parameter at a time and use a control situation.One further consideration is the usage of the word 'ancient.' This word is traditionally reserved in the context of the intron debate for the period before the divergence of eukaryotes from prokaryotes, and this distinction has become all the more important with the mounting evidence for significant intron presence in early eukaryotes. One way to distinguish the two epochs is between 'ancient' and 'ancient/early eukaryotic'.This is an important distinction and has been made more clear in the article. Although it is difficult to make a distinction between ancient and ancient/early eukaryotic with the experiments presented, the data is in line with ancient origin (before pro/eukaryotic split). I assume, however, that eukaryotes were not derived from prokaryotes, but that they both share an ancestor whose genome was eukaryotic-like.Following a long line of previous results, de Roos identifies intron positions that are shared between broad eukaryotic groups. Previous statistical analyses of the numbers and distributions of these patterns have concluded that a substantial majority of these represent ancestral positions, indicating that there were already substantial intron numbers by the time of divergence. While the eukaryotic groups used here (and elsewhere) may not allow for inferring an intron's presence in the last ancestor of all extant eukaryotes, we can now be confident that there were already a very large number of introns in the last ancestor of eukaryotes, since the spliceosome is broadly shared among eukaryotes, and large numbers of intron positions are shared between potentially early diverging eukaryotes and others .I bring up this in order to point out two things. First, the debate over the timing of introns is now over whether introns arose in a universal ancestor or between the eukaryotic-archaeal ancestor and the last eukaryotic ancestor. Although findings of conservation of introns or splicing-associated features across eukaryotes underscores the fact that introns are very old within eukaryotes, it does not help us to distinguish the timing of origin of spliceosomal introns. Second, it is important to point out that the findings of large number of introns in early eukaryotes are not fundamentally transformative for the introns early/late debate. Though substantial intron presence is clearly resonant with introns-early (and perhaps nearly necessary for the model), it does not contradict the introns-late model. The fundamental tenet of introns-late is that introns arose after the last common universal ancestor, thus intron presence in early eukaryotes only refines the timing of the origin on that model.I rewrote sentences that implied a contradiction of introns-late. Instead I focused on results that are in line with an Exon theory of genes. On the other hand, the patterns of conserved introns in ancient protein modules, phase distributions and correlation with ancientness of genes as seen in this article, may also make a mechanistic model for introns-late more difficult and is therefore relevant to the debate.I think that the most important question in unravelling the origins of introns is whether introns were inserted into preformed genes, or were at the basis of the genes themselves. Insertion of introns at the DNA level seems extremely difficult given the fact that an intron should be excised at the RNA level perfectly in order to be functional. I have not seen a mechanism for gradual spliceosome evolution and intron insertion that could address the negative fitness effects of such a scenario. Moreover, intron insertion models should explain how complex genes arose in the first place without the help of exons. So, as long as the mechanistic puzzles have not been solved, I consider the debate still open.In light of this, we can ask whether tests such as those undertaken here are likely to shed light on the introns early/late debate. The presence of shared introns in shared eukaryotic genes does not shed light on whether these introns date to earlier epochs. One possible approach would be to compare intron densities between ancient (shared eukaryotic-prokaryotic) gene and ancient eukaryotic genes. However, even in this case a difference would not be conclusive support for ancient introns, since the origin of many ancestral eukaryotic genes may postdate the origin of introns. And this is leaving aside the differential rates of intron loss across genes, the fact that genes that arise by gene duplication (and then diverge beyond recognition) may retain ancestral introns, and difficulties with distinguishing truly eukaryotic-specific genes from the inability of programs like BLAST to detect homology.Another approach may be to first unravel the mechanistic steps in an Exon theory of genes for building a genome, and then compare the construction of eukaryotic genes with those of prokaryotes and see if they are assembled in a similar way. In this respect, the proto splice-sites as proposed by Dibb and Newman[could as well represent the remnants of the ancient exon concatenations (see[nd Newmancould as ions (see).Sandro de Souza, Ludwig Institute for Cancer Research, Sao Paulo, Brazil. Nominated by Manyuan LongAlbert de Roos developed a new way to identify conserved intron positions between taxa from different kingdoms of life. Instead of looking for conserved intron positions in know related genes, de Roos searches for conserved "splice sites" in an intron database. By "splice site", he means a segment of protein sequence (10 aa residues) flanking an intron position. He considered an intron position conserved when this "splice site" was conserved (6 out of 10 residues in that window) between two distantly related protein sequences. The strategy may be interesting since it can identify cases missed by other approaches. Although I recognize the efforts of the author in trying to look at this problem with a different, creative strategy, I found the manuscript hard to follow and highly speculative with many assumptions not supported by the data. Furthermore, since this is a new strategy, the author should give more details about the methodology as well as provide more analyses for his dataset. I will list some issues below in a point-by-point basis to make it easier for the author to reply.I have changed the article to focus more on the data that support a 'very early' origin of introns, but does not prove introns-early, and can by itself not disprove introns-late. The article was too strong in that aspect.Overall, I have many problems with the "Introduction" section, in which many important references were misquoted or ignored.• For instance, the original quotation for the introns-early theory is given to Gilbert's 86 paper on RNA world and to a review from Gilbert and colleagues in Cell . Here the papers cited should be the Nature 1978 paper from Ford Doolittle and the "Exon Theory of Genes" paper from Gilbert in 1987, which best represent the early phases of the introns-early theory.• Furthermore, the synthetic theory of intron evolution, developed by myself, Scott Roy and Wally Gilbert is completely ignored.• In a particular point, the papers cited as evidence for a correlation between intron positions and module boundaries is certainly outdated. Papers from de Souza et al (1996), de Souza et al (1998) and Fedorov et al (2001) were ignored but best represent the conceptual aspect explored by the author.I changed the references and rewrote the introduction.....one would expect to find conserved introns specifically in ancient proteins". In the way is presented, this sounds wrong. An intron conserved between vertebrates and invertebrates does not necessarily be located in an ancient gene. I think the author should be more explicit by saying that this conservation has to be deep among the four kingdoms that he later will mention.1) In the last paragraph of the "Introduction" section, the author state that the rationale for this manuscript was based in the assumption that "I rewrote the introduction and the articles focuses on the identification of possible ancient introns and their characteristics.2) It would be nice to have a more detailed description of the 251 (or 250?) conserved intron positions. The entire list of positions should be available. It was not clear as well the level of stringency used in the analysis. As far as I understood a simple match involving proteins from, for example, human and Arabidopsis was suffice to flag that as conserved.I included the entire result set of the potentially conserved introns, as well as the larger set with strongly homologous genes that was cleaned to obtain the 251 potential ancient introns. The flag 'shared between major eukaryotic kingdoms' was indeed based on the occurrence in plants as well as animals. In order to further qualify for 'conserved', the splice site sequence and intron position should be conserved, as well as show more homology further away from the splice site. The stringency is also discussed in the article.3) Since this is a new method, the author should compare his dataset with other datasets. For example, the dataset reported by Rogozin et al  was analyzed by these authors and by Roy and Gilbert . The datasets are apparently significant different. They should be since the methodology is different but the author needs to explore these differences. For instance, I am surprised by the low number of conserved intron position identified by de Roos. In the dataset mentioned above, there were almost a thousand introns conserved between animals and A. thaliana or P. falciparum.I included a comparison of the different method used and the results that were obtained in both mentioned articles. The method used in my article is quite stringent in the way that it requires high sequence similarity in a short region. The basic concept of an ancient intron, i.e. an intron at the same position within a similar sequence was used, so not only the position but also the splice site sequence had to match. I expect that a more BLAST like search with gap-alignment of homologous amino acids would yield more conserved introns. The scope of the first part of the article was not to get an exhaustive set, but to confirm that they exist and to study some characteristics.4) Many of the interpretations seem to me quite speculative and not supported by the data. For example, by looking at so many introns, it is expected that some matches will occur just by chance. Some simulations aimed to draw a threshold for this numbers would be welcomed.As now more explicitly mentioned in the article, there is a potential bias in the search method, which was investigated and shown in Fig. 5 and in Fig. 6 which was quantified. The results supported the hypothesis of the Exon theory, and I have been careful not to suggest that they prove introns-early. See also discussion of dr. Roy's review.5) The analysis on the occurrence of conserved intron positions in ancient genes is presented in a confusing way. I strongly recommend the author to re-write this. Nevertheless, I have serious concerns about the analysis. First, it seems that de Roos call a gene ancient when there is some conservation in a window of 10 residues. This window size should be increased to at least the average size of a protein domain. Furthermore, it does not seem that the CDD database was used here.I rewrote the analysis. I call an intron ancient when it is located in a conserved sequence, i.e. there is splice site similarity (6/10) as well a overall gene similarity as measured over an additional 20 (2 × 10) amino acids, overall a similarity of at least 12/30. In my approach this yielded a strong selection of conserved genes, and most matched sequences were a member of a conserved protein domain and shared a similar phase.There was no a priori assumption of gene relatedness and the CDD database was indeed not used to find the conserved set, but was used to confirm the ancientness of the set. As Fig. 6 shows, when only an 8/10 splice site similarity was used, most sequences were conserved as shown by the sequence similarity further up and downstream. Lowering this to 6/10 but requiring an additional 6/20  selected only conserved genes. Even with only a similar splice site  and no further similarity, the sequences were in 42.1% genes shared with prokaryotes and 52.6% were member of a conserved domain (see text).6) The interpretation derived from the analyses on the ancientness of the genes identified is kind of circular. Of course, if a condition was established in the early stages of the pipeline that a match would be considered a match only if involved kingdoms that split a long time ago, I would expect to have my dataset enriched with ancient proteins.The article consists in a way of two parts. In the first, I tried to get a set of conserved introns by specifically looking for them. Since I query for similar intron positions between sequences that diverged in the eukaryotic tree, the dataset will be enriched for ancient proteins. I then looked whether this enrichment is due to the presence of an intron, or solely intrinsic to the method used. As can be seen in the controls of Figs the presence of an intron makes a difference both in conservation of phase and overall sequence. I conclude that the enrichment of these sequences is due to the presence of the introns, and not a result of the method. In the Results as well as in the Discussion section, I made this more clear.Gáspár Jékely, European Molecular Biology Laboratory, Heidelberg, GermanyThis paper describes a new method to identified conserved intron positions in distantly related eukaryotic taxa. It is based on looking at short protein sequence stretches around a splice site and finding similar sequences having an intron at the corresponding position, rather then defining and aligning orthologs and then mapping intron positions onto the alignment. Using this method the author identifies 218 introns with the same phase at homologous sites that probably trace back to early eukaryote evolution. The method is novel and interesting. I have some comments on how to improve the presentation and analysis of the results.1) One problem is that the phylogenetic context is not defined explicitly. It is not enough to refer to kingdoms, especially in the case of protist. If an intron is shared by animals, fungi (and in addition e.g. by choanoflagellate protists), it does not mean, that it was there in the eukaryotic common ancestor. The phyletic distribution of each identified intron shared by two or more kingdoms should be checked against a simplified but rooted eukaryote tree including the species under study (best would be to take a tree rooted between Unikonts and Bikonts). Only this way can the author reliably reconstruct conserved intron positions that have most likely been already present in the last eukaryotic common ancestor.In line with the comments of the other reviewers, I have weakened the conclusions about the timing of the origin of introns based on the results presented here. Instead, the article is placed more as additional support for the Exon theory of genes, by the demonstration of conserved introns in ancient genes such as phosphatases.2) All the identified conserved introns should be shown in a word or excel sheet as supplemental information.I now made the entire set available for download, including a set that still includes homologous genes  "Shared introns in ancient protein domains"When the results of the CDD searches are presented, they should be presented along with a control, preferably as a table or graph. Great care should be taken when designing a control set (or sets). It should be selected based on the same criteria, including the phyletic distribution. It is quite tricky, given that the 'result set' is heterogeneous in this regard. The control should be of similar sample size as well, and given the heterogeneity and the small sample the best would be to sample a control many times independently (e.g. to have five control sets of ~ 200 sequences that show the same degree of sequence similarity across the same phyletic distance as the 'result set'). The selection for highly conserved sequences will obviously result in an enrichment of conserved domains. The design of the controls is crucial for the correct interpretation of the results, and it should be explained in detail in the text.I have added paragraphs explaining the method and results in more detail. The sample size was not correctly given and the sample size for the control was not included, and this was changed. The results in Figs. 3 and 4 do not have a direct control, they represent potential conserved introns and represent a different set used in Figs 6 and It was investigated whether this was the result of a bias in the query and just represent a subset of conserved introns in conserved genes. The quantitative experiments in Figs. 6 and 7 show this potential bias in a controlled situation, where the only difference between the sets is the presence of an intron. The positive correlation between presence of an intron and ancientness of the sequence is taken as support for the hypothesis that introns were indeed ancient.4) "Preferred occurrence of conserved introns in ancient genes"In the analysis to identify prokaryotic hits, only sequences having "a number of identical amino acids residues of 6 (2 × 3) around a  splice site" were used. This means 19 sequences. The analysis should best be repeated for the whole set (218 sequences), with the appropriate control set (see above).As can be seen in the graph of Fig. there is a small window where are difference can be seen in ancientness of the genes. A lower similarity  would mainly select false-positives, a higher number will select only conserved sequences in both sets. If the whole set is taken, then any effect will disappear in the noise. This represents one of the problems in analyzing intron data: given the enormous introns loss and possibly gain during evolution, together with possible intron sliding and the continuous protein diversification, it is difficult to discern an ancient signal in this noise.On page 2 the author writes: "The strongest prediction of an introns-early scenario is the presence of conserved introns between orthologous proteins that diverged early in the eukaryotic lineage." I don't think it is correct. If introns originated early during eukaryote evolution, but still later then prokaryotic genes, i.e. not as building blocks of the first genes to be assembled, one would also expect to find many conserved introns between eukaryotic orthologs.I changed the introduction. Although expected in introns-early, conserved introns are compatible with introns-late. As now mentioned in the article in the last paragraph of the Discussion, I believe that the ancestral genome of prokaryotes and eukaryotes was a eukaryotic-like genome with characteristics such as introns and the RNA relics (e.g. the ribosome and the spliceosome). In that respect, early eukaryotic and ancient would be synonymous.Page 2/3: "It was found that conserved introns are frequently and specifically found in ancient protein domains" the word 'specifically' is too strong here, it would mean that conserved introns are only found in ancient domains, which is not the case (it is 53%).Changed to 'positively correlated with sequence ancientness'.The word 'ancient' is often used as a synonym of 'conserved' but it is of course not the same thing. E.g. page 5 "Thus, based on these results, introns seem to be preferentially located into ancient genes, in line with an introns-early scenario" these are rather conserved, then necessarily 'ancient' genes.Checked for inappropriate references to ancient and conserved.page 7: "In conclusion, the data presented here indicate a high occurrence of introns in ancient genes, followed by a massive loss of introns later in evolution. " High occurrence sounds too strong, a total of 218 introns were identified. Intron loss was not addressed in the paper, this would require mapping the distribution of conserved introns onto a phylogenetic tree.I changed this. My interpretation of the data, with an Exon theory point-of-view, is that the ancient domains contained many introns since multiple were found in phosphatases for example  indicate that either many were lost during evolution, or the sequence diverged and were not picked up by the current method. In the article there are two sets shown. The first consists of a set of conserved introns, based on the requirement that any 6 out of 10 residues should be identical, the second set consists of 5 separate queries in which the exact position of the identical residues was determined.ple Fig. . The relPage 7: "These results support the idea of ancient introns and are difficult to reconcile with an origin of introns late in evolution, although an insertion directly after the eukaryote-prokaryote split cannot be excluded based on the current results." I would rather say that the results nicely support the ancestral presence of introns in the eukaryote common ancestor. This is interesting enough, and the whole paper and discussion would be much better if the author didn't try to argue too strongly for a model, that is not strongly supported by the data, but rather discuss what one can really conclude from the results.I have changed the focus on the distinction between introns-late and introns-early (which was not well-supported by the data presented) towards a support for the Exon theory of genes. I made more explicit that, seen in the light of introns-late, these data would not exclude an introns-late scenario.Sets of conserved intron positions. This file contains the two sets of conserved introns in Excel format used in this article and a Word document containing a short description of field names.Click here for file


The role of spliceosomal introns in eukaryotic genomes remains obscure. A large scale analysis of intron presence/absence patterns in many gene families and species is a necessary step to clarify the role of these introns. In this analysis, we used a maximum likelihood method to reconstruct the evolution of 2,961 introns in a dataset of 76 ribosomal protein genes from 22 eukaryotes and validated the results by a maximum parsimony method. Our results show that the trends of intron gain and loss differed across species in a given kingdom but appeared to be consistent within subphyla. Most subphyla in the dataset diverged around 1 billion years ago, when the “Big Bang” radiation occurred. We speculate that spliceosomal introns may play a role in the explosion of many eukaryotes at the Big Bang radiation. Their role in the genome, however, remains poorly understood. From the view of eukaryotic evolution, it is very important to know why exon/intron structures of genes differ across species and what the effects of intron gain and loss are. In order to clarify these issues, we must first reconstruct the process of intron gain and loss during eukaryotic evolution. This task became possible recently with the availability of many completely sequenced genomes. In a representative study, Rogozin Giardia lambliaWe have recently compiled a dataset of ribosomal protein (RP) genes We compiled a dataset of 76 RP gene orthologs from 22 eukaryotes. The phylogenetic tree of these 22 species is depicted in We first used our recently developed maximum likelihood (ML) method Coprinus cinerea and pezizomycotina . Likewise, introns increased in the unicellular species Cryptococcus neoformans but decreased in saccharomycotina (subphylum 6) and schizosaccharomycetes (subphylum 7). Similar results were also obtained in an analysis using 13 additional fungal species (data not shown).The most significant feature in Drosophila melanogaster and Anopheles gambiae were very strong during the period from 1.0 to 0 Ga (∼151 losses versus ∼20 gains on average).Stagonospora nodorum (275 introns), and the species with the fewest gains was Dictyostelium discoideum (26 introns). During this period, the species with the most intron losses was Saccharomyces cerevisiae (255 introns), and the species with the fewest losses was Homo sapiens (57 introns). The patterns of intron gain and loss were most complicated in fungi. The number of intron losses in Magnaporthe grisea since the crown ancestor was 220, whereas the number of gains was 230. During the period from 1.5 to 0 Ga, fungal species in average experienced 2.2 intron gains and 2.6 intron losses per gene (as 168 gains and 198 losses in 76 genes). In contrast, humans experienced 2.0 intron gains and 0.8 intron losses per gene (as 155 gains and 57 losses in 76 genes).During the period from 1.5 to 0 Ga, the species with the most intron gains was In this study, we have analyzed intron dynamics in 76 RP gene families from 22 species. So far, there has been no analysis that used such a large dataset with many gene families and species. On one hand, the results inferred from the RP genes are mostly in-line with previous studies using fewer species. For example, when the same maximum likelihood was used, the trends of intron gains and losses in seven species of the 684-ortholog dataset When the intron dynamics in RP genes from 22 species were viewed together, it becomes clear that the trends of intron gains and losses differ across species in a given kingdom but are similar within subphyla. It has been proposed that most subphyla in our dataset diverged around 1 Ga Several roles have been proposed for spliceosomal introns. One of these is to increase the variation of proteins through alternative splicing D. melanogasterIf alternative splicing is not the only role of introns, what are other roles? We speculate that there is a relationship between introns and non-coding RNAs (ncRNAs) and, through the process of intron gain and loss, introns may affect the variation of expression of ncRNAs to become a force for the speciation of eukaryotes. There are several lines of supporting evidence for this speculation. First, some ncRNAs are transcribed from introns in protein coding genes H. sapiens, Ciona intestinalis, D. melanogaster, Caenorhabditi elegans, Schizosaccharomyces pombe, S. cerevisiae, Arabidopsis thaliana, and Plasmodium falciparum) were taken from the manually curated Ribosomal Protein Gene database  H. sapiens, A. gambiae, and Apis mellifera ; D. melanogaster ; C. elegans ; S. pombe (http://www.sanger.ac.uk/Projects/S_pombe/); S. cerevisiae ; M. grisea, Fusarium graminearum, S. nodorum, C. cinerea, C. neoformans, Ustilago maydis, and Rhizopus oryzae ; Yarrowia lipolytica ; D. discoideum ; A. thaliana ; Oryza sativa ; C. intestinalis, Chlamydomonas reinhardtii, and Fugu rubripes, ; and P. falciparum . When a gene existed in multiple copies in a given species, the copy with the most introns was used. The sequences of the 76 orthologs of RP genes are available at http://ribosome.miyazaki-med.ac.jp/.The RP genes of 8 eukaryotes . In a previous analysis of intron evolution P. falciparum). Therefore, the cost function in the maximum parsimony method was modified as follows:gains and losses are the numbers of intron gains and losses; K is the ratio between rate of loss and rate of gain; λ shows the fraction of introns present at the root of the phylogenetic tree; and rs = 0 or 1 means intron absence or presence at the root. The values K = 500 and λ = 0.01 were used here.Two methods, maximum likelihood Dataset S1Matrix of the Entire Set of Intron Presences and Absences.(0.03 MB TXT)Click here for additional data file.


Metarhizum anisopliae is currently used as a biocontrol agent (BCA) of insects. In the present work, we analyzed the sequence data obtained from group I introns in the large subunit (LSU) of rDNA genes with a view to determining the genetic diversity present in an autochthonous collection of twenty-six M. anisopliae isolates selected as BCAs.The entomopathogenic anamorphic fungus M. anisopliae isolates were amplified by PCR. The amplicon sizes ranged from 0.8 to 3.4-kb. Four intron insertion sites, according to Escherichia coli J01695 numbering, were detected- Ec1921, Ec2066, Ec2449 and Ec2563- after sequencing and analysis of the PCR products. The presence/absence of introns allowed the 26 isolates to be distributed into seven genotypes. Nine of the isolates tested showed no introns, 4 had only one, 3 two, and 10 displayed three introns. The most frequent insertion sites were Ec1921 and Ec2449. Of the 26 isolates, 11 showed insertions at Ec2563 and a 1754-bp sequence was observed in ten of them. The most-parsimonious (MP) tree obtained from parsimony analysis of the introns revealed a main set containing four-groups that corresponded to the four insertion sites.DNA fragments corresponding to the 3'-end of the nuclear LSU rDNA genes of 26 M. anisopliae. Intron insertions at the Ec2563 site were observed for first time in this species.Four insertion sites of group I introns in the LSU rDNA genes allowed the establishment of seven genotypes among the twenty-six biocontrol isolates of  Metarhizium have been shown to efficiently control several insect species [M. anisopliae, initially described as a pathogen of the wheat cockchafer Anisoplia austriaca. This fungus is commonly known as "green muscardine fungus" [M. anisopliae spores have come into contact with the outer surface of the insect, they germinate. After penetrating the insect exoskeleton, they rapidly grow inside the insect and cause its death.The use of entomopathogenic microorganisms to combat insects is currently considered to be a viable supplement or alternative to synthetic chemical insecticides, which are known to have toxic effects on non-target organisms . Strains species ,3. The m fungus"  and can  fungus" ,3. Once Metarhizium [Traditionally, the classification and typing of anamorphic entomopathogenic fungi have mainly been based on morphological characteristics . Nevertharhizium  or for marhizium .M. anisopliae and other entomopathogenic fungi in order to identify strains of interest, determine the origin of the isolates, study populations, or carry out phylogenetic analyses. Thus, a useful polymorphism for fingerprinting M. anisopliae isolates was detected using restriction fragment length polymorphism (RFLP) analysis of mitochondrial (mt) DNA [M. anisopliae isolates tested [The application of molecular techniques in mycology has shed new light on the systematics, biochemistry, and ecology of entomopathogenic fungi . Molecul(mt) DNA . A high s tested ,12. Thiss tested .Beauveria brongniartii. RFLP analysis of PCR products revealed the presence of insertional elements of about 350–450 bp within the LSU rDNA. Several authors have reported the usefulness of group I introns, inserted after specific sites in SSU or LSU rDNA genes for genotyping species and strains in genera such as Beauveria or Cordyceps [M. anisopliae has been identified at three different positions within the LSU rDNA [Group I introns are autonomous genetic elements characterized by their ability to "self-splice", or to splice due to their particular topology. These introns are found in eukaryotic and (eu)bacterial domains ,14. In eordyceps -20. The LSU rDNA ,12,17.M. anisopliae isolates selected as biocontrol agents against insects. Twenty-six biocontrol isolates, most of them obtained from different locations in the Iberian Peninsula, were examined, based on the analysis of sequence data from group I introns in the LSU rDNA genes. The usefulness of group I introns in LSU rDNA genes for the characterization and identification of M. anisopliae strains was evaluated.The present study was undertaken to determine any genetic diversity existing in an autochthonous collection of M. anisopliae isolates  were 1270 bp long, with one intron at position Ec1921.M. anisopliae isolates analyzed allowed a distribution into the following genotypes: A1B2A3A4, B1B2A3A4, A1B2A3B4, B1B2A3B4, B1A2B3B4, A1B2B3B4 and B1B2B3B4. Letters A and B indicate the presence and absence of introns, respectively, and the insertion sites are numbered from 1 to 4; that is, Ec1921 (1), Ec2066 (2), Ec2449 (3) and Ec2563 (4). These genotypes and their distribution frequencies are shown in Table The presence/absence of introns at the 3'-end of the nuclear LSU rDNA of the twenty-six M. anisopliae 33  [M. anisopliae IMNST 9601 .A 440-bp intron was identified in fourteen isolates at position Ec1921. Alignment of these sequences revealed the existence of two variants with a few bp differences for this intron. The divergences were due to nucleotide substitution at different positions. BLAST analysis of these sequences revealed high sequence identity with an intron inserted at the same position in the LSU from F197122)  and withM. anisopliae isolates 11 and 33 .Isolate Ma17 from Seville was the only one of the twenty-six isolates selected for study that showed evidence of an intron, 343 bp long, at position Ec2066. This intron had 95 and 98 % identity with two sequences previously detected at the same position in the LSU of M. anisopliae 33 (AF197124).Fourteen isolates displayed one intron at position Ec2449. Five sequence variants, ranging from 409 to 417 bp in size, were identified at this site. These five sequences were very similar and showed a 96–98 % identity with the Ec2449-position intron sequence reported for M. anisopliae isolates. One of them was 1754-bp in size sequence and was detected in ten of these isolates. A 161-bp fragment at the 3' end of this sequence showed a BLASTn identity of 83% with a Cordyceps intron sequence from position 1398 to 1585 (AB044641). The rest of this 1754-bp sequence did not show either BLASTn or BLASTx identity with any of the genome sequences deposited in the public databases. An open reading frame (ORF) coding for 360 amino acids, located at position 49-1084-bp, was identified in this sequence. No homing endonuclease (HE) motifs were detected. The second type was observed in only one isolate, Ma12, corresponding to a 435-bp long intron showing a sequence of 172-bp with a 84% of identity, from position 125 to 297, with the same intron sequence of Cordyceps (AB044641).Two sequence types, very different in length, were detected at position Ec2563 of eleven of the M. anisopliae, the comparison was performed with the B. bassiana sequence a. n. AF430699. P, Q and R elements were conserved at the four insertion sites. However, the element S was also identical in introns located at the Ec1921, Ec2066 and Ec2563 sites, but was not conserved for introns inserted at position Ec2449. At Ec2563 site the four elements were located in the 1754-bp intron at the following positions: 1428 to 1440 (P), 1505 to 1519 (Q), 1455 to 1466 (R) and 1553 to 1564 (S). In the 435-bp intron, these elements were located at positions 141 to 153 (P), 168 to 179 (Q), 218 to 232 (R) and 265 to 276 (S).In order to explore the different intron sequences obtained in the present work in greater detail, a comparative study with elements P, Q, R and S, previously reported in group I introns , was carMetarhizium, Beauveria and Cordyceps, and the subsequent phylogenetic analysis is shown in Figure The MP tree obtained after the alignment of 11 distinct intron sequence types identified from 26 isolates and GenBank-deposited sequences, that represent intron sequences from three entomopathogenic genera Beauveria and Metarhizium [Beauveria by PCR were used to characterize our M. anisopliae isolates. The polymorphism observed in this region of M. anisopliae genome was high, due to the presence/absence of introns at the four conserved sites. In the 26 isolates of M. anisopliae, we identified a total of 40 group I introns, which were inserted at any of the four specific sites in the LSU rDNA. This degree of polymorphism is in agreement with the high number of group I introns detected in Cordyceps, in which 28 representative members of this genus have been studied [B. brongniartii and 125 of B. bassiana were distributed within 9 and 13 genotypes, respectively. A high number of introns inserted at the four specific sites was detected for both species, and only 7% of isolates tested did not display intron insertions [M. anisopliae a low intron number was detected; only 5 and 2 introns were described from 30 and 40 strains studied, respectively [M. anisopliae results were surprising since a high degree of polymorphism, due to vertical transmission events, would be expected, and an even higher degree if the high polymorphism discovered in the rDNA genes of Cordyceps, the teleomorph of these species, was considered [The variability in group I introns from LSU rDNA genes can be used as a robust molecular tool for the identification of polymorphisms in entomopathogenic fungi such as arhizium ,16,21. T studied . The latsertions . Howeverectively ,12. Bearnsidered .Cordyceps, proposing a cyclic model of invasion, degeneration, loss, and reinvasion [B. bassiana strains and isolates with three-, two- and one-intron insertions, representing 16%, 48% and 28% frequency, respectively [Phylogenetic studies have been used to explain the sporadic distribution of these elements in invasion . Howeverectively .M. anisopliae sequences obtained in this work and some other Metarhizium, Beauveria and Cordyceps representative sequences from public databases. In general, the MP tree shows that introns inserted at the same site are closely related. This is expected, considering that horizontal transmission is one of the pathways along which group I introns can move [M. anisopliae isolates are separated in an independent clade (bootstrap of 53%) from the Beauveria and Cordyceps isolates. In spite of the sequence polymorphism observed at this insertion site, the five variants of M. anisopliae grouped together. In this sense, a sequence comparison of the P, Q, R and S motifs in introns inserted at the four specific sites was carried out (data not shown). Compared with AF197124 sequence of M. anisopliae, only P, Q and R motifs were conserved.A phylogenetic analysis based on intron sequences in the LSU rDNA was carried out with the 11 different can move ,23. Thiscan move ,20. At tB. bassiana [Naegleria sp. [M. anisopliae ORF, suggesting the ORF is not an HE.An intron at insertion point Ec2563 was identified in eleven isolates. Nevertheless, the intron of the ten isolates grouped in genotype 1 had an uncommon size of 1754-bp. This intron type is usually small, ranging in size from 250–500 bp . Intronseria sp. . HE consMetarhizium and Beauveria have been differentiated by RAPD analysis, but little evidence of a correlation between RAPD groups and the geographical origin or host has been gathered [M. anisopliae have described that similar RAPD profiles tend to be obtained from isolates of the same geographic area [B. bassiana [Species and strains of gathered . Other shic area . LSU intbassiana . In the Metarhizium includes entomopathogenic isolates used as BCAs. To predictably and successfully apply BCAs to combat insects in the field, it is essential that their biology and ecology should be well understood. The release of biocontrol agents into the environment demands the development of methods to monitor their presence or absence in the field [B. bassiana strains were discriminated in co-formulation studies [M. anisopliae strains and soil samples with a view to generating SCAR markers and developing quantitative real-time PCR assays.The genus he field ,30. Usin studies . In thisM. anisopliae. These introns exhibited various insertion patterns and this small and selected collection of 26 isolates was distributed into 7 genotypes. Phylogenetic analysis of these intron sequences demonstrated a general correlation between specific insertion sites and sequence homology. P, Q, R and S elements were conserved at insertion positions Ec1921, Ec2066 and Ec2563, and only P, Q and R elements were conserved at Ec2449 site.A total of 40 group I introns related to four insertion sites detected at the 3' end in LSU rDNA genes have been identified in 26 biocontrol isolates of M. anisopliae were used in this study. The cultures were obtained from the collection at the CRAF  Department of the University of Cordoba . Their habitat/host and geographical origins are shown in Table Twenty-six strains of et al. [Mycelia for DNA extraction were obtained by inoculating MEA plates that contained a sterile cellophane sheet with spores from MEA cultures after incubation at 25°C for 4 days. Mycelia were collected by scraping the cellophane sheets and were then frozen, lyophilized and ground. Total DNA was extracted using the method described by Möller et al. .Beauveria [M. anisopliae included in this study was carried out with the primer pair E24 (5'- GCTGAATTACCATTGCGGAG-3') and I29 (5'-CTGCCCAGTGCTCTGAATGTC-3'), using the Taq polymerase system , following the manufacturer's instructions. PCR was performed in a total volume of 50 μl containing 25 ng of genomic DNA and 0.20 μM concentrations of each primer described above. The amplification included an initial denaturing cycle of 5 min at 94°C, followed by 35 cycles of 1 min 30 sec at 94°C, 2 min 30 sec at 57°C and 3 min at 72°C, and a final extension step of 5 min at 72°C in a PCR System 9700 Genetic Thermal Cycler . The PCR products were electrophoresed on 1% agarose gels buffered with 1X TAE [The primers used for the amplification of the 3'-end of the LSU rDNA region have been reported previously for the amplification of this region in the genus eauveria ,20. Amplh 1X TAE  and staiThe PCR products were purified from agarose gels using the GenClean II kit  according to the manufacturer's protocol. DNA sequences were obtained using an automated ABI 377 Prism Sequencer (Applied Biosystems) with fluorescent terminators. All PCR products were sequenced in both directions. Sequences of internal primers were as follows: M1 , 5'-GGTAAAACTAACCTGTCTCACG-3'; M2 , 5'-GATCCTTCCCAGGGAGGTG-3'; M3 , 5'-CGGGTAGGTATTGTAAGGCG-3'; M4 , 5'-GGGACCTCGAGGTTTGTG-3' and M5 , 5'-CCAGGTCTGACTTACATG-3'.M. anisopliae was analyzed by detecting previously described target sequences [Metarhizium, Beauveria, Cordyceps or Naegleria were retrieved from GenBank and included in the alignments. The GenBank accession numbers were: B. bassiana, AF322942 and AF430706 for insertion site Ec1921; AF430703 and AF322939 for insertion site Ec2066; AF430700 for insertion site Ec2449, and AF336302 and AF322936 for insertion site Ec2563; M. anisopliae, AF197120, AF197122 and AF363479 for insertion site Ec1921; AF197121 and AF197123 for insertion site Ec2066, and AF197124 for insertion site Ec2449, and Cordyceps prolifica, AB044640, and Cordyceps sp., AB044641, for insertion sites Ec1921, Ec2066, Ec2449 and Ec2563. The Naegleria sp. AM167886 sequence was used as an outgroup. Phylogenetic analysis was carried out with PAUP . Gaps, encoded as missing data, and uninformative characters were excluded from the analysis. The MP trees were obtained from heuristic searches using TBR branch swapping [The presence or absence of introns at the 3'-end of the nuclear LSU rDNA of equences . Sequencequences . Publishswapping , and allM. anisopliae obtained in this study were deposited in the GeneBank database under the accession numbers given in Table The sequences corresponding to the introns from M. anisopliae isolates. In addition, EQM participated in genomic DNA extraction. EM conceived the design of the study and helped to review the manuscript. RH participated in the design and coordination of the study, carried out sequence phylogenetic analyses and helped to write the manuscript. All authors have read and approved the final manuscript.MM carried out laboratory work, participated in sequence alignments, and wrote the draft of manuscript. EAI reviewed the laboratory work. EQM and CSA provided the