The Paramecium Germline Genome Provides a Niche for Intragenic Parasitic DNA: Evolutionary Dynamics of Internal Eliminated Sequences
[['Paramecium belongs to the ciliate phylum , a deep radiation of highly diverse unicellular eukaryotes .', 'The hallmark of ciliates is nuclear dimorphism: each unicellular organism harbors two kinds of nuclei with distinct organization and function .', 'A diploid “germline” micronucleus ( MIC ) undergoes meiosis and transmits the genetic information to the next sexual generation but is not expressed .', 'A polyploid “somatic” macronucleus ( MAC ) contains a version of the genome streamlined for gene expression and determines the phenotype .', 'A new MAC is formed at each sexual generation by programmed rearrangements of the entire zygotic , germline-derived genome , and the maternal MAC is lost .', 'The MAC genome of P . tetraurelia has been sequenced [1] revealing a series of whole genome duplications ( WGDs ) in the lineage that provide a unique tool for evolutionary analyses .', 'Ciliate genome rearrangements and their epigenetic control by non-coding RNAs have been recently reviewed [2]–[4] .', 'In Paramecium , genome rearrangements involve', '( i ) endoreplication of the DNA to about 800 haploid copies ,', '( ii ) imprecise elimination of genomic regions that contain , in particular , transposons and other repeated sequences , usually leading to chromosome fragmentation and', '( iii ) elimination of Internal Eliminated Sequences ( IES ) by a precise mechanism .', 'The accuracy of this process is crucial for IESs located within coding regions , to correctly restore open reading frames .', 'The characterization of fewer than 50 IESs identified by cloning MIC loci [5] showed that they are short ( 26–883 bp ) , unique copy elements that are located in both coding and non-coding regions of the genome .', 'The IESs are invariably flanked by two TA dinucleotides whereas only one TA is found at the MAC chromosome junction after IES excision ( Figure 1 ) .', 'IESs have also been discovered by cis-acting mendelian mutations that prevent their excision , conferring a mutant phenotype [6]–[10] .', 'The mutations in almost all cases were found in one of the flanking TA dinucleotides , which seem to be an absolute sequence requirement for IES excision .', 'Extrapolation of the number of IESs found mainly in surface antigen genes led to the estimation that there could be as many as 50 , 000 IESs in the Paramecium genome .', 'Such massive presence of unique copy IESs inserted in genes is not a characteristic of all ciliates .', 'The estimated 6 , 000 IESs of the related oligohymenophorean ciliate Tetrahymena [11] are excised by an imprecise mechanism [12] , are usually multi-copy including recognizable transposons [13]–[15] and are rarely found in coding sequences [16] , [17] .', 'Klobutcher and Herrick [18] first reported a weak consensus at the ends of 20 IESs from Paramecium surface antigen genes ( 5′-TAYAGYNR-3′ ) that resembles the extremities of Tc1/mariner transposons .', 'These authors hypothesized a “transposon link” to explain the origin of IESs , suggesting that they are the decayed relics of a Tc1/mariner transposon invasion and that they are excised from the MAC DNA by a Tc1/mariner transposase encoded by a gene that has become part of the cellular genome [19] .', 'In this model , IES excision represents the exact reversal of Tc1/mariner transposon integration into its TA target site with duplication of the TA dinucleotide , an evolutionary novelty that may have appeared more than once in the ciliate phylum .', 'One problem with the model is that transposition catalyzed by Tc1/mariner transposases usually leaves a 2 or 3 bp “footprint” at the donor site [20] while IES excision is precise .', 'A decisive step towards understanding the mechanism of IES excision and validating a transposon link for the origin of the IES excision machinery was the identification of a domesticated piggyBac transposase in Paramecium [21] .', 'Baptized PiggyMac ( Pgm ) , the protein is encoded by the PGM gene which is expressed only late in sexual processes , at the time of genome rearrangements .', 'Pgm , localized in the developing new MAC , was found to be required for the excision of all IESs tested and for the imprecise elimination of several regions containing transposons or cellular genes [21] .', 'A similar piggyBac-derived transposase is found in Tetrahymena and is required for heterochromatin-dependent DNA elimination [22] .', 'Since the Paramecium and Tetrahymena proteins appear to be monophyletic , based on a broad phylogeny of piggyBac transposases ( L . Katz and F . Gao , personal communication ) , the domestication event may have preceded the divergence of these two ciliates , estimated at 500–700 Ma ( million years ago ) [23] .', 'Most significantly , the in vivo geometry of IES excision , initiated by staggered double-strand breaks ( DSBs ) that generate 4-base 5′ overhangs centered on the TA at both ends of the IES [24] , is fully compatible with the in vitro reaction catalyzed by a piggyBac transposase isolated from an insect [25] , whose target site is a 5′-TTAA-3′ tetranucleotide .', 'piggyBac elements leave behind no scar when they jump to a new location: only ligation is required to join the fully complementary 5′ overhangs .', 'Limited processing of 5′ and 3′ ends is further required for precise closure of the Paramecium IES excision sites since only the TA dinucleotides at the center of the 4-base 5′ overhangs are always complementary [24] , [26] .', 'We report here a genomic approach to exhaustively catalogue the IESs in the Paramecium tetraurelia germline genome in order to study their evolutionary dynamics and seek evidence for a transposon origin of these elements .', 'We obtained DNA highly enriched in un-rearranged germline sequences , from cells depleted in Pgm by RNA interference .', 'Deep-sequencing of this DNA ( hereafter called “PGM DNA” ) allowed us to identify a genome-wide set of nearly 45 , 000 IESs , by comparing contigs assembled using the PGM DNA ( hereafter called “PGM contigs” ) with the MAC reference genome [1] .', 'The hypothesis that Pgm is required for excision of all IESs was tested by genome-scale sequencing of a source of DNA from purified MICs [27] , providing validation of the IES catalogue .', 'The evolutionary dynamics of the IESs was studied by exploiting the series of WGDs that have been characterized in Paramecium [1] .', 'The study provides , to our knowledge , the first genome-wide set of IESs , in Paramecium or any ciliate , and provides new evidence that IESs have deleterious effects on fitness and that at least a fraction of IESs do derive from Tc1/mariner transposons that have decayed over time .', 'The IES sequences evolve rapidly .', 'The constraints we could detect concern their size distribution , suggestive of the assembly of a transpososome-like excision complex and a weak consensus at their ends , which resembles the extremities of Tc1/mariner elements .', 'We discuss the possibility that ancient domestication of the Pgm transposase favored subsequent propagation of transposons throughout the Paramecium germline genome , by providing a mechanism for their precise somatic excision , therefore allowing insertions in coding sequences .'], ['An overview of the strategy for identification of a genome-wide set of IESs is presented in Figure S1 .', 'The first step was next-generation deep sequencing of DNA enriched in un-rearranged sequences , isolated from strain 51 cells that had undergone the sexual process of autogamy after depletion of Pgm protein by RNAi ( Figure S2 ) .', 'In the absence of Pgm , the zygotic DNA is amplified but rearrangements are impaired .', 'The sample that was sequenced contained a mixture of 60–65% un-rearranged DNA from the developing new MACs and 35–40% rearranged DNA from the fragments of the maternal MAC still present in the cytoplasm , as judged by Southern blot quantification of MIC and MAC forms at one locus ( Figure S3 ) .', 'The PGM sequence reads ( Table', '1 ) were mapped to the MAC reference genome of strain 51 ( see Materials and Methods ) , and putative IES insertion sites were defined as sites with a local excess of ends of read alignments ( pipeline MIRAA for “Method of Identification by Read Alignment Anomalies” ) .', 'This excess of ends of alignments arises when a read contains a MIC IES junction , since only part of such a read can align with a MAC chromosome , either starting or ending at the IES insertion site , expected to be a TA dinucleotide .', 'Using MIRAA , we identified 45 , 739 potential IES insertion sites .', 'Essentially all ( 99% ) of the insertion sites contained a TA dinucleotide , even though this was not assumed by the pipeline .', 'In order to obtain the sequence of the IESs , the paired-end PGM DNA sequence reads were assembled into contigs ( cf . Table S1 for assembly statistics ) and compared to the MAC reference genome assembly ( pipeline MICA for “Method of Identification by Comparison of Assemblies” ) .', 'We looked for insertions in the PGM contigs with respect to the MAC reference assembly .', 'Any insertion bounded by TA dinucleotides after local realignment was considered to be an IES .', 'Using this pipeline we identified 44 , 928 IESs .', 'The fact that 96% ( n\u200a=\u200a43 , 220 ) of the IESs identified by MICA correspond to an IES insertion site identified by MIRAA ( Figure S1 ) testifies to the overall reliability of the procedure .', 'Experimental validation of 6 IESs identified only by MICA and 17 insertion sites identified only by MIRAA was carried out by PCR amplification of an independent preparation of PGM DNA .', 'The results ( Table S2 ) show that the 6 IESs and at least 12 of 17 insertion sites tested do correspond to the presence of an IES .', 'Interestingly , among the IES sites identified only by MIRAA , we found 8 examples of a pair of IESs separated by one or only a few nucleotides ( in 5/8 cases , these tandem IESs are located in exons , a proportion similar to that found for the genome-wide IES set , see below ) .', 'This case is not handled by the MICA pipeline since the initial global alignment with BLAT would have detected a single large insertion that would have been rejected by the local realignment filter , which requires the insertion to be flanked by TA dinucleotides .', 'This is the first report of such closely spaced IESs , although nested IESs ( Figure', '1 ) have been previously documented [8] .', 'In order to see whether the set of 44 , 928 IESs is likely to be exhaustive , we looked for the 53 previously characterized IESs identified directly by cloning MIC loci in P . tetraurelia strain 51 cells ( Table S3 ) .', 'All 53 previously cloned IESs were found , with the exception of one IES that had been assembled into the MAC reference genome and one IES form that represents use of an alternative boundary .', 'In addition , two small IESs , each of which is nested within a larger IES , were found in PGM DNA but were not identified by our pipeline as IESs .', 'Indeed , nested IESs can only be identified by time-course experiments or if the outer IES is retained in the MAC e . g . as the result of a point mutation [8] .', 'Since 49 of 51 non-nested IESs were identified by MICA , the IES identification procedure has a sensitivity of at least 96% .', 'The entire IES identification approach is based on the assumption that the excision of all IESs in Paramecium requires the Pgm domesticated transposase activity .', 'In order to test this assumption , we sequenced inserts from a lambda-phage library constructed some 20 years ago [28] , using DNA from MICs that had been separated from MACs by Percoll gradient centrifugation [27] .', 'This library has been extensively used to clone MIC loci with specific probes .', 'Although the contigs assembled from the phage DNA reads only partially covered the MAC reference genome ( Table 1 ) , 98 . 5% of the 13 , 377 IESs that could be identified using the phage DNA and the MICA pipeline had also been identified using the PGM DNA .', 'The difference of 1 . 5% is within the estimated sensitivity of the MICA pipeline .', 'We conclude that all Paramecium IESs very likely require Pgm for excision , and that our data set does represent a genome-wide set of P . tetraurelia IESs .', 'The genome-wide set of IESs has an overall G+C content of 20% , significantly lower than the 28% G+C content of the MAC reference genome [29] but comparable to the G+C content of intergenic regions ( 21% ) .', 'The IESs are found in exons ( 76 . 8% ) , introns ( 5 . 4% ) and intergenic regions ( 17 . 8% ) , suggesting a nearly random distribution of IESs with respect to genes , since the MAC reference genome is composed of 76% exons , 3 . 2% introns and 20 . 8% intergenic DNA [1] .', 'However , IESs are not randomly distributed along the chromosomes .', 'Intriguingly , as shown in Figure S4 for the 8 largest MAC chromosomes , IESs tend to be asymmetrically distributed along MAC chromosomes .', 'The MAC assembly ( 188 scaffolds >45 Kb constitute 96% of the 72 Mb assembly ) contains 115 telomere-capped scaffolds , varying in size from ∼150 Kb to ∼1 Mb , that are considered to represent complete MAC chromosomes .', 'For 70 of these telomere-capped scaffolds , IESs display non-uniform distributions ( p<0 . 002 , median scaffold size 417 Kb ) while for the remaining 45 telomere-capped scaffolds , the IES distribution is uniform ( median scaffold size 275 Kb ) .', 'Thus the larger the MAC chromosome , the greater the chance of observing a non-uniform IES distribution .', 'The distributions for all scaffolds are easily visualized using the ParameciumDB [30] Genome Browser .', 'The significance of the asymmetry in IES distribution is not clear , but might be related to the global organization of MIC chromosomes , currently unknown ( discussed in [29] ) .', 'The genome-wide set of IESs covers 3 . 55 Mb ( mean IES size 79 bp ) , compared to 72 Mb for the MAC reference genome assembly .', 'The IESs thus add about 5% to the sequence complexity of the part of the MIC genome that is collinear with MAC chromosomes .', 'The total complexity of the PGM contigs ( after elimination of contigs with low PGM read coverage and high G+C content , assumed to represent bacterial contamination as confirmed in many cases by BLASTN matches against bacterial genomes ) is ∼100 Mb , however the use of a single paired-end sequencing library with small inserts ( ∼500 bp ) may have perturbed assembly of repeated sequences , possibly leading to underestimation of repeated sequence content .', 'We infer that ∼25 Mb of germline-specific DNA corresponds to the imprecisely eliminated regions located outside of the MAC-destined chromosomes i . e . the part of the MIC genome that is not collinear with MAC chromosomes .', 'We have not further characterized this fraction of the PGM DNA .', 'However , we did identify the first germline P . tetraurelia Tc1/mariner transposons ( Figure S5 ) , by using the phage-lambda library of MIC DNA [28] to walk past the end of MAC scaffold_51 , which bears the subtelomeric 51G surface antigen gene [31] .', 'In all , 5 phage inserts and 4 cloned PCR products corresponding to part or all of different copies of the element downstream of the 51G surface antigen gene , named Sardine , were sequenced ( EMBL Nucleotide Sequence Database accession numbers HE774468–HE774475 ) and a consensus for the ∼6 . 7 Kb transposon was constructed ( Figure S5 and Text S1 ) .', 'The ends of the Sardine copies contain intact or partially deleted 425 bp terminal inverted repeats ( TIRs ) which are themselves palindromic , containing a unique , oriented region nested within outer inverted repeats ( Figure S5 ) .', 'Sardine contains up to 4 ORFs .', 'One ORF is a putative DD35E transposase of the IS630-Tc1 family , like the DDE transposases of the TBE and Tec transposons found in stichotrich ciliates [32] .', 'Another ORF , as in Tec transposons [33] , encodes a putative tyrosine recombinase .', 'The other two ORFs are hypothetical , though ORF2 shows some similarity ( 31 . 7% identity and 55 . 4% similarity over 202 aa ) to the hypothetical ORF1 of the Tennessee element from P . primaurelia [34] .', 'One of the Sardine copies ( copy S6 ) is interrupted by the insertion , within the putative tyrosine recombinase gene , of a different but similar element , named Thon ( French for “tuna” ) , which also contains a DD35E transposase , a tyrosine recombinase , possibly the two hypothetical ORFs , and palindromic TIRs of ∼700 bp ( Figure S5 ) .', 'For a handful of IESs , it has been shown experimentally that they are single copy elements [5] .', 'In order to see whether this is generally the case , we looked for all IESs present in more than 1 fully identical copy ( 100% sequence identity ) .', 'We found 44 , 210 IESs to be unique copy ( 98 . 4% ) .', 'We examined all IESs present in 2 or more identical copies and found 39 cases of duplicate IESs as a result of errors in assembly of the MAC reference genome that had led to small , partially redundant scaffolds ( 4% of the MAC assembly is contained in scaffolds <45 Kb and some of these are partially redundant with the chromosome-size scaffolds [1] ) .', 'The rest of the 319 IESs found in 2 copies were inserted in homologous genomic sites and appeared to be the result of recent segmental duplication or gene conversion .', 'The 23 cases of IESs found in 3 to 6 copies correspond to expansion or recombination of repeated sequences such as tetratricopeptide repeat ( TPR ) domains or WD40 repeats .', 'We performed an all by all sequence comparison of the IESs and of their flanking sequences to see whether we could identify homologous IESs inserted at non-homologous sites in the genome .', 'As shown in Table 2 , we were able to identify 8 clusters of 2 to 6 IESs that share significant homology ( BLASTN E-value <10−10 ) over at least 85% of their length , inserted in non-homologous sites ( cf . Text S2 for the alignments ) .', 'Moreover , we found significant nucleotide identity ( E-value 9×10−57 for the best match; nucleotide identity between 68 and 78% for the HSPs ) between the IESs of cluster 5 and one of the Tc1/mariner-like transposons identified using the phage library ( Thon , Figure S5 ) .', 'This is a strong indication that these IESs are derived from recently mobile elements .', 'However , the IES sequences of this cluster correspond to a single palindromic TIR .', 'This might reflect assembly problems given use of a single insert size for the paired-end sequencing , either because these IESs contain sequences repeated elsewhere in the genome or because the Thon TIRs are large ( ∼700 bp ) and palindromic so that the assembly might have jumped from one TIR to the other deleting the rest of Thon .', 'We therefore used a long-range PCR strategy capable of amplifying large DNA fragments containing each of the IESs to verify their size and attempt to obtain sequences ( detailed in Text S3 ) .', 'Amplification products of the expected sizes were obtained for all of the IESs from cluster 5 , making it unlikely that these IESs correspond to a complete Thon element that had failed to be assembled from the paired-end sequencing reads .', 'Three IESs were chosen for sequencing , and the sequences of the corresponding PCR products confirmed the IESs , indicating that they had been correctly assembled .', 'Identification of 6 IESs ( at non-homologous genomic sites ) that share sequence identity with a P . tetraurelia Tc1/mariner solo TIR argues that at least a fraction of IESs do originate from Tc1/mariner-like elements .', 'We therefore adopted a complementary strategy , using the PFAM-A library of curated protein domains to search for domain signatures in the genome-wide set of IESs .', 'Matches at a BLASTX E-value cutoff of 1 were inspected visually to filter out matches with PFAM-A protein domains from Paramecium and matches owing to compositional bias ( high A+T content ) .', 'This left 6 IESs , ranging in size from 2416 to 4154 bp , with a DDE_3 ( PFAM accession number 13358 ) DDE superfamily endonuclease domain characteristic of IS630/Tc1 transposons .', 'The peptides encoded by the IESs were subjected to an HMM search of the PFAM-A hmm profiles ( http://pfam . sanger . ac . uk/search ) for confirmation of the conserved residues and to validate the statistical significance of the match ( E-values of 0 . 02 to 2 . 1×10−15 for the 6 peptides ) .', 'The IESs were aligned with MUSCLE and a neighbor-joining tree grouped 4 of them together with good bootstrap values ( not shown ) .', 'The 4 IESs were used to search for sequence similarity with the genome-wide set of IESs and this allowed identification of 28 IESs ranging in size from 1251 to 4154 bp ( Table S4 ) .', 'The IESs were aligned to provide the consensus sequence for 2 distinct Tc1/mariner-like 3 . 6 kb transposons from the same new family , baptized Anchois ( Anchovy ) .', 'Manually adjusted alignments used to reconstruct the AnchoisA and AnchoisB elements , consensus sequences and annotation are provided in Text S1 .', 'Alignment of the DDE domains of the reconstituted Anchois transposons with the DDE domains from bacterial IS630 elements , invertebrate Tc1 transposons and all known ciliate Tc1/mariner elements indicates that the Anchois transposase belongs to the IS630/Tc1 subfamily ( Figure 2A ) .', 'Unlike Thon and Sardine but like the P . primaurelia Tennessee element , Anchois TIRs are short and lack internal palindromes , moreover Anchois does not contain a putative tyrosine recombinase .', 'Anchois has 2 hypothetical ORFs in addition to the DDE transposase ( Figure 2B; Text S1 ) .', 'The ORF2 of Anchois displays homology to ORF2 of Sardine ( 36 . 2% identity and 56 . 2% similarity over 210 aa ) and to ORF1 of Tennessee .', 'Interestingly , for 6 of the 28 IESs that initially identified the copies of Anchois , the Anchois TIRs do not correspond to the extremities of the IES , raising the possibility of Anchois insertions within pre-existing IESs .', 'The discovery of the Anchois elements and the fact that several IESs appear to be full-length copies , provides a strong , direct link between IESs and transposons .', 'The size distribution of the genome-wide set of IESs is shown in Figure 3A , for the 93% of the IESs that are shorter than 150 bp .', 'The most remarkable feature is a periodicity of ∼10 bp , which corresponds to the helical repeat of double-stranded DNA .', 'The first peak of the size distribution has maximal amplitude at 26–28 bp and includes 35% of all identified IESs .', 'The abrupt cutoff at 26 bp represents the minimum IES size .', 'A second peak appears to be forbidden and contains only a few IESs .', 'The following peaks are centered at approximately 45–46 , 55–56 , 65–66 bp etc . and the distance between these peaks is best fit by a 10 . 2 bp sine wave ( not shown ) .', 'At the far end of the spectrum , 95 of the IESs are between 2 and 5 Kb in size .', 'Similar periodic size distributions are found for IESs inserted in coding sequences and for IESs inserted in non-coding sequences ( Figure S6 ) .', 'This indicates that the constraint on the distance between IES ends is an intrinsic property of the IESs and is not related to the locus in which they are inserted in the genome .', 'Whatever their size , the IESs adhere to the weak , Tc1/mariner-like end consensus first reported for 20 IESs located in surface antigen genes [18] , as illustrated in Figure 3B for the whole set .', 'Differently sized subsets of the IESs all display essentially the same end consensus ( data not shown ) .', 'We further examined constraints on IES size and sequence by evaluating IES conservation with respect to the 3 WGDs in the Paramecium lineage .', 'We used the large number of paralogs ( hereafter termed “ohnologs” ) of different ages ( Table 3 ) that could be identified for each of the WGD events [1] to ask whether IESs are present , at the same position relative to the gene coding sequences , in ohnologs of the different WGD events .', 'This analysis makes the assumption that IES insertions are rare events so that if IESs are present at the same position in ohnologous genes , then they must have been acquired before the WGD and can be considered to be “ohnologous” IESs .', 'As shown in Table 3 , we found 84 . 5% , 23 . 2% and 5 . 9% conservation of IESs with respect to the recent , intermediate and old WGDs respectively .', 'For comparison , more than 99% intron conservation was found for 1 , 112 pairs of genes related by the recent WGD [35] .', 'This indicates that the dynamics of IES insertion or loss over evolutionary time is relatively fast compared to that of introns .', 'The only phylogenetic study of IESs , carried out for two loci in a few different stichotrich ( formerly called hypotrich ) ciliates , which are very distantly related to Paramecium , also concluded that the intragenic IESs in those species evolve very rapidly [36] .', 'We found that the ohnologous IESs related by the recent WGD are highly divergent in sequence .', 'In more than 90% of cases , the sequence identity was too low for detection by BLASTN ( E-value threshold of 10−5 ) .', 'This high level of sequence divergence is consistent with the pattern expected for neutrally-evolving non-coding regions , since the average synonymous substitution rate measured between ohnologous genes derived from the recent WGD is about 1 substitution per site [1] .', 'However , if we compare the lengths of IESs that are conserved with respect to the recent WGD ( Figure 4A ) , for ∼55% of the pairs , both IESs are found in the same peak of the IES size distribution .', 'The honeycomb appearance of the plot ( Figure 4A ) , with off diagonal cells that result from ohnologous IESs in different peaks of the distribution , underscores the strong evolutionary constraint that is exerted on IES size .', 'In order to investigate the rate of IES insertions and losses during the evolution of the Paramecium lineage , we examined gene families , which we call “quartets” , for which all 4 ohnologs issued from duplication of an ancestral gene at the intermediate and then the recent WGD are still found in the present day genome .', 'Of the 1350 such quartets identified in the MAC genome [1] , 878 contain at least one IES in at least one of the 4 duplicated genes .', 'We evaluated the conservation of IESs at the same position with respect to the coding sequence for all members of each quartet ( Figure S7 ) , and identified 2126 IES groups , each group containing an IES conserved either in all 4 genes ( N1111\u200a=\u200a190 ) , in 3 genes ( N1110\u200a=\u200a64 ) , in 2 genes on the same intermediate WGD branch ( N1100\u200a=\u200a1304 ) , in 2 genes on different branches ( N1010\u200a=\u200a10 ) or in only one of the 4 genes ( N1000\u200a=\u200a558 ) .', 'Under the assumption that two IESs present at the same location in ohnologous genes derive from a single ancestral IES ( i . e . the probability of two insertion events occurring at the same site after a WGD is considered negligible ) , and that the rate of IES losses has remained constant , it is possible to estimate the rate of IES gain during the evolution of the Paramecium lineage ( the model is developed in Text S4 ) .', 'The quartet analysis is fully consistent with a model whereby IES acquisition has been ongoing since before the intermediate WGD ( 15% of the IESs predating this WGD ) , with a peak in the period between the intermediate and the recent WGD events: 69% of IESs were acquired during the interval between these two WGDs , vs . 16% during the period since the recent WGD , which corresponds to about the same evolutionary time .', 'Genome-wide IES data for other Paramecium species will be necessary in order to test the assumption of a constant rate of IES losses .', 'However , even if we relax this assumption ( i . e . rates of IES losses are allowed to vary over time ) , the model still strongly rejects the hypothesis that all IESs were acquired before the intermediate WGD ( cf . Text S4 ) .', 'Thus , with the presently available data and biologically reasonable assumptions , we conclude that IESs have been acquired in all 3 of the time periods delimited by the intermediate and recent WGD events .', 'We compared the cumulative size distributions of the N1111 , N1100 and N1000 IESs ( Figure 4B ) .', 'The N1111 IESs , which must have been acquired before the intermediate WGD , are much shorter than the IESs of the two other samples , with almost 80% of the IESs in the first peak , compared to 20% for N1100 IESs , which may mainly result from IES acquisition after the intermediate but before the recent WGD , and only 16% for N1000 IESs , at least some of which may have been acquired since the recent WGD .', 'In addition , the curves are significantly shifted with respect to each other , in particular , 30% of the N1000 IESs are larger than 150 nt , compared to scarcely any IESs larger than 150 nt for the two other samples .', 'This analysis shows that the older an IES , the shorter it is likely to be , consistent with a decay process involving progressive shortening of IESs by accumulation of small deletions , in addition to the accumulation of point mutations .', 'Quartet analysis is restricted to IESs in genes that have been retained in 4 copies ( fewer than 10% of all IESs ) .', 'Similar distributions of IES size are found if we consider all ohnologous IESs ( 45% of all IESs , cf . Table 3 ) .', 'IESs conserved with respect to the intermediate WGD ( 76% of IESs in first peak ) are significantly shorter than IESs conserved only with respect to the recent WGD ( 30% of IESs in the first peak ) ( data not shown ) .', 'The size distribution of IESs conserved with respect to the old WGD is poorly determined because of the small number of conserved IESs ( Table 3 ) , which are moreover often in genes that have undergone recent gene conversion judging from the nucleotide divergence of the ohnologs ( data not shown ) .', 'It is therefore uncertain that IESs were present in the genome before the old WGD , consistent with the absence of TA-bounded IESs in Tetrahymena , which diverged from Paramecium after the old WGD event [1] .', 'Since we found essentially no IESs shorter than 26 bp , it seems likely that some mechanism ( s ) other than decay of the sequence through internal mutations and deletions is responsible for the complete loss of an IES .', 'In order to explore this question , we examined case by case , using both nucleotide and conceptual protein alignments , all of the N1110 quartet IES groups ( n\u200a=\u200a64 ) , which are most parsimoniously explained by insertion of an IES before the intermediate WGD followed by loss of an IES after the recent WGD .', 'We examined the raw read alignments and PGM and phage contigs in order to be sure that there was sufficient read coverage and no evidence suggesting presence of an IES at any site of putative IES loss .', 'We found 4 different explanations for the quartet triplets: precise loss of the fourth IES ( n\u200a=\u200a17 ) , gain of the third IES by gene conversion between intermediate WGD ohnologs ( n\u200a=\u200a1 ) , recruitment of the fourth IES into the exon sequence ( n\u200a=\u200a6 ) , and deletion of the region that encompasses the fourth IES ( n\u200a=\u200a23 ) , often testifying to the formation of a pseudogene .', 'In addition , we found 5 errors in IES detection ( the fourth IES probably exists as it can be found in the phage contigs or is predicted by the MIRAA pipeline ) .', 'In the remaining cases ( n\u200a=\u200a12 ) , annotation or alignment problems made it difficult to conclude .', 'The observation of 17 cases of precise loss of an IES from the germline DNA raises the possibility that there is a mechanism for conversion of a MIC locus to the IES-free form using a MAC genome template .', 'However , we cannot rule out the possibility that IESs can be precisely excised from the MIC DNA , and therefore lost , by the same Pgm-dependent mechanism as that involved in MAC genome assembly .', 'The analysis of sequence variability in the polyploid ( 800n ) MAC genome , carried out by comparing the MAC assembly representing a “consensus” sequence with the 13× Sanger sequencing reads used to build the assembly , revealed nearly 2000 “TA-indels” that were presumed to be produced by the IES excision machinery and to reflect excision errors [29] .', 'As shown schematically in Figure 5 , “residual” TA-indels ( n\u200a=\u200a739 ) , that were suggested to represent occasional retention of IESs on some macronuclear copies , were absent from the assemb
['Introduction', 'Results', 'Discussion', 'Materials and Methods']
['Insertions of parasitic DNA within coding sequences are usually deleterious and are generally counter-selected during evolution .', 'Thanks to nuclear dimorphism , ciliates provide unique models to study the fate of such insertions .', 'Their germline genome undergoes extensive rearrangements during development of a new somatic macronucleus from the germline micronucleus following sexual events .', 'In Paramecium , these rearrangements include precise excision of unique-copy Internal Eliminated Sequences ( IES ) from the somatic DNA , requiring the activity of a domesticated piggyBac transposase , PiggyMac .', 'We have sequenced Paramecium tetraurelia germline DNA , establishing a genome-wide catalogue of ∼45 , 000 IESs , in order to gain insight into their evolutionary origin and excision mechanism .', 'We obtained direct evidence that PiggyMac is required for excision of all IESs .', 'Homology with known P . tetraurelia Tc1/mariner transposons , described here , indicates that at least a fraction of IESs derive from these elements .', 'Most IES insertions occurred before a recent whole-genome duplication that preceded diversification of the P . aurelia species complex , but IES invasion of the Paramecium genome appears to be an ongoing process .', 'Once inserted , IESs decay rapidly by accumulation of deletions and point substitutions .', 'Over 90% of the IESs are shorter than 150 bp and present a remarkable size distribution with a ∼10 bp periodicity , corresponding to the helical repeat of double-stranded DNA and suggesting DNA loop formation during assembly of a transpososome-like excision complex .', 'IESs are equally frequent within and between coding sequences; however , excision is not 100% efficient and there is selective pressure against IES insertions , in particular within highly expressed genes .', 'We discuss the possibility that ancient domestication of a piggyBac transposase favored subsequent propagation of transposons throughout the germline by allowing insertions in coding sequences , a fraction of the genome in which parasitic DNA is not usually tolerated .']
['Ciliates are unicellular eukaryotes that rearrange their genomes at every sexual generation when a new somatic macronucleus , responsible for gene expression , develops from a copy of the germline micronucleus .', 'In Paramecium , assembly of a functional somatic genome requires precise excision of interstitial DNA segments , the Internal Eliminated Sequences ( IES ) , involving a domesticated piggyBac transposase , PiggyMac .', 'To study IES origin and evolution , we sequenced germline DNA and identified 45 , 000 IESs .', 'We found that at least some of these unique-copy elements are decayed Tc1/mariner transposons and that IES insertion is likely an ongoing process .', 'After insertion , elements decay rapidly by accumulation of deletions and substitutions .', 'The 93% of IESs shorter than 150 bp display a remarkable size distribution with a periodicity of 10 bp , the helical repeat of double-stranded DNA , consistent with the idea that evolution has only retained IESs that can form a double-stranded DNA loop during assembly of an excision complex .', 'We propose that the ancient domestication of a piggyBac transposase , which provided a precise excision mechanism , enabled transposons to subsequently invade Paramecium coding sequences , a fraction of the genome that does not usually tolerate parasitic DNA .']
['genome evolution', 'evolutionary biology', 'genome sequencing', 'dna recombination', 'molecular genetics', 'dna', 'sequence analysis', 'transposons', 'comparative genomics', 'biology', 'molecular biology', 'nucleic acids', 'dna transposons', 'dna repair', 'genomics', 'molecular cell biology', 'genomic evolution', 'computational biology', 'genetics and genomics']