It is often supposed that a protein's rate of evolution and its amino acid content are determined by the function and anatomy of the protein. Here we examine an alternative possibility, namely that the requirement to specify in the unprocessed RNA, in the vicinity of intron–exon boundaries, information necessary for removal of introns  affects both amino acid usage and rates of protein evolution. We find that the majority of amino acids show skewed usage near intron–exon boundaries, and that differences in the trends for the 2-fold and 4-fold blocks of both arginine and leucine show this to be owing to effects mediated at the nucleotide level. More specifically, there is a robust relationship between the extent to which an amino acid is preferred/avoided near boundaries and its enrichment/paucity in splice enhancers. As might then be expected, the rate of evolution is lowest near intron–exon boundaries, at least in part owing to splice enhancers, such that domains flanking intron–exon junctions evolve on average at under half the rate of exon centres from the same gene. In contrast, the rate of evolution of intronless retrogenes is highest near the domains where intron–exon junctions previously resided. The proportion of sequence near intron–exon boundaries is one of the stronger predictors of a protein's rate of evolution in mammals yet described. We conclude that after intron insertion selection favours modification of amino acid content near intron–exon junctions, so as to enable efficient intron removal, these changes then being subject to strong purifying selection even if nonoptimal for protein function. Thus there exists a strong force operating on protein evolution in mammals that is not explained directly in terms of the biology of the protein. Intron-exon boundaries, once fixed in proteins, are found to be subject to purifying selection, even if they are not optimal for protein function.  Most of the DNA in our genes is actually not involved in the specification of proteins. Rather, the bits with the protein-coding information (exons) are separated from each other by noncoding bits, introns. Before a gene can be translated into protein these introns are removed and the exons are spliced back together to be translated into protein. While information about which DNA to remove is largely in the introns themselves, parts of the exons near the intron–exon boundary can, for example, function as splice enhancer elements. In principle, then, these parts of exons have two functions: to specify the amino acids of the resulting protein and to enable the correct removal of introns. What impact might this have on a gene's evolution? We show that near intron–exon boundaries, amino acid usage is biased towards nucleotides involved in splice control. Moreover, these parts of genes evolve especially slowly. Indeed, we estimate that a gene with many exons would evolve at under half the rate of the same gene with no introns, simply owing to the need to specify where to remove introns. Likewise, genes that have lost their introns evolve especially fast near the former intron's location. Thus, human proteins may not be as optimised as they could be, as their sequence is serving two conflicting roles. Why do some parts of proteins evolve more slowly than others? Why, in turn, do some proteins evolve more slowly than others? Intragenic conserved regions are typically considered to reflect domains of functional importance to the protein . SimilarIn mammalian genes, which are rich in introns , correctMight the same forces also act to cause skews in amino acid usage in the vicinity of intron–exon junctions? In a preliminary analysis, we showed that there is a tendency for enrichment near boundaries of an amino acid whose codons are common in splice enhancers: lysine is coded by AAA and AAG, both of which are common in splice enhancers, and at both 5′ and 3′ ends of exons, lysine's proportional usage increases . Is it mp < 0.001 indicates significance). For all plots for individual amino acids see r = 0.96, p < 0.0001).For 178,382 human exons we considered the trends in amino acid composition as one approaches the intron–exon boundary, as assayed by the rank correlation, rho, between distance from the boundary and proportional usage of the amino acid. Considering the 2-fold and 4-fold blocks of the 6-fold degenerate amino acids as different groupings, we found that of 46 independent comparisons (23 amino acid groups 5′ and 3′ prime), 34 showed significant trends for enrichment or avoidance near intron–exon boundaries . After BDo these effects necessarily relate to the nucleotide content of the codons, as the splice-regulation model requires? One might conjecture instead that these effects reflect some coincidence of exon boundaries with protein substructures having unusual amino acid contents. Several facts strongly support the hypothesis that the trends seen are at least in part driven by effects at the nucleotide level. Notably, while the 2-fold block of arginine . As an alternative to rho, we can employ the slope of the best-fit regression line between proportional usage of an amino acid and distance from intron–exon junctions. A negative slope, like a negative rho, indicates preferential usage near junctions. Using this slope on the best-fit regression line revealed, as expected, the same trend .If splice-enhancer domains impact amino acid usage near intron–exon boundaries, we expect that those amino acids preferred in splice enhancers should be preferred near junctions . To test this we developed a metric of involvement of codons in splice-enhancer hexamers, which we term the hexamer preference index (HPI). Using hexamers found both in mouse and human to define the HPI (and ignoring 3′ and 5′ differences), we found a striking predictability of patterns of preference near boundaries .These results are not greatly affected by considering 5′ and 3′ ends separately  . Neither thymine nor guanine content showed any trends (p >> 0.05). These results suggest that the general profile of enhancers and the specifics employed to define HPI are about equally good predictors of patterns of preference/avoidance.One might suppose that our measure of HPI might be biased by incomplete knowledge of enhancers. We can control for this, in part, by recognizing that splice enhancers tend to be adenine rich and cytosine poor. Consider then the composite measure AC bias = frequency of adenine in synonymous codon set – frequency of cytosine. For example, in the 4-fold degenerate set for alanine (GCN), of the 12 bases in four possible synonymous codons, adenine and thymine both featured 1/12 of the time, and guanine and cytosine both featured 5/12 of the time. So AC bias for alanine is 1/12 − 5/12 = −1/3. This AC bias was a robust predictor of preference/avoidance near boundaries  . AvoidanKA] and exon length, rho = 0.15, p < 0.0001). This might, however, be owing to a trend for genes with small exons to be disproportionately in functional classes of protein that have intrinsically low rates of evolution. To control for this we considered, for all genes with more than two internal exons, the Spearman rank correlation between exon KA and the size of the exon. As each correlation coefficient is derived from a given gene, between-gene variation in KA (and indeed the number of synonymous substitutions per synonymous site [KS]) is controlled for in any such analysis. If splice control impacts rates of exon evolution we expect that on the average this correlation should be positive, while the null hypothesis, that small exons have low rates of evolution because they derive from classes of genes with intrinsically low KA, predicts a mean rho of zero. The distribution of rho was very strongly skewed to positive values . Restricting analysis to genes with ten or more exons only strengthened this conclusion .The above results suggest that selection acts to prefer nucleotides that permit efficient intron removal. Does this in turn affect rates of protein evolution? Were there such an effect, we should expect that smaller exons should evolve more slowly, as a higher proportion of sequence is near  boundaries. Indeed, from a set of 36,683 mouse–human aligned exons, we found that small exons do tend to have low rates of evolution (Spearman rank correlation between the number of nonsynonymous substitutions per nonsynonymous site ). This is not an artefact of contamination by collagen transcripts (GPXn), as the distribution of proline indicates no such trend. This pattern in glycine usage is still strong over 500 residues away from the reference point.(79 KB DOC)Click here for additional data file.Table S1(36 KB DOC)Click here for additional data file.


Giardia lamblia (a diplomonad). This intron is only 35 nucleotides in length and is unusual in possessing a non-canonical 5' intron boundary sequence, CT, instead of GT.Only one spliceosomal-type intron has previously been identified in the unicellular eukaryotic parasite, G. lamblia, in the ribosomal protein L7a gene (Rpl7a), that possesses a canonical GT 5' intron boundary sequence. A comparison of the two known Giardia intron sequences revealed extensive nucleotide identity at both the 5' and 3' intron boundaries, similar to the conserved sequence motifs recently identified at the boundaries of spliceosomal-type introns in Trichomonas vaginalis . Based on these observations, we searched the partial G. lamblia genome sequence for these conserved features and identified a third spliceosomal intron, in an unassigned open reading frame. Our comprehensive analysis of the Rpl7a intron in other eukaryotic taxa demonstrates that it is evolutionarily conserved and is an ancient eukaryotic intron.We have identified a second spliceosomal-type intron in Rpl7a intron suggests its utility as a phylogenetic marker to evaluate particular eukaryotic groupings. Additionally, analysis of the G. lamblia introns has provided further insight into some of the conserved and unique features possessed by the recently identified spliceosomal introns in related organisms such as T. vaginalis and Carpediemonas membranifera.An analysis of the phylogenetic distribution and properties of the  Giardia lamblia  appeared abnormally truncated at the C-terminal end relative to other eukaryotic and archaeal sequences, terminating at position 171 of S. cerevisiae L7a  resides in a putative [2Fe-2S] ferredoxin gene and begins with CT rather than GT at the 5' intron boundary , where B = C, G or T; Y = C or T; N = any nucleotide and the branch-point sequence is in bold italics. Remarkably, only two matches to this pattern were found in the entire T. vaginalis genome data set and neither of these sequence patterns appears to be an intron candidate. Furthermore, neither of these two sequences contains ACTGAC in the predicted position for the intron branch-point sequence. This result further emphasizes the importance of the ACTAAC sequence motif in the T. vaginalis introns and the differences in sequence constraints apparent between these introns and those of G. lamblia. If additional T. vaginalis spliceosomal introns are found (<519 nt in size) that contain alternative sequences at the 3' intron boundary, sequence differences must also be present in the 5' intron boundary that prevent the search algorithm from finding these intron candidates using the parameters we have employed.To investigate whether an additional group of  PatScan , we searG. lamblia partial genomic data set for intron candidates. As expected, the Rpl7a and ferredoxin introns are detected by this method, as is an additional set of 15 matches to this sequence pattern. Of these matches, only one was a likely intron candidate that had the potential to disrupt a predicted ORF, among other criteria. Since the ORF in which this candidate intron resides (or that it disrupts) does not encode a conserved protein – unlike the Rpl7a intron – further experimentation was required to prove whether or not it is an intron. Removal of the intron candidate sequence, nt positions 1263 to 1482 of the G. lamblia contig [GenBank:AACB01000025], extends the predicted protein encoded by the unassigned ORF [GenBank:EAA41257] by an additional 118 amino acids at its amino terminus. Further, when the intron sequence is removed, the inferred initiation codon is now positioned directly adjacent to an AT-rich motif that Yee et al. , which lacks any introns. This situation is in stark contrast to other hexapod Rpl7a sequences, which contain introns in addition to the conserved intron discussed here. The A. gambiae case may be an example of intron loss mediated by a reverse transcription mechanism [Within Opisthokonta and Amoebozoa, several groups appear to lack the  outside ,17 of thechanism ,19 and iechanism .Rpl7a intron. Only a single member of Rhizaria (B. natans) was investigated here so it is premature to conclude that the Rpl7a intron is absent altogether from this supergroup. Our survey failed to detect the Rpl7a intron in representatives of the Euglenozoa, Heterolobosea or Parabasalia, all eukaryotic groups for which inclusion in a larger Excavata assemblage is only weakly supported [E. gracilis is well endowed with spliceosomal introns [Spironucleus barkhanus does not have the Rpl7a intron, likely a result of intron loss in this organism given the presence of the intron in other related Excavata.None of the examined members of Plantae, Chromalveolata or Rhizaria contained the conserved upported . Whereas introns -23, intr introns . The dipRpl7a intron could be explained by independent events of intron gain or by lateral gene transfers. We note that the Rpl7a exon boundary sequences conform poorly to the conserved proto-splice site sequence AG/G [Rpl7a intron resulting from multiple intron gains at proto-splice sites. Supporting the argument against intron gain is the apparent very low density of introns in G. lamblia. This is also in agreement with recent data suggesting that few shared intron positions between distantly related taxa are due to parallel gain  at proto-splice sites [We considered other (less probable) explanations that the phylogenetic distribution of the A,C)AG/G   Rhizaria, but maintained in Opisthokonta, Amoebozoa and Excavata. Alternatively, if one assumes a specific relationship among Opisthokonta, Amoebozoa and Excavata, it is only necessary to invoke a single loss of the Rpl7a intron in a common ancestor of Plantae/Chromalveolata/Rhizaria, to explain the apparent absence of this intron in these three supergroups.Depending on one's views of the eukaryotic tree, there are two possibilities to explain the observed distribution of the tic tree , the RplRpl7a intron, supporting a grouping of Opisthokonta, Amoebozoa and Excavata.While multiple cases of intron gain seem unlikely (as discussed above), a single intron gain at the base of Opisthokonta, Amoebozoa and Excavata would result in the observed distribution, assuming the Plantae/Chromalveolata/Rhizaria had already diverged. While additional intermediate possibilities exist with regards to various intron loss and/or gain events, we propose that a single loss or gain is the most parsimonious explanation for the observed distribution of the Rpl7a intron does not position the root of the eukaryotic tree, it may help to resolve some of the basal branches. It is important to note that Excavata is a tenuous grouping that may in fact be polyphyletic: thus, members of Excavata without the intron may fall on either side of a putative loss/gain event. In this instance, it is possible that one or more members of Excavata will be found to group with the Plantae/Chromalveolata/Rhizaria consortium. Conversely, if additional evidence robustly groups the organisms in question within Excavata, this result would imply that they lost the intron independently, as is evidently the case for Fungi within Opisthokonta.Although the distribution of the Rpl7a intron in other representatives of Plantae, Chromalveolata and/or (particularly) Rhizaria than those listed in Table Rpl7a intron in these other eukaryotic supergroups would only strengthen the argument that this intron is indeed ancient. At present, the simplest explanation for the observed distribution of the Rpl7a intron is a specific relationship uniting Opisthokonta, Amoebozoa and Excavata to the exclusion of Plantae, Chromalveolata and possibly Rhizaria. However, additional characters will need to be found in order to strengthen this proposed assemblage.New eukaryotic genome sequences may well reveal the G. lamblia spliceosomal-type intron (35 nt in length) raised questions regarding the importance of its non-canonical 5' intron boundary and possible constraints on the size of Giardia introns. Furthermore, it was clear that the identification of additional spliceosomal introns would be required to assess the degree of conservation of predicted functional sequence elements within these introns [G. lamblia two larger introns (109 and 220 nt) in genes encoding, respectively, L7a and a non-conserved, unassigned protein. Both of these newly identified introns exhibit a canonical GT 5' intron boundary. Evolutionary conservation of the exact position and phase of the Rpl7a intron (within a highly conserved region of the L7a protein sequence in members of diverse eukaryotic groups) indicates that this particular intron was likely present in the ancestor of these lineages. The Rpl7a intron exhibits all of the hallmark features of an ancient intron, such as widespread distribution, phase 0 positioning, location in an ancient eukaryotic gene, and lack of a proto-splice site sequence; thus, this intron can be considered to be a meaningful phylogenetic marker. Currently, no singular genomic marker definitively resolves the branching order of the eukaryotic supergroups. We propose that patterns of conservation of ancient introns, when larger data sets are examined, may provide such information.The properties possessed by the first identified  introns . In thisR. americana, ATCC 50394; S. ecuadoriensis, ATCC 50688; M. jakobiformis, ATCC 50310; M. californianus, ATCC 50740); A.J. Roger ; A.J. Lohan ; D.F. Spencer (E. gracilis strain Z); and J.M. Archibald . Genomic DNA from J. libera (ATCC 50422) and P. polycephalum (strain M3C) was obtained by lysing cells in 1% SDS followed by phenol extraction and ethanol precipitation.Samples of genomic DNA from various protists (full organism names are listed in Table Rpl7a from genomic DNA (100–200 ng), using Invitrogen Taq DNA polymerase. PCR primers used are listed in Polymerase chain reaction (PCR) was used to amplify S. cerevisiae sequences as queries. Protein alignments were generated with ClustalX 1.82 [Rpl7a sequences. Searches for spliceosomal-like introns within the preliminary genome sequence data of T. vaginalis and G. lamblia were performed using the sequence pattern search program PatScan [Ribosomal protein L7a gene and protein sequences were identified by searching relevant sequence databases using TBLASTN or BLASTP with the alX 1.82  applying PatScan , recompiG. lamblia Rpl7a intron, performed genomic searches for additional introns in G. lamblia and T. vaginalis and carried out the RT-PCR experiments to confirm the presence of the third G. lamblia spliceosomal intron. TES examined the phylogenetic conservation of the Rpl7a intron in currently available eukaryotic genome databases, performed all the PCR experiments to determine the prevalence of the intron, and participated in the genomic searches for additional G. lamblia introns. RFW performed the phylogenetic analysis of L7a sequences. MWG made substantial intellectual contributions to this work. All authors participated in the assembly and editing of the manuscript. All authors read and approved the final manuscript.AGR analyzed the L7a protein sequences, discovered the G. lamblia ORF. Evidence for a spliceosomal intron in an unassigned This file (PDF format) presents gel electrophoretic data documenting the results of PCR and RT-PCR experiments to confirm the existence of a putative spliceosomal intron in a non-conserved, unassigned ORF in G. lamblia, as described in the text.Click here for fileSupplemental Tables. This file (PDF format) contains two tables. Table 1 contains the sequences of the oligonucleotide primers used for amplifying Rpl7a sequences from various eukaryotic taxa. Table 2 is a complete list of all the organisms and sequence sources that were examined for the presence of the Rpl7a intron.Click here for fileL7aTree. This file (PDF format) presents a maximum likelihood tree generated using L7a sequences from representative eukaryotic and archaeal taxa. Also included are the methods used to generate the tree, the sources of the sequences and the alignment that was used.Click here for file


The evolution of spliceosomal introns remains poorly understood. Although many approaches have been used to infer intron evolution from the patterns of intron position conservation, the results to date have been contradictory. In this paper, we address the problem using a novel maximum likelihood method, which allows estimation of the frequency of intron insertion target sites, together with the rates of intron gain and loss. We analyzed the pattern of 10,044 introns  in the conserved regions of 684 sets of orthologs from seven eukaryotes. We determined that there is an average of one target site per 11.86 base pairs (bp) . In addition, our results showed that: (i) overall intron gains are ~25% greater than intron losses, although specific patterns vary with time and lineage; (ii) parallel gains account for ~18.5% of shared intron positions; and (iii) reacquisition following loss accounts for ~0.5% of all intron positions. Our results should assist in resolving the long-standing problem of inferring the evolution of spliceosomal introns. When did spliceosomal introns originate, and what is their role? These questions are the central subject of the introns-early versus introns-late debate. Inference of intron evolution from the pattern of intron position conservation is vital for resolving this debate. So far, different methods of two approaches, maximum parsimony (MP) and maximum likelihood (ML), have been developed, but the results are contradictory. The differences between previous ML results are due predominantly to differing assumptions concerning the frequency of target sites for intron insertion. This paper describes a new ML method that treats this frequency as a parameter requiring optimization. Using the pattern of intron position in conserved regions of 684 clusters of gene orthologs from seven eukaryotes, the authors found that, on average, there is one target site per ~12 base pairs. The results of intron evolution inferred using this optimal frequency are more definitive than previous ML results. Since the ML method is preferred to the MP one for large datasets, the current results should be the most reliable ones to date. The results show that during the course of evolution there have been slightly more intron gains than losses, and thus they favor introns-late. These results should shed new light on our understanding of intron evolution. Twenty-eight years have passed since the discovery of spliceosomal introns , but theAlthough introns have high mutation rates, making it difficult to trace lineages through sequence homology, their positions are well conserved ,3. ThereTwo main approaches, maximum parsimony (MP) and maximum likelihood (ML), have been applied to determine intron evolution from the pattern of intron position conservation. The MP approach, based on the assumption that intron gain and loss events occur rarely in evolution, infers the most parsimonious scenario (measured as a function of gains and losses). Conversely, the ML approach infers a scenario with the highest probability of producing the observed data, for a given model of intron evolution.In a large-scale study of intron evolution, Rogozin et al.  applied Roy and Gilbert ,7 applieThe results of the two ML methods clearly contradict each other and also differ significantly from results of the MP methods. This may be because of the different assumptions they made about the number of target sites; a target site is defined here as a possible position for intron insertion in the multiple-sequence alignment of all species. The number of target sites thereby includes all observed sites, plus possible sites that had introns in the past or may have introns in the future. The method of Roy and Gilbert ,7 assumeIn this paper, we propose a new ML method, the underlying basis of which is the treatment of target site number as a parameter requiring optimization. A log-likelihood function was formulated to allow optimization of the number of target sites, and an expectation-maximization (EM) algorithm was developed to optimize the log-likelihood function. Our results paint a different picture from those provided by previous ML methods.Drosophila melanogaster and Anopheles gambiae as arthropods, Caenorhabditis elegans as a nematode, and Homo sapiens as a deuterostome), we first tested both hypotheses using the pattern of intron position conservation. In our method, the maximum log-likelihood values for each tree configuration are computed, after which a χ2 test with one degree of freedom is performed for the value −2logΛ, where Λ is the likelihood ratio. The maximum log-likelihood value for the ecdysozoa hypothesis was −255.48 (p < 10−9), a result consistent with that of another proposed method . The. The9].  −255.48  and 3, w −255.48 . Thus, −θ (see θ) was 0.071  of H. sapiens, and the terminal branch leading to Arabidopsis thaliana, experienced many more gains than losses. In contrast, all other branches except that leading to C. elegans experienced many more losses than gains. Losses along the terminal branch leading to C. elegans slightly outnumbered gains. In total, there were 6,382 gains versus 5,099 losses.H. sapiens, and increased until the bilateral ancestor appeared, after which it decreased in other lineages, while continuing to increase in H. sapiens. The intron density also increased in the terminal branch leading to A. thaliana, but decreased in the terminal branch leading to Schizosaccharomyces pombe. Although our method cannot infer the intron density at the root node of the phylogenetic tree, it inferred that 107 intron positions . The probability that more than three introns would be gained in parallel at the same position was close to zero. Therefore, the total number of parallel intron gains accounted for ~18.5% of the shared intron positions. This result is slightly higher than the predicted range of 5% to 10%, but still less than the upper bound of 20% reported in a study using a different method on the same dataset [We also measured the frequencies of parallel intron gain and reacquisition following loss. Using our model of intron evolution, and incorporating the rates of intron gain and loss depicted in 2 test cannot be used directly. Therefore, we grouped 75 intron patterns showing values of <3 into five new equally sized categories, and the result of the χ2 test was not statistically significant .The observed and expected (using the MLEs of the parameters) numbers of cases for each external intron pattern are presented in Since the log-likelihood function is highly complex and no closed form is available for calculating the MLEs of the parameters, three numerical methods were applied. The first was based on an EM algorithm, the second on a downhill simplex (DS) method, and the third on a genetic algorithm (GA). See the θ = 100, produced almost the same results as a previous ML method [The present results were obtained using the EM algorithm, the advantage of which is its rapid convergence to a solution . HoweverL method  providesH. sapiens and A. thaliana. Therefore, we believe that the proto-splice site hypothesis is the more likely explanation for our results. However, the estimated frequency of the pattern MAG|R in the whole genomes of the six species we examined, excluding A. gambiae, is 2%–3% (unpublished data) and therefore too small to explain the optimal value of 8.4%. Consequently, the consensus pattern of proto-splice sites may need to be re-determined.To the best of our knowledge, ours is the first estimate of frequency of target sites, and can be accounted for by two hypotheses. The first is the exon theory of genes, which postulates that present-day exons are remnants of primordial mini-genes (~45–60 bp long) from the time of the last common ancestor of prokaryotes and eukaryotes . The secOur results show a slight excess (~25%) of intron gains over losses . However, there was an excess of intron-lost branches over intron-gained branches (five versus four). Since these results may change upon addition of more species, the conclusions about the relative importance of intron gain and intron loss will depend upon the balance of future datasets. Assuming that the seven species studied here represent a balanced dataset, our conclusion is that intron gain has played a slightly larger role than intron loss during the course of eukaryotic evolution, but that the relative importance may vary with time and lineage.Note that the results shown here were determined using the intron patterns in conserved alignment regions. We predict that the relative importance of intron gain and loss would tip more toward intron gain if complete alignments were used, as suggested by the results of Rogozin et al. . HoweverD. melanogaster and A. gambiae). Moreover, except for the ecdysozoa–C. elegans branch in which intron gains and losses are approximately in balance, the other branches experienced either many more gains than losses, or losses than gains. There seems to be an inverse relationship between rates of gain and loss on each branch. One possible explanation for this pattern is that the rates of intron gain and loss may be controlled by selective pressure exerted through genome compaction and expansion. This pressure should vary significantly among distantly related species, but only slightly among those more closely related.D. melanogaster, intron gains were dominant during the period from the crown ancestor to the bilateral ancestor, and intron losses were dominant after this point. There are two scenarios for intron evolution during the period between the last common ancestor of eukaryotes and the crown ancestor: either an increase or a decrease in intron density. As there would be only one turning point in the former, but two in the latter, the parsimony principle favors the former scenario in which intron density increased during the earliest stage of eukaryotic evolution.Two theories, introns-early and introns-late, have been proposed for the origin of spliceosomal introns. The introns-early theory asserts that introns were required to facilitate the assembly of early genes and were already present in the last common ancestor of prokaryotes and eukaryotes. Thus, this theory suggests that intron loss is the main driving force for intron evolution –17. In cH. sapiens, Roy and Gilbert's method predicted that gains and losses were approximately in balance. In contrast, our method predicted a notable excess of intron gains over losses. A possible explanation for these differences is that their model assumes that parallel intron gains do not occur [θ set to 100 , where θ = 0 indicates that the number of target sites is equal to the number of observed splice sites, and θ = 1 indicates that the number of target sites is equal to the total length of the alignments. Therefore, the value θ = 100 is unrealistic, since it implies that the number of target sites is approximately 100 times the total length of the alignments. Consequently, their results appear to underestimate intron gains and to overestimate losses.Our results differed greatly from those obtained by Roy and Gilbert ,7, whoseot occur ,7. This t to 100 , and theθ = 0 in our method). This assumption may lead to an overestimation of the rate of intron gain, which in turn leads to an overestimation of parallel gains. When we used θ = 0, our method produced results in which the ratio of parallel gain reached ~98.4%, thus supporting our explanation of the differences between the two methods. Additionally, since their dataset contains an average of 68 sequences per gene family, the total number of intron patterns is 268. This is much larger than 49, which is the average number of intron positions per gene family. Thus, it is possible that their sample dataset was insufficiently large for a valid statistical inference.Our results also contradicted the findings of Qui et al. , who detθ = 100 and θ = 0) and were outside the 95% CI (0.055–0.096). Their log-likelihood values were −282.28 and −2199.16, respectively. Thus, they were both rejected in favor of the MLE of θ .Note that assumptions about the target site frequency of both previous ML methods represented extreme versions of our method . These variations are believed to be caused by the differences between ML and MP. In an MP method, the most parsimonious scenario is always chosen, even though the next-most parsimonious scenario has a close probability of occurrence. Let us consider an intron pattern in which the chances of the most and the next-most parsimonious scenarios occurring are 51% and 49%, respectively. In the MP method, 100% of cases will be assigned to the most parsimonious scenario, while none will be assigned to the next-most parsimonious. In the ML method, 51% and 49% of cases will be assigned to the most parsimonious and the next-most parsimonious scenarios, respectively.Rogozin et al.  were theAlthough the results obtained by the method of Nielsen et al.  are not Although the debate between ML and MP is vigorous ,24, muchT as a parameter needing to be optimized, and find the MLE of T over all tree configurations; for the latter, we would need to construct a gene presence/absence matrix, rather than an intron presence/absence matrix.The method presented here may also be applied to at least two other problems: (i) determination of the phylogenetic tree of eukaryotic species based on the conservation of intron positions; and (ii) inference of rates of gene gain and loss on a genomic scale. For the former, we would consider the phylogenetic tree ftp://ftp.ncbi.nlm.nih.gov/pub/koonin/intron_evolution [D. melanogaster, A. gambiae, C. elegans, H. sapiens, S. pombe, Saccharomyces cerevisiae, A. thaliana, and P. falciparum. Multiple protein sequence alignments and intron presence/absence matrices of these KOGs were downloaded from the above site. Following Roy and Gilbert [S. cerevisiae due to its sparse intron distribution. The total length of conserved alignment regions in the dataset was 488,157 bp. The number of intron positions in these conserved regions was 7,221; the number shared across two or more species was 1,787. When the outgroup P. falciparum was excluded, the number of intron positions was 7,049 and the number shared was 1,722.We used the 684 eukaryotic clusters of orthologous genes (KOGs), which are available at  Gilbert , we onlyOur model of intron evolution assumes that introns are inserted independently and uniformly at all available target sites. Similarly, intron loss occurs with equal probability at all sites possessing introns, regardless of when they were inserted. Rates of gain and loss are assumed to be constant along each branch of the phylogenetic tree, but they can vary between branches. As suggested by the results of Rogozin et al.  and StolN be the number of species, G be the total length of all alignments in bp, and T be the phylogenetic tree of these species. Since the phylogenetic tree T is binary, it contains N external nodes, each corresponding to a species, M = N − 1 internal nodes, with each node corresponding to a divergent event, and B = 2M branches . An external intron pattern is a binary sequence s1s2...Ns of length N. When is = 0 or 1 , this means that an intron is absent or present at the ith external node. The definition of an internal intron pattern is similar, but with length M instead of N. There are 2N (indexed from 0 to 2N − 1) possible external intron patterns, and 2M (indexed from 0 to 2M − 1) internal intron patterns. For the ith external/internal intron pattern, the binary code of i will give the intron states of the external/internal nodes. For example, for N = 4, the binary code of external intron pattern i = 3 will be “0011.” Thus, an intron will be absent at the first and second species, yet present at the third and fourth species in this pattern. We denote in  as the number of cases for the ith external pattern, and kg and kl  as the number of gains and losses along the kth branch. We then have to estimate kg and kl for a given set of in. Note that n0  cannot be observed, and must be treated as a parameter requiring optimization.Given the model of intron evolution described above, our problem can be defined as follows: Let S and P as the numbers of observed splice sites (intron positions) and target sites in the dataset, respectively, we have:Denoting n0 = P − S. Since P must ≤G , n0 must be constrained by: 0 ≤ n0 ≤ G − S. If we define It follows from Equation 2 that k = 1, 2, …, B) as the probabilities of changing state from 0 to 1 (intron gain) and from 1 to 0 (intron loss) along the kth branch, respectively; kb and ke  as the node indexes of the beginning and end nodes of the kth branch, respectively; and hs  as the intron state of the hth node. The expected number of cases for the complete intron pattern ij , which is the combination of the ith external pattern and the jth internal pattern, is calculated by:We denote where:s1 is the intron state of the root node.Here ip  for the ith external pattern is:Then the probability Finally, the likelihood function is:and the log-likelihood function is:We have to find the MLEs Proposition 1: There are infinite sets of MLEs Proof: See Proposition 1 indicates that the number of introns at the root node, as well as the numbers of gains and losses along the two deepest branches, cannot be determined without additional information. Therefore, these values will not be reported in the N−2 sets of MLEs k = 3, 4, …, B).Proposition 2: There are 2Proof: See N−2 sets of MLEs k = 3, 4, …, B) as the one having the least sum of variances for intron gains and losses among all branches, i.e., having the smallestWe define the most biologically meaningful solution in these 2Numerical Recipes in C [An EM algorithm was proposed for calculating the MLEs of pes in C . Howeverpes in C . Our impij  given in is calculated as follows:E-Step: In our problem, the complete dataset comprises the number of cases for all complete intron patterns . The conditional expected number of cases for the complete intron pattern ijm is calculated based on Equation 3 using the current set of parameters where k of the phylogenetic tree by:M-Step: In this step we must find a new set of parameters that maximize the likelihood of the complete data conditioned by the observed data. First, we calculate the conditional expected numbers of gains and losses for each branch h is calculated by:Next, the conditional expected number of introns at each node Finally, the new parameters are calculated by the following formulas:oni1| is the conditional expected number of introns at the root node.where S/2P, so that the introns are present in half of the observed intron positions at the root node; then set the values of k = 1, 2, …, B). The two steps described above are then repeated until the difference −8 in our algorithm).First, we set the value of λ to The source code of the EM algorithm was written in C language and is available on request to the corresponding author.To implement the DS method, we used the C code in  with onl. In this GA, only one offspring solution is produced, either from two parental solutions by crossover, or from one parental solution by mutation in each generation. The offspring is immediately inserted into the population, and if it is fitter it replaces the worse parent. Linear ranking selection with a bias of 1.25 was used for selecting the parents. The population size was set to 400 individuals, which were divided equally into eight subpopulations. The mutation rate was set to 0.5.Our GA is based on a multi-population steady-state GA  and can Figure S1(11 KB PDF)Click here for additional data file.Figure S2(17 KB PDF)Click here for additional data file.Figure S3(9 KB PDF)Click here for additional data file.Protocol S1(80 KB PDF)Click here for additional data file.Protocol S2(90 KB PDF)Click here for additional data file.


Ever since the discovery of 'genes in pieces' and mRNA splicing in eukaryotes, origin and evolution of spliceosomal introns have been considered within the conceptual framework of the 'introns early' versus 'introns late' debate. The 'introns early' hypothesis, which is closely linked to the so-called exon theory of gene evolution, posits that protein-coding genes were interrupted by numerous introns even at the earliest stages of life's evolution and that introns played a major role in the origin of proteins by facilitating recombination of sequences coding for small protein/peptide modules. Under this scenario, the absence of spliceosomal introns in prokaryotes is considered to be a result of "genome streamlining". The 'introns late' hypothesis counters that spliceosomal introns emerged only in eukaryotes, and moreover, have been inserted into protein-coding genes continuously throughout the evolution of eukaryotes. Beyond the formal dilemma, the more substantial side of this debate has to do with possible roles of introns in the evolution of eukaryotes.I argue that several lines of evidence now suggest a coherent solution to the introns-early versus introns-late debate, and the emerging picture of intron evolution integrates aspects of both views although, formally, there seems to be no support for the original version of introns-early. Firstly, there is growing evidence that spliceosomal introns evolved from group II self-splicing introns which are present, usually, in small numbers, in many bacteria, and probably, moved into the evolving eukaryotic genome from the α-proteobacterial progenitor of the mitochondria. Secondly, the concept of a primordial pool of 'virus-like' genetic elements implies that self-splicing introns are among the most ancient genetic entities. Thirdly, reconstructions of the ancestral state of eukaryotic genes suggest that the last common ancestor of extant eukaryotes had an intron-rich genome. Thus, it appears that ancestors of spliceosomal introns, indeed, have existed since the earliest stages of life's evolution, in a formal agreement with the introns-early scenario. However, there is no evidence that these ancient introns ever became widespread before the emergence of eukaryotes, hence, the central tenet of introns-early, the role of introns in early evolution of proteins, has no support. However, the demonstration that numerous introns invaded eukaryotic genes at the outset of eukaryotic evolution and that subsequent intron gain has been limited in many eukaryotic lineages implicates introns as an ancestral feature of eukaryotic genomes and refutes radical versions of introns-late. Perhaps, most importantly, I argue that the intron invasion triggered other pivotal events of eukaryogenesis, including the emergence of the spliceosome, the nucleus, the linear chromosomes, the telomerase, and the ubiquitin signaling system. This concept of eukaryogenesis, in a sense, revives some tenets of the exon hypothesis, by assigning to introns crucial roles in eukaryotic evolutionary innovation.The scenario of the origin and evolution of introns that is best compatible with the results of comparative genomics and theoretical considerations goes as follows: self-splicing introns since the earliest stages of life's evolution – numerous spliceosomal introns invading genes of the emerging eukaryote during eukaryogenesis – subsequent lineage-specific loss and gain of introns. The intron invasion, probably, spawned by the mitochondrial endosymbiont, might have critically contributed to the emergence of the principal features of the eukaryotic cell. This scenario combines aspects of the introns-early and introns-late views.this article was reviewed by W. Ford Doolittle, James Darnell (nominated by W. Ford Doolittle), William Martin, and Anthony Poole. Reviewed by W. Ford Doolittle, James Darnell (nominated by W. Ford Doolittle), William Martin, and Anthony Poole. For the full reviews, please go to the Reviewers' Comments section.th century biology  a wise father that knows his own child.I think that from the history of science perspective, Koonin has the early days of Author response: Sadly, I was unaware of the American Naturalist review as well (not in PubMed). To keep the record straight, I am not citing it in the body of the article – here is the citation for the interested reader, in this review, from the author himself.I recall the theory's history. In his 1978 "Why genes-in-pieces" Wally Gilbert famously argued that the mosaic structure of eukaryotic genes allowed them to evolve by a subtler mode and faster tempo than could the genes of prokaryotes, through what came to be called exon shuffling. This was not yet his exon theory of genes, which was named by him in 1987 and incorporated much of his and others' subsequent thinking about the "RNA World" into a general view about gene origins. Indeed, "Why genes-in-pieces" did not address origins, or seek to explain the presence of spliceosomal introns in  eukaryotic nuclear genomes and their absence from (any) prokaryotic genomes. The prevailing notion at the time, however, was that eukaryotes emerged from within prokaryotes, through the coming together of several lineages  and that the eukaryotic nuclear genome is a complexified descendant of a prokaryotic one. Parsimony would thus have dictated that introns were introduced into once-intact prokaryotic genes at the time of this prokaryote-eukaryote transition.So here's how Why genes-in-pieces" addressed intron origins within the then reigning phylogenetic paradigm, it would have been most akin to introns late. But it didn't, and origin issues arose first in attempts to explain how and why (by what mechanism and in response to what selective forces) mosaic gene organization first came into being. After all, exon shuffling is only useful after many introns have been introduced. And even then the benefit is a long term one: evolution does not look ahead and the short-term cost would have been prohibitive. So their presence was an evolutionary mystery.That is to say, had "Why genes-in-pieces". A few of us had already been thinking along just those lines even before the discovery of introns. Jim Darnell, in particular, had already speculated that the bizarre and seemingly very wasteful things that eukaryotes do with mRNA bespeak retained primitivity. To him (and to me) the elegant efficiency of prokaryotic machinery for gene expression seemed the more evolutionarily refined, with the loss of introns being but part of this refinement. For me a crucial additional component in making this "eukaryotes early" notion seem reasonable was the articulation of the three domain concept by George Fox and Carl Woese, and their redrawing of the Tree of Life . In the first versions of this tree and until the late 1980s, the three domains emerge independently from a primitive ancestral state ("the progenote").A way around this was to imagine that eukaryotes had not emerged from within prokaryotes after all, and were instead a parallel line of descent anciently diverged from them at some inchoate stage of cellular and genomic evolution. Then we might with equal respect for parsimony see introns as a primitive genomic feature, since lost from prokaryotes (through "streamlining"), but retained in eukaryotes, where they could indeed confer the evolutionary advantages mapped out in "introns early was no more "a decidedly unparsimonious scenario" than was introns late. Indeed, if introns played the role in gene assembly first envisioned or later integrated into the RNA World hypothesis by Darnell and me (PNAS 83: 1271–1275) and by Gilbert in his "Exon Theory of Genes", their presence in the progenote and before may even have been a precondition for the rapid evolution of complex proteins. Really we know nothing about how genes arose, and to suppose that they sprang full blown and full length from noncoding polynucleotides seems to me more of a stretch than to imagine that they were cobbled together from smaller oligopeptide-encoding modules. Parsimony is often in the eye of the beholder, and its relevance in reconstructing evolution is in any case questionable. Surely we do not require of real historians that a primary criterion for constructing narratives about the human cultural or political past be that they invoke only the minimum possible number of historical forces and events! Yet what are we biological evolutionists but historians of Life?So the absence of introns in prokaryotes was not as Koonin asserts "a potentially embarrassing complication" needing to be "explained away" but rather an important element in the development of the theory! In the context of the unrooted three-domain tree, Author response: To get the history at least closer to the real picture, the introductory text was slightly modified to emphasize that the full-blown exon theory is a later development than the original introns-early. With regard to the more substantial aspect of this comment, I must agree that we know (next to) nothing about the emergence of proteins. Assembly from short peptides is one of the possible routes and  recombination between RNA molecules very well could have contributed to that. However, this is not the same as introns-early. Inasmuch as there is no evidence of an evolutionary relationship between such putative non-coding sequences and modern introns, this seems to be a different scenario. Origin of proteins is, generally, outside the scope of this paper (it is relevant only inasmuch as introns are implicated) but a clarifying comment on this issue was added to the Conclusions.On a more general note, the status of parsimony in reconstructions of the past and the comparison to the historians of the human civilization  seems to beg for some comment. I believe the role of parsimony depends on the depth of history one considers. Surely, it would be a little preposterous to claim that, in February of 1917, Lenin's Bolshevik party snatched the power in Russia directly from Nicholas II, on the grounds that considering any intermediate between the two systems would violate parsimony. That is because we know for a fact that, in February of 1917, the power was transferred to the pluralist Interim Government, and it was only in October of that year that the Bolsheviks took over, through an improbable combination of events. And yet, what guiding principle do "real historians" have when it comes to early stages of the history of civilization, where the only evidence available is archaeological ? I believe one has no choice other than to rely on parsimony or, put another way, Occam razor. The same applies to the reconstructions of biological evolution, so I do not really agree that the role of parsimony is questionable. It is, indeed, another and, I think, more difficult matter that parsimony is "in the eye of the beholder", i. e., we often effectively apply weighted parsimony, with the weight of different characters determined either on pure intuition or with some rationale. Herein seem to lie many problems in deep reconstructions, and much caution is needed.introns early – for me anyway – was nevertheless a powerful parsimony argument, based on a revision of the phylogenetic consensus. By the late 1980s, we had come generally to believe in a rooted three-domain tree, with archaea and eukaryotes sisters, and bacteria more deeply diverging. So now we introns early advocates would have to imagine two independent episodes of intron loss through streamlining (one in the line leading to Bacteria and the other on the line to Archaea) rather than the arguably more parsimonious single gain early in eukaryote evolution imagined for introns late. More importantly, by then we'd come to believe that the (seemingly) most deeply diverging eukaryote lineages  lacked both mitochondria and introns. Independent loss episodes would have to be imagined for each of these several eukaryotic lineages in order to hold on to introns early. John Logsdon and Jeff Palmer were the first to make us (or me anyway) aware of the serious unparsimoniuosness of this scenario. And if, as then seemed likely, eukaryotes with mitochondria had introns and those which had never acquired such organelles didn't, why not suppose that the latter were introduced along with the former, as Tom Cavalier-Smith did, in 1991. Introns late acquired a vector for the introduction of introns, and a specific scenario for their origins as genetic entities, from Group II introns in bacteria.What tipped the balance against introns early has shifted again, returning to the neutral position! All eukaryotes likely have introns and likely all have, or once had, mitochondria. So Koonin argues that the door is open again for something like introns early. However, it does matter what we take that term to mean. Rather early on in the introns early – introns late debate, it became clear to most protagonists that the notion that all introns are primoridial relics, with a history exclusively of loss, would not wash. Lining up all the positions at which introns are known in at least one of all the available orthologs of a given gene forces the conclusion that ancestral exons were far shorter than needed to code for any credible protein structural module. For me, the "weaker" or "revised" version of introns early, the one under dispute for most of the two decades before the turn of the millennium, and in my judgement the only legitimate child of the 1978 version of the theory, was this: the first genes were assembled from smaller protein-coding modules, introns marked the positions at which such modules were joined, and at least some introns (or intron positions) in some modern eukaryotic nuclear genes are direct relics of that assembly process. So even if LECA's genes were chockablock full of Group II introns or their degenerate spliceosome-dependent products, as long as we see these introns as having been "introduced into" the nuclear genome , we're dealing with an introns late scenario.But, since the turn of the millennium, the balance of phylogenetic arguments for or against But do we absolutely have to see it that way? We believe that eukaryotes and archaea are sisters because we believe that the genes that produce this sisterhood relationship, which are mostly those of translation and other components of the information processing machinery, tell the true story of eukaryote origins. And we see the non-archaeal genes in eukaryotic genomes as having been "introduced into" the eukaryotic cellular lineage because we still see the pre-mitochondrial  cellular lineage, from which many of these genes likely derive, as having been introduced (as an "endosymbiont") into some post-archaeal host cellular lineage. Or most of us see it that way, anyway.at al.  that suggest that the majority of yeast nuclear genes are of bacterial ancestry, then surely we must seriously entertain their ironic conclusion that "yeast shares a sister-group relationship with eubacteria, not with archaebacteria".But eukaryotic origin scenarios in which the complexity of eukaryotic cells and their ability to engulf other cells arise only after the tight integration of the bacterial and archael parental lineages are increasingly popular . In these scenarios, designation of one lineage as host and the other as symbiont (and the notion that one set of genes was "introduced into" the other), seems pretty arbitrary. Indeed, if we chose to go with the majority of genes and accept the analyses of Esser de novo events of transposition of such Group II introns. Put another way, there is no reason not to assume that some of the Group II introns in genes that are part of the bacterial heritage were simply converted, in situ, without moving so much as a nucleotide to left or right, into the pitiful degenerates we now call spliceosomal introns. Such a scenario is, mirabile dictu, perilously close to introns early in what Koonin calls "its modified form" but considers untenable, favoring "a massive invasion of Group II introns escaping from the mitochondrial endosymbiont into the host".The bacterial lineage and many its genes presumably came already modestly freighted with introns, albeit Group II introns. There is no reason to assume that all spliceosomal introns that now dot the eukaryotic nuclear genomic landscape landed there as the result of as introns early, unless we in addition suppose that some individual bacterial Group II introns that have survived as spliceosomal derivatives in eukaryotic nuclear genes were themselves relics of primitive stages of gene assembly. This may seem unlikely, given that in bacteria Group II introns appear to avoid the interior regions of protein coding genes. But who knows? Maybe bacterial genomes were once riddled with relict introns, some indeed primordial leftovers. Maybe these were removed one at a time as translation got increasingly fast and waiting for splicing increasingly a burden, and remain mostly where interference with translation is not a big issue. The imposition of the nuclear membrane, far from being a defense against invading introns, might have allowed the relatively few such parasites that were part of the bacterial heritage to run amuck. So it would not be that the nuclear compartmentalization arose as a barrier to the "attack of bacterial retroelements" but rather that it arose for some other reason, and permitted the subsequent proliferation of introns.But this scenario is indeed not the same introns early theory is judged to be parsimonious (and thus more or less believable) depends on what one takes to be the relative likelihoods of the inferred genetic processes (streamlining versus massive intron infestation) and the accepted phylogenetic framework (the Tree of Life) against which its parsimoniousness should be evaluated. The former is unknowable and the latter has varied radically over the life of the theory, and still varies radically between individual theorists in the field.None of us can present his or her evolutionary hypotheses without embedding them in a story of the relevant thinking that has gone before, else we would be plagiarists . But each of us has a different take on that past, and each of us recounts it so as to make his/her own contribution seem to be the product of inexorable logic – not just for rhetorical purpose but because this is how we come to understand it, ourselves. Koonin's arguments about the biology are interwoven with his recounting of the history of thought in the area, in service of his goal to determine whether a 38 year old theory is still worthy of consideration because it most parsimoniously explains the present data. Whether or not the Koonin's evolutionary narrative, as I understand it, is this:introns early and later elaborated in the "exon theory of genes".• Genes were probably first assembled from pieces, as envisioned in the early versions of • Group II introns, as a class of transposable selfish elements, may well date from this time of gene assembly. They continue to infest bacterial genomes, albeit at modest level, and the (endosymbiotic) bacterial genome that became the mitochondrial genome was so infested.• The host  genomic lineage was (for some reason) devoid of such elements, which were introduced into it by transposition from this (endosymbiotic) bacterial genome. The defenseless host genome became the scene of a transpositional orgy.• Compartmentalization of the nuclear genome and several other aspects of eukaryotic cell biology arose as defenses against this "intron catastrophe".Author response: This is an important point, and clarification is due. The first statement among these four is not an integral part of my narrative. I think that the issue of the origin of first proteins is wide open (see above) and recombination between RNA molecules is one of the likely contributed mechanisms . However, this is not where I see the link to – and partial vindication of – introns-early. This connection, mostly, stems from points (ii) – the evolutionary antiquity of the (precursors of) introns and (iV) – the crucial role of introns in eukaryogenesis.introns early and introns late views" which it does, and that there may be "no losers in the introns early vs. introns late debate as both views capture important aspects of the evolutionary reality." I'm not sure, however, that "aspects" are enough to legitimize Koonin's version of introns early as the natural child of the 1978 theory. In my opinion that theory, even in its weak form, requires that "at least some introns (or intron positions) in some modern eukaryotic nuclear genes are direct relics" of primordial gene assembly process.  That so many introns have been added since, from whatever source and by whatever mechanism, may mean that the signal is too small to be detected and the theory thus formulated is unfalsifiable. This doesn't mean that it is false, or even useless, although it may no longer be a scientific theory. There are many interesting ideas out there about how life itself first arose. Few of them  are falsifiable, but one or more of them may have elements of truth, and we do know that life did, somehow, arise. And thinking about such theories stimulates the collection of much data and the generation of many new ideas, as have introns early and introns late, in their 38-year struggle for the hearts and minds of molecular evolutionists.He claims that this scenario "combines aspects of Author response: Parts of the manuscript, including the title (as per Bill Martin's suggestion), have been modified to de-emphasize the claim that the debate ended in a "draw". I really do not insist that the current view of the evolutionary history of introns articulated in the present paper is a true, legitimate child of the original introns-early. I only would like to maintain that there is a connection, i.e., some heritage of that view factors in the currently emerging picture. If the latter is a true child of introns-late, but just a nephew of introns-early, that's fine with me.James E. Darnell, Laboratory of Molecular Cell Biology, The Rockefeller University, New York 10021, USA (nominated by W. Ford Doolittle)3believe2 (too strong and irrelevant a word) that eukaryotes arose carrying with them somehow the ability to get along with fragmented nucleic acid , an ability with which the whole pre-cellular pool must have been endowed and which has been maintained in all multi-cellular eukaryotes and by inference the one (or more?) single-celled precursors to multi-cellular eukaryotes. Surely small bits of information predated large chunks of information and somehow tacking together sufficiently large pieces of information to make usable peptides was an early (? primary) task  that 'could serve as a genetic melting pot.' . Lacking more insight into this stage leaves me unwilling to swallow a primitive self-sustaining eukaryote devoid of a mechanism for recruiting useful information from disjointed stretches that then swallowed and used non-nucleated cells as plastids and gene donors. The latter swallowing surely occurred but by whom? I realize that this position is not particularly helpful since it generates few (? no) experiments/measurements/observations, but to discuss eukaryotic origins as obligatorily arising from either bacteria or archaea or both seems to me wrong-headed. Eugene Koonin's paper is reasonable, written well and highly informative on many up-to-date details. But its diagrammatic simplicity embracing the 'later' dependent origin of eukaryotes doesn't convince me that a reasonable proposal is a right proposal.I think speculation on the origin of eukaryotic cells that does not go back to the pre-cellular phase of evolution unlikely to unlock any secrets and at worst to be vacuous. Therefore I warm to Koonin's last section, "Self-splicing introns as heritage of the primordial gene pool." To quote from Carl Woese, "Next comes the evolution of the eukaryotic cell itself. While biologists have traditionally seen this as a step  beyond the stage of bacterial cells, I do not."  He then goes on to develop the vast difference in cell structure and organization . His objection to eukaryotic cell development AFTER non-nucleated cell development might be thought to dissolve with the discovery of proteins in bacteria that have the same three-dimensional structure as tubulin and actin . However that does not undercut the more general argument for pushing eukaryotic origins back to a very early stage of cell evolution. What all this suggests to me is that essentially all of the protein folds existed by the time sustainable 'life' as we know it arose. If this is the case, then the platform from which eukaryotes arose could be the same as that from which the other two kingdoms arose. And then the question is what the pools looked like from which the extant genomes were constructed. What strikes me now  is the unlikelihood of already highly developed, single, non-nucleated cells whose aim in life is to grow and divide being the entities from which eukaryotes derived. It seems much more plausible to me to back up and admit that pre-cellular 'life' had passed the Rubicon of coded protein (peptide) synthesis and probably elementary nucleic acid duplication (even DNA synthesis). In this gemisch the quite different polymer synthesis machines, archaeal and bacterial, must also BOTH have existed before functioning cells. And if this pre-cellular stage provided the platform from which the two (and only two?) non-nucleated cell types arose, multiplied and remained distinct (ignoring LGT), it is from this same pre-cellular but elaborate stage that I Author response: I appreciate the thoughtful comments from one of the founding fathers of our current understanding of eukaryogenesis. I think there are, actually, many points of agreement with respect to the evolution of many complex features of life, including the two distinct DNA replication machineries  within a precellular genetic pool . What we seem to disagree on is the early (from within the same pool) vs late  origin of (pro)eukaryotes. For sure, a reasonable proposal is not necessarily a right proposal. But should we not try to stick to a reasonable proposal and explore its implications until it is shown to be false?James E. Darnell, Laboratory of Molecular Cell Biology, The Rockefeller University, New York 10021, USA. (nominated by W. Ford Doolittle)You have reason on your side.William Martin, Institute of Botany III, University of Dusseldorf, D-40225 Dusseldorf, Germany20, 1–38). Too bad that there is not a 2006 SGM symposium volume, this would have fit nicely.This is an interesting and informative paper, although I don't think that the current title is very good. Introns early and introns late are irreconciliable views, they can't both be right so it is not really a draw. And if they are both wrong it is not a draw either. The paper is basically an essay on "some aspects of eukaryotic introns and their possible evolutionary significance" and a title along those lines might better advertise its content. That title would paraphrase one of Ford Doolittle's more famous titles , which was intentionally identical to Stanier's famous 1970 SGM title  aspects of the present paper. For the reader's understanding: the original title was 'Introns-early versus introns-late: is it a draw?' I agree that this is not a "real" draw, and if we formulate the debate as an 'either/or' question, we have to accept that introns-late has won. However, the version of introns-late that is currently taking shape has very strong reverberations with introns-early, hence the original title. The same thought is expressed in the revised title in a more cautious manner. Several changes to the wording in the Abstract also have been made, to go along with the new title. Having changed the title, we could decide to dispense with all this discussion but I think it might be of certain interest to the reader and worth keeping in the published record.better understood (under these premises) as defense responses in the broad sense against invading mobile elements . Ideas that aid our understanding need not require that the ideas are correct (how can we prove anything about early evolution anyway?). But if they help us to structure the problem (and the prokaryote-to-eukaryote transition needs considerable restructuring with the demise of archezoa) by linking phenomena that appear otherwise unrelated in a temporal and mechanistic manner that is tangible, then that constitutes progress. This paper fills that bill.As its main thrust (from my viewpoint), the text extends and further develops the idea in ref. 59 that introns may have precipitated the origin of the eukaryotic nucleus (as a consequence of the origin of mitochondria) and NMD by the compatible notion that introns might have precipitated ubiquitinylation as well. This suggests that several otherwise puzzling eukaryotic novelties can be Author response: I appreciate this comment and, by and large, agree. This is what I was trying to do, basically, to advance, even if just a little bit, our understanding of the wondrous series of events associated with eukaryogenesis. A full-scale philosophical discussion certainly is out of place here but it is tempting to throw in a brief comment. I agree that it is impossible to "prove" anything about early evolution but, then, this applies to most general statements about the physical world, even those that have nothing to do with remote past. Typically, such statements are accepted as "correct" inasmuch as they i) are compatible with a large body of diverse empirical data and ii) explain diverse aspects of the world in an economical and plausible manner . With regard to early evolution, it is, most of the time, particularly hard to properly satisfy criterion (i) although we certainly must keep trying. Accordingly, criterion (ii) is becoming particularly important.At the same time, the paper looks further back in evolution towards the origin of genes and aims to reconcile aspects of introns-early with aspects of introns-late. The important evidence for intron stasis in animals indicates that one aspect of introns late in some of its original formulation is wrong: introns are apparently not continuously mobile over time. There was a time when they were actively mobile, but that time has apparently past. I still find it interesting that, as far as I know, nobody has ever observed the same intron in two places in genomes, that is, recent spliceosomal intron transposition does not seem to occur at an observable rate. Introns late was saying that it should be ongoing today. One point for introns late, but probably for the wrong reason. One could probably calculate the maximum rate at which introns with significant sequence similarity move, that would be interesting.Author response: Generally, true: unlike intron loss that is, more or less, clock-like, intron gain does not at all seem to occur uniformly in time, which is bad news for a strong version of introns-late. This being said, there are indications of recent intron gain in some lineages, and the reference to Coghlan and Wolfe (Ref. 53) on intron gain in nematodes was added in support of this point.If one accepts the view of an RNA world, then recombination is splicing, so the idea that splicing was around very early fits well. The idea that spliceosomal introns are in situ holdovers from that phase of gene invention (the exon theory of genes) is part of the eukaryotes early (or thermoreduction) idea still today, but beyond the New Zealand and French sections, I don't know that very many folks are card-carrying members in that club anymore. Ref. 98 distinguishes introns first from the exon theory, that could be debated because except for snoRNAs they are saying the same thing. Ref 98 has "intron gain" in the title, suggesting that there is some evidence for recent intron gain, I wonder what that evidence is, in detail.Author response: I tend to agree that the difference between 'introns first' and the good, old 'introns early' is minor, so to avoid confusion, I do not really discuss 'introns first' as a separate concept. As for evidence of gain, even apart from the Coghlan-Wolfe observations on the recent gains in nematodes, I believe reconstructions provide enough evidence in support of intron gain, at least at some stages of eukaryotic evolution. Granted, this is not the same as a "smoking gun", which would be a pair of highly similar introns in unrelated genes indicating a recent gain . However, if intron gain was, indeed, episodic in eukaryotic in eukaryotes as suggested in this paper and previously elsewhere (ref. 50), such unequivocal cases of gain are likely to be very few and far between, and this seems to be so. Complete analysis of this issue, obviously, is beyond the scope of the present paper. Very hopefully, elsewhere.The history aspects of the introns early vs late debate are more easily dealt with accurately by someone who was in the thick of it. I'm not convinced that the whole introns debate is needed to understand the novel aspects of this paper, getting to the point more quickly might keep readers on track more effectively. Introns early vs introns late is over and I would not recommend warming back up. The present title as a subtitle of a more informative title might get the early introns players back into the arena again, though, which would be interesting. But this paper is suggesting that they would need to be debating other ideas, which is unlikely to occur.Author response: Maybe it would be easier and faster to cut to the chase right away and only discuss the current ideas on the role of introns in eukaryotic evolution without reheating the old debate...but I thought revisiting it was interesting. Ford Doolittle's review comes from the very thick of it and corrects my historical aberrations where necessary.nd paragraph: the exon theory of genes came much laterBackground, 2Author response: Technically, yes, but the main ideas were there, I think. In any case, modified to reflect history more accurately.th paragraph: exon theory of genes, but one that operates in eukaryotes only.Results and Discussion, section "Multiple, pivotal roles of ancient introns in eukaryogenesis...". 4Author response: Yes, this is the gist of it, more or less, as noted in the text.William Martin, Institute of Botany III, University of Dusseldorf, D-40225 Dusseldorf, GermanyThe replies to all comments, in particular the comments of referees 1, 2, and 4, add quite a lot to this interesting paper that was missing in the first version. This document now contains the best and most accurate summary of whence introns early that there is, but as a published referee report, that's something new for the literature. I wonder how to cite it.is RNA recombination, introns were probably involved in the primordial assembly of genes. But (NB: none of) the introns of eukaryotes were present at any of their current positions as those first genes arose, because spliceosomal introns are a secondary invention of the eukaryotic lineage via degeneration of group II introns that were acquired via the mitochondrion. This is not what either introns early or introns late was saying, as far as I can recall. But that debate structured the problem in a way that gave us concepts to sort the current observations from genomes into a reasonable temporal sequence of imaginable events. So I still think that the compromise offered by the title is not delivered. The present view is not introns early or the exon theory of genes, it is more the intron theory of nuclear origins. Any way one cuts the cake, introns still seem to carry burgeoning evolutionary significance; that comes closer to what introns early was saying than to what introns late was saying.What is the consensus of what has been said here? I think it goes like this: Because RNA recombination was probably involved in the origin of genes, and because splicing Proc Natl Acad Sci USA 95:6239–6244, 1998; Nature 431:152–155, 2004), Dennis Searcy , and others . The significance of this concept is substantial because it implies that there was no lineage of eukaryotic-type gene or cell organization that goes all the way back to the origin of life (an assumption implicit in most formulations of introns early). The view of an archaebacterial host suggests that eukaryotes arose directly from fully-fledged and free-living prokaryotes, not directly from the same collection of organic molecules that gave rise to prokaryotes. It is thus mutually exclusive with regard to the progenote concept, which is closely related to introns early, but is compatible with some  symbiogenic views of eukaryogenesis. An historian of science specialized in the area of endosymbiosis, Jan Sapp, has gotten this aspect of the modern history of endosymbiosis and early cell evolution wrong on several occasions, so it was nice to see it spelled out more clearly by the authors and referees of this contribution. In 1981, Woese published an interesting paper in the June issue of Scientific American (p 98ff) that put his progenote view of eukaryote origins against Margulis's view of eukaryote origins head-to-head in figures. The progenote view was the view held by introns early, I suppose, or introns early might have even been an edifice of the progenote concept, who knows. Twenty five years later, neither the Woese camp nor the Margulis camp has indicated any willingness to find the kind compromise that Koonin has suggested here, as inspection of their more recent papers will attest. Maybe refugees from those two camps can someday find a popular solution – not a compromise! – that squares off well with the observations from genomes.The view expressed here that the host for the origin of mitochondria was a garden variety archaebacterium  is a significant part of the present synthesis. This is what some of us have been saying for quite a while, the idea can be found in Ford Doolittle's 1996 SGM paper and in papers by Jim Lake Nature 440:41).This article comes in two parts; the first provides a thorough and up-to-the-minute review of the existing literature on the introns-early/late debate, is easy to follow and refreshingly even-handed for a field where one is too often subjected to vehemently partisan advocacy. This part of Koonin's article is well written and, while there's always something to disagree with in this field, I have no particular axe to grind. The second half of the article offers some new material, extending ideas Koonin developed with Martin  brief and I do not address all the points, just those I felt were the most important ones, the reason is my wish to avoid the specter of infinite regression that, indeed, plagues some philosophy journals.There is plenty in the second part of the paper to take issue with. In an attempt to avoid the phenomenon in philosophy journals where nit-picky rebuttals sometimes run longer than the original argument, I will confine myself to two main ones. The first is that, to buy any of this, one has to accept two main assumptions, both of which one can take issue with. If these do not hold, the entire argument comes crashing down. The second is that I do not think a good argument can be made that introns proliferated in the host that Koonin invokes in his model.Curr Opin Genet Dev 8:637). The assumption of common ancestry is not really debated, since both introns-early and introns-late proponents accept this as given, albeit in different forms. In the former case, it has been argued that group II introns are relics of the RNA world  and interrupted RNA genes, and that excision of the group II intron was required for production of functional RNAs. Group II introns would thus be early parasites, and, contrary to the historical situation Koonin alludes to where introns-early and the exon theory of genes were one and the same, this is not so in the revised introns-early scenario. However, in this scenario a satisfactory explanation for how group II introns could have evolved into a trans-splicing system comprising 5 snRNAs is not given, though, aside from an issue of timing, the problem is one and the same as for introns-late: the origin of the spliceosome.So, what are the assumptions we must accept? The first is that group II introns are related to the spliceosomal snRNAs and spliceosomal introns, and that the evolution of the these from group II introns is established, as per the mitochondrial seed hypothesis (Logsdon 1998 Chlamydomonas reinhardtii), and this three-piece self-splicing intron does at least provide a plausible intermediate in an organelle of bacterial endosymbiotic origin (see Stoltzfus 1999 J Mol Evol 49:169 for a model). The importance of this observation in the context of the mitochondrial seed hypothesis cannot be denied; other RNA genes in pieces are found in mitochondria , even if no split group II introns have thus far been found in this type of organelle.Mechanistically, support for introns-late rests on the observation that group II introns-in-pieces are found (admittedly in a chloroplast genome – that of Cell 72:161 for a critique). More significant is the demonstration that U5 snRNA can substitute for the ID3 domain of a group II intron . Another piece of evidence comes from recent structural comparisons of the D5 domain of a group II intron and U6 snRNA , though finding similarities between two hairpins, while suggestive, is unfortunately not strong evidence for a common origin.The evidence for a common origin for snRNAs and group II introns is, by necessity, circumstantial; a common origin is invoked on the basis of the chemistry of reaction, which is in itself a weak argument (see Weiner 1993 Mol Biol Evol 22:1053), whereas the exon theory of genes was never a good explanation for the origin of introns. It was quickly realised that this suffered from evolutionary foresight; introns would have had to have evolved in order to promote shuffling of domains, thereby producing new protein variants that would, possibly, be useful at some future point . That the exon theory failed to explain the origins of introns does not however mean that introns must by implication be late however .Overall, Koonin is being fair to the literature in so far as these similarities have all been used to argue for a group II intron origin for the spliceosome, and, as noted above, group II introns-in-pieces might well be expected to emerge in the mitochondrion. There is no question in my mind that, compared to models offered by the introns-early camp, the mitochondrial seed hypothesis is the better-developed hypothesis. It can potentially explain the stepwise emergence of a trans-splicing system comprised of several RNAs from a single cis-splicing element  scenario under which such a stage was a transient one triggered by endosymbiosis. I believe, though, that the latter version has distinct advantages in terms of compatibility with the available data and explanatory power. Firstly, organisms with numerous Group II introns in protein-coding genes  are not known , so it is, at least, less of a stretch to postulate that condition as a transient one. Secondly, I believe there is a very good reason why such an organism has never been discovered: it never existed because having those multiple (even if self-splicing) introns under the transcriptional-translational coupling mode of expression would be too much of a disadvantage. If so, one would think that such an organism would already have transcription and translation uncoupled. Again, we are unaware of such organisms – other than eukaryotes, of course. I think the hypothesis that we proposed with Bill Martin (ref. 59) and that I expand somewhat in the present paper offers a plausible chain of causation to tie it all together.Debate over the timing of the evolution of introns, and the relative contributions of intron gain and loss, is still raging, and, on balance, Koonin is perfectly entitled to assume the mitochondrial seed hypothesis as a starting point for discussions on the origin of spliceosomal introns, even if not everyone would agree. It is the second assumption with which I take issue.The second assumption, again, is a case of assuming we know the ancestral state, and in this case concerns the nature of the endosymbiosis that gave rise to the eukaryote cell. This is the more difficult assumption to defend, and also that which is most important with regard to the novel ideas outlined in this paper.Koonin's assumption is that the host of the endosymbiont that evolved into the mitochondrion is, as he puts it, a 'garden variety archaeon'. This is a commonly held assumption, but fails on closer inspection. I will not develop a full argument here, as I have recently submitted an article elsewhere on this , but here are two important issues. First, if archaea had already become a distinct domain prior to the origin of eukaryotes, eukaryote nuclear genes of archaeal origin should group specifically within the diversity of modern archaea, in exactly the same way as genes of mitochondrial origin fall within the diversity of modern bacteria, showing a specific relationship to alpha-proteobacteria. We see this clearly for mitochondrial-origin genes, but this has not been shown to be the case for supposedly archaeal-origin genes. That many archaeal genes are similar to eukaryotic genes supports a common origin for the two domains, but does not demonstrate that eukaryotes evolved from archaea. For this to be supported requires an explicit phylogenetic affinity between eukaryote genes and orthologous genes from a specific group of archaea, for instance methanogens.Author response: I maintain that the notion of an archaeal host for the mitochondrial endosymbiont remains the null hypothesis that we cannot reject. The argument that the 'archaeal' genes of eukaryotes should cluster within a specific branch of archaea is straightforward and substantial at first glance but fails upon closer consideration. Firstly, even with the mitochondria, the α-proteobacterial origin is demonstrable by phylogenetic trees for a frustratingly small number of genes [see Esser at al. ]. It is, mostly, because some of these genes remained in the mitochondrial genome that we have no doubts as to the origin of the mitochondria. The genes that moved to the nucleus, in all likelihood, have experienced a substantial acceleration of evolution which complicates phylogenetic analysis. The same acceleration, probably, affected the host, archaeal proteins, hampering assignment to a specific group of archaea. On top of that, just as is the case with the mitochondria, we do not know the actual gene set of either the host of the endosymbiont, and given the amount of HGT in prokaryotes, this is far from being a moot issue. Some of the 'archaeal' proteins of eukaryotes do show phylogenetic affinity with a specific archaeal lineage, it is just that the signals are somewhat conflicting. Furthermore, the host did not even have to belong to one of the presently characterized groups of archaea. It very well could have been an archaeal branch that was outside the tree we are aware of and has either gone extinct or is still lurking somewhere . It still would be an archaeon even if the word 'garden-variety' might be risky in such a case. In summary, the nature of the relationship between eukaryotes and archaea deserves further, careful investigation but at present I cannot see how we can reject the straightforward hypothesis of an archaeal host for the mitochondrial endosymbiont.IUBMB Life 57:539), and modern examples of endosymbioses involving eukaryotes are widespread. While there is one example of a bacterium within a bacterium within a eukaryote  this is not equivalent to a bacterial-archaeal endosymbiosis. Consequently, the entire thesis rests on the unproven ability for archaea to be capable of hosting endosymbionts.The second criticism is that there are no known cases modern archaea housing bacterial endosymbionts. All endosymbioses that have generated organelles  subsequent to the mitochondrion clearly involve eukaryotes . This is not unlike the ultimate origin of cells: an enormously complex evolutionary transition for which we may not have a convincing scenario but we would not question the origin of cells from "some" kind of precellular (pre)life form because...what would be the alternative?With this point in mind, I turn now to the speculative part of Koonin's paper, in which he argues that group II introns entered an archaeon via the alpha-proteobacterial endosymbiont. The consequence is an explosion in intron-numbers, compensatory evolution of a number of mechanisms of transcript quality control , nonsense-mediated decay (NMD), ubiquitinylation), and side-effects such as the evolution of linear chromosomes with telomeres and telomerase. The major point that needs to be discussed here is the expansion of group II introns, but before I come to that, I will deal with several minor points.Genome Biol 4:R81), and extends this speculation by suggesting a mitochondrial origin for this toxin-antitoxin system. The original paper demonstrates similarity in so far as a universal domain, the PIN-domain, is found in both proteins involved in NMD. However, Koonin's hypothesis is phylogenetically testable – NMD genes which are homologous to bacterial genes should group with alpha-proteobacterial examples of these genes in gene trees. One can envisage complications, in that toxin-antitoxin systems are horizontally transferable, but without phylogenetic analysis the position Koonin takes cannot develop past the speculative stage. Again with ubiquitin, similarity to bacterial counterparts alone cannot establish the direction of evolution – more specific tests must be made if these suggestive links are to form the basis for hypotheses for the origin of key eukaryotic processes from bacterial or archaeal 'progenitors'. I am open to these possibilities, but feel it is insufficient to determine the direction of evolution based on similarity; this smacks of the Great Chain of Being, and we should be able to do better. One might equally argue that any such similarities are due to reductive evolution in bacteria and archaea (as I pointed out for group II introns above), but without more specific tests, the two opposing hypotheses are at loggerheads. We may not always be able to resolve the precise evolutionary history, but we can at least formulate alternative hypotheses and attempt formal tests.Koonin cites a paper wherein it was suggested that eukaryotic NMD evolved from a bacterial toxin-antitoxin system . This is a key point, and one that the reader must accept in order to buy into any of the ensuing speculation. The argument builds upon the assertion that the small effective population size of this new 'prekaryotic chimera' precluded elimination of invading group II mobile elements by purifying selection. The host in this case is an archaeon  with a small population size, but I am not sure we have any good examples to compare the model to.Methanosarcina acetovorans &M. mazei , and seem to have become established as the result of horizontal gene transfer from bacteria . Here it seems that none of the group II introns are inserted in archaeal open-reading frames, and have a tendency to insert into the reverse transcriptase genes encoded by other group II introns, generating nested introns. I have no idea as to the effective population size of these two archaea, but that these elements have neither gone berserk, nor inserted into archaeal protein-coding genes does not serve to strengthen the model presented by Koonin.Group II introns have recently been found in the archaea Author response: The case of Methanosarcina is, indeed, quite interesting. Surely, their genomes are full of acquired bacterial genes, and there are some Group II introns as well but all this has not made them eukaryotes. On the one hand, that does not strengthen the model I discuss in this paper...but, I do not think this weakens it either. What this case does confirm, is that eukaryogenesis is no small feat (see above) – in a way, one might be tempted to think of Methanosarcina as a failed eukaryote although we have no idea whether or not some sort of transient endosymbiosis was involved.Buchnera are more clearly analogous to the endosymbiont, not the host, and here it seems that explosive expansion of existing selfish elements is not a feature of these lineages; the opposite is true. Population size could well play a role, and is in principle testable, but to my knowledge the only examples of massive expansion of selfish elements are in organisms with meiotic sex. Here it has been argued that selfish elements expand at the expense of the host because outcrossing permits spread, provided that the cost to the host is less than 0.5 (Hickey 1982 Genetics 101:519). Species with smaller effective population sizes do seem to have a higher density of elements (e.g. complex multicellular organisms), but these are all sexual. Conversely, in asexual systems, the theoretical expectation is that selfish elements should be rapidly eliminated, because they impart a selective disadvantage on the host, meaning that element-free lineages will outcompete those with elements .Examples of asexual lineages with small population size, such as Under Koonin's scenario, mitochondrial group II introns inserted into the host genome sweep to fixation in the population via drift, and subsequently expand in numbers. It is of course not impossible for drift to sweep a selfish element to fixation, even under Hickey's model. However, should these then expand significantly in number over a longer evolutionary period? One key point that emerges from Hickey's original treatment of this problem is that there should be attenuation of selfish element activity because in an asexually reproducing lineage the reduction in fitness imparted on the host by a given element is also the fitness reduction on the element. There is therefore selection on the element to become less virulent. This is exactly the opposite of what Koonin's model (and indeed Martin and Koonin's model) requires.Bioessays 27:76). So, in my mind, for Koonin's 'berserk introns' theory to work over a long enough time scale for several complex systems of quality control to emerge, I would argue that he should at least have invoked the emergence of facultative meiotic sex. The problem with this is that sex, with its two-fold reproductive cost, must be invoked under a scenario where there is a population of primed selfish elements 'waiting' to spread. While the level of sexuality can be increased in a facultatively sexual population, this is not so for an asexual population (Johnson & Brookfield op. cit.). As the ever-present difficulty with models for the origin of sex is accounting for the short-term selective advantage for sex, this would represent a rather backwards way of approaching the problem!It has recently been argued that element overload is one probable cause of extinction of obligately asexual lineages that have evolved from sexual lineages (Arkhipova & Meselson 2005 Curr Biol 15:185) for recent discussion on the timing of the evolution of meiosis). One has to accept the necessity of stem-lineage eukaryote ancestors irrespective of the absence of extant primitively amitochondriate eukaryotes.  This is because, even if, as Koonin argues, the ancestors of eukaryotes were archaea bearing alpha-proteobacterial endosymbionts, there are no intermediate stages between this hypothetical ancestor and modern eukaryotes . By invoking a sexual, eukaryotic host, the spread of introns in eukaryotes can better be accounted for under our current understanding of selfish element spread in sexual populations.A further point worth considering is that Koonin's archaeon model weakens the introns-late stance because it eliminates the possibility of the mitochondrion entering an early eukaryote stem lineage that had already evolved meiosis , which I extend a little here, goes some way to propose a plausible chain of causation for eukaryogenesis  without invoking mysterious, extinct creatures. This does not mean such creatures have never existed, just that, following Occam razor, we probably should try to explain eukaryogenesis as best we can without their help.Methanosarcina have not suffered from massive intron expansion, seems to confirm this suspicion.In summary, I am not convinced that the host in the endosymbiosis leading to modern eukaryotes was an archaeon. This should be detectable phylogenetically, picking out a specific group of archaea as the closest relatives of eukaryotes in exactly the same way as this is possible for the mitochondrion. Second, no archaea have been identified which carry endosymbiont bacteria, so accepting Koonin's assumption would require that all extant archaea have subsequently lost this capacity. Nor am I convinced that the transfer of group II introns into an archaeal host from its bacterial endosymbiont would have led to massive expansion of group II elements. This does not seem to fit with our current knowledge of selfish element spread under an asexual reproductive mode. That modern methanogenic archaea of the genus