Molecular Biology and Evolution

MBE Advance Access originally published online on August 30, 2006
Molecular Biology and Evolution 2006 23(12):2259-2262; doi:10.1093/molbev/msl098

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Letters

Smoke Without Fire: Most Reported Cases of Intron Gain in Nematodes Instead Reflect Intron Losses

Scott William Roy and David Penny

Allan Wilson Centre for Molecular Ecology and Evolution, Massey University, Palmerston North, New Zealand

E-mail: scottwroy{at}gmail.com.

	Abstract

TOP Abstract Methods Supplementary Material Acknowledgements References

 
Identification of recently gained spliceosomal introns would provide crucial evidence in the continuing debate concerning the age and evolutionary significance of introns. A previously published genomic analysis reported to have identified 122 introns that had been gained since the divergence of the nematodes Caenorhabidits elegans and Caenorhabditis briggsae 100 MYA. However, using newly available genomic sequence from additional Caenorhabditis species, we show that 74% (60/81) of the reported gains in C. elegans are present in a C. briggsae relative. This pattern indicates that these introns represent losses in C. briggsae, not gains in C. elegans. In addition, 61% (25/41) of the reported gains in C. briggsae are present in the more distant C. briggsae relative, in a pattern suggesting that additional reported gains in C. elegans and/or C. briggsae may in fact represent unrecognized losses. These results underscore the dominance of intron loss over intron gain in recent eukaryotic evolution, the pitfalls associated with parsimony in inferring intron gains, and the importance of genomic sequencing of clusters of closely related species for drawing accurate inferences about genome evolution.

Key Words: intron gain • genome complexity • genome annotation • genome sequencing • genome evolution • parsimony

The origin of the splicesomal introns of eukaryotes constitutes a 30-year-old mystery (de Souza 2003; Jeffares et al. 2006; Martin and Koonin 2006; Rodríguez-Trelles et al. 2006; Roy and Gilbert 2006). In 1998, Logsdon et al. laid out conditions for determining the source of a recently gained intron: 1) strong evidence for the intron's recent gain, derived from "dense phylogenetic sampling"; and 2) the "molecular smoking gun"—an intronic sequence whose clear similarity to another genetic element betrays the intron's origin. The subsequent years have been an extremely active time for the study of intron evolution (Tarrío et al. 1998, 2003; Venkatesh et al. 1999; Sakharkar et al. 2001; Seo et al. 2001; Wolf et al. 2001; Fedorov et al. 2002; Llopart et al. 2002; Wada et al. 2002; Bon et al. 2003; Fedorov et al. 2003; Rogozin et al. 2003; Nielsen et al. 2004; Slamovits and Keeling 2006). However, until 2004 only a single clearly characterized intron gain had been reported (Iwamoto 1998). Then finally, 2 years ago Coghlan and Wolfe (2004) reported the cases of 81 potentially recently gained introns in Caenorhabditis elegans and 41 in Caenorhabditis briggsae. Each of the 122 introns was not found in the other Caenorhabditis species or in various outgroups (apparently fulfilling the first criterion; fig. 1), and 28 of the introns showed sequence similarity to other Caenorhabditis introns (apparently fulfilling the second). These results have been widely discussed (e.g., Roy 2004; Rodríguez-Trelles et al. 2006) and widely cited (>35 citations) and were hailed by Logsdon (2004) as the long-awaited "smoking gun."

View larger version (11K): [in this window] [in a new window] [Download PowerPoint slide]   FIG. 1.— Some previously reported intron gains instead represent intron losses. Coghlan and Wolfe (2004) previously aligned homologous sequences from Caenorhabiditis elegans and Caenorhabditis briggsae, the distantly related nematode Brugia malayi, and other available outgroups (but not Caenorhabditis remanei or Caenorhabditis sp. 4). They identified introns specific to one of the two Caenorhabiditis species and absent in outgroups (e.g., intron 2), as recent intron gains. However, although 85% of introns are shared between C. elegans and C. briggsae (Kent and Zahler 2000), intron conservation between Caenorhabditis and available outgroups is 50% or less (Guiliano et al. 2002; Rogozin et al. 2003), thus some introns present in the C. elegans–C. briggsae ancestor may not be represented in outgroups (e.g., intron 3). We assessed presence of reportedly gained introns in C. remanei and C. sp. 4. As shown in the figure, most reported C. elegans gains are present in one or both species (e.g. intron 2), suggesting intron presence in the C. elegans–C. briggsae ancestor and subsequent loss in C. briggsae. In addition, we assessed intron presence/absence of reported C. briggsae intron gains in the 2 C. briggsae relatives (not shown). Species relationships are from Cho et al. 2004 and Kiontke et al. 2004.

 
The first point of contention concerned interpretation of the intron sequences themselves (the second criterion). Coghlan and Wolfe (2004) as well as Logsdon (2004) interpreted observed sequence similarity between the reported apparent gains and other introns as evidence of intron gain by transposition of existing introns into new positions in the same or different genes. However, sequence similarity between introns often spanned only a fraction of the introns' lengths, evidence against a simple intron transposition event (Roy 2004; Roy SW, unpublished data). Also, regions of similarity were often limited to many-copy repetitive elements, which were also found in intergenic regions, leading one of us to suggest that the reported gains might instead be due to transposable element (TE) insertions (Roy 2004).

Here, we report evidence that many of the reported intron gains are not even true intron gains (criterion 1) but instead reflect intron losses. We examined putatively orthologous sequences from newly available genomic sequences from 2 relatives of C. briggsae: Caenorhabditis remanei and Caenorhabditis sp. 4 (fig. 1). If the 81 C. elegans introns reported to be recent gains are in fact just that, they should clearly be absent from these species. Instead, 74% (60/81) were found to be shared with one or both species (table 1, see e.g., in fig. 2; a more detailed summary is available as Supplementary Material Online). This implies that these introns' absence in C. briggsae is due to intron loss and not due to recent gain in C. elegans. The remaining 21 possible C. elegans gains may either be actual gains in C. elegans or losses in the C. briggsae–C. sp. 4 ancestor (table 1, branch ii). Thus, in most cases (at least for C. elegans), there is no smoking gun.

View this table: [in this window] [in a new window]   Table 1 Observed phylogenetic patterns among 122 Caenorhabditis introns previously reported to be recently gained (Coghlan and Wolfe 2004)

 

View larger version (17K): [in this window] [in a new window] [Download PowerPoint slide]   FIG. 2.— Example of a reported intron gain in Caenorhabditis elegans, which instead appears to be an intron loss in Caenorhabditis briggsae. The first intron in C. elegans gene F02E11.1 was reported by Coghlan and Wolfe (2004) to have been recently gained since the C. elegans–C. briggsae ancestor. The first and second C. elegans exons (uppercase) and intervening intron (lowercase) are shown, along with putatively orthologous sequences for C. briggsae, Caenorhabditis remanei, and Caenorhabditis sp. 4. The intron is clearly present in the C. briggsae relatives C. remanei and C. sp. 4, suggesting intron loss in C. briggsae. Typically, that the intervening C. remanei and C. sp. 4 sequences are in fact intronic is reinforced by the "gt...ag" boundary structure (underlined), the presence of in-frame stop codons in both sequences (bold), and the fact that the intervening sequence in C. sp. 4 is not a multiple of 3 bp. The sequences are from C. elegans gene F02E11.1, C. briggsae gene CBG04332, C. remanei contig Cont107.34, and C. sp. 4 read CPAA-aga86e12.b1, respectively. Cele, Cbri, Crem, and Csp4 indicate C. elegans, C. briggsae, C. remanei, and C. sp. 4, respectively.

 
Among the 41 reported C. briggsae gains, fully 61% (25) are present in C. sp. 4 and, thus, could represent gains in the C. briggsae–C. remanei–C. sp. 4 ancestor (branch ii) or losses in C. elegans. If all 25 of these introns and all 21 C. elegans–specific introns were true intron gains, there would be no losses but numerous gains in both C. elegans (branch i) and in the C. briggsae–C. sp. 4 ancestor (branch ii). This would be surprising in light of the observation of 3.75 losses per gain (60/16) in the sample occurring in C. briggsae since the C. briggsae–C. sp. 4 ancestor (branch iii). This suggests that some of the remaining possible gains may also represent unrecognized losses. Though direct estimation of the loss/gain numbers here is not possible, the ratio of losses to gains in both the C. briggsae–C. sp. 4 ancestor and in C. elegans would also equal 3.75, if there were 5.6 gains and 20.9 losses in C. elegans and 4.1 gains and 15.4 losses in the C. briggsae–C. sp. 4 ancestor. In this case, only 21% (16 + 5.6 + 4.1 = 25.7 out of 122) of the reported intron gains would represent true intron gains.

Most biases identified by Coghlan and Wolfe (2004) among the 122 reported gains are not strong for the remaining 21 possible C. elegans gains and 16 probable C. remanei gains. Only 4/37 are found in genes involved in mRNA splicing. Only 27.0% (10/37) show sequence similarity to other introns, similar to the C. briggsae losses (25.0%, 15/60). Only the reported bias toward oocyte expression remains: among genes that contain C. elegans gains and/or probable C. briggsae gains for which oocyte expression is available (Hill et al. 2000), 70% (16/23) are present in oocytes, more than 42% for all genes assessed (P 0.01 by a Fisher's Exact test). This bias could reflect additional undetected oocyte-biased intron losses, a higher frequency of insertion of intron-creating TE insertions into germline-expressed genes (perhaps due to more accessible chromatin structure), or a dependency of intron gain on an mRNA intermediate, as suggested by Coghlan and Wolfe (2004).

The suggestive biases among the introns reported by Coghlan and Wolfe (sequence similarity to other introns/TEs, gene biases toward germline expression and involvement in mRNA processing) are thus apparently a case of smoke without fire (or evidence without a crime), as these introns are primarily derived from cases of intron loss, not gain. Why should introns in one species that are lost in the other show sequence similarity to other introns/TEs? It seems likely that these sequence similarities are due to independent intronic and intergenic insertions of the same TE. If rates of intron loss and intronic TE insertion were correlated due to the dependence of both processes on local recombination rate or general DNA accessibility (chromatin structure) in the germline, the same introns that are lost in one species might tend to experience TE insertion in the other. Preferential intron loss from germline-expressed genes could reflect mRNA-mediated intron loss (Mourier and Jeffares 2003), as could the bias toward mRNA splicing-related genes, though why splicing-associated transcripts, as opposed to proteins, should associate with the spliceosome is unclear (Coghlan and Wolfe 2004). These surprising biases in intron loss deserve further attention.

These results highlight 3 important points. First, along many eukaryotic lineages, recent evolution has been characterized by a dominance of intron loss over intron gain (e.g., Roy et al. 2003; Cho et al. 2004; Kiontke et al. 2004; Lin et al. 2006; Roy and Hartl 2006; Stajich and Dietrich 2006): in this case, even the introns that appeared most likely to represent cases of recent gains are instead mostly due to loss. Second, these results provide an important case study of the utility of parsimony in the face of high degrees of evolutionary change. Third, these results demonstrate the importance of greater taxonomic sampling and the indispensability of sequencing additional genomes for answering even seemingly straightforward questions about genome structure and evolution.

The general dearth of clear recent intron gains continues to frustrate attempts to understand mechanisms and causes of intron creation (Roy et al. 2003; Lin et al. 2006; Roy and Hartl 2006; Roy et al. 2006; Stajich and Dietrich 2006). These observed low intron gain rates are curious as huge numbers of introns in various eukaryotic genomes attest to substantial intron creation at some point in evolution. One possible explanation is improved policing of genome insertions in modern eukaryotes relative to early/pre-eukaryotic evolution. Investigation is ongoing.

	Methods

TOP Abstract Methods Supplementary Material Acknowledgements References

 
From the text and supplementary materials of Coghlan and Wolfe 2004, we extracted amino acid sequences flanking apparent intron gains, gene functions, and names of introns exhibiting sequence similarity to other introns from the same genome. We performed TBlastN searches against the assembled C. remanei genome (version 1, downloaded from Wormbase [http:www.wormbase.org]). Intron presence/absence was determined by either 1) the presence of a gain in the resultant alignment, almost always with stop codons in the gapped C. remanei sequence or 2) the presence of 2 independent HSPs to the same contig, one upstream and one downstream of the intron position, with the sequence stopping abruptly at the intron position, and with a significant intervening gap. Both the best hit and other highly significant hits were surveyed to determine intron presence in all possible orthologous or closely related sequences. An analogous search was made of the 2,714,032 available genomic shotgun sequencing reads for Caenorhabditis sp. 4, downloaded from TraceDB (www.ncbi.nlm.nih.gov). Following Coghlan and Wolfe, genes that are always or sometimes expressed in oocytes were determined from the oligonucleotide studies of Hill et al. (2000)

	Supplementary Material

TOP Abstract Methods Supplementary Material Acknowledgements References

 
Summary of results including for each intron, the gene name, intron number, and presence (+), absence (–), or uncertainty (?) in the C. briggsae relatives is available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/).

	Acknowledgements

TOP Abstract Methods Supplementary Material Acknowledgements References

 
We thank Manuel Irimia for constructive comments and helpful discussions during the preparation of this manuscript.

	Footnotes

 
William Martin, Associate Editor

	References

TOP Abstract Methods Supplementary Material Acknowledgements References

 

Bon E, Casaregola S, Blandin G, et al. (11 co-authors). (2003) Molecular evolution of eukaryotic genomes: hemiascomycetous yeast spliceosomal introns. Nucleic Acids Res 31:1121–1135.[Abstract/Free Full Text]

Cho S, Jin SW, Cohen A, Ellis RE. (2004) A phylogeny of ceanorhabditis reveals frequent loss of introns during nematode evolution. Genome Res 14:1207–1220.[Abstract/Free Full Text]

Coghlan A and Wolfe KH. (2004) Origins of recently gained introns in Caenorhabditis. Proc Natl Acad Sci USA 101:11362–11367.[Abstract/Free Full Text]

de Souza SJ. (2003) The emergence of a synthetic theory of intron evolution. Genetica 118:117–121.[CrossRef][ISI][Medline]

Fedorov A, Merican AF, Gilbert W. (2002) Large-scale comparison of intron positions among animal, plant, and fungal genes. Proc Natl Acad Sci USA 99:16128–16133.[Abstract/Free Full Text]

Fedorov A, Roy S, Fedorova L, Gilbert W. (2003) Mystery of intron gain. Genome Res 13:2236–2241.[Abstract/Free Full Text]

Guiliano DB, Hall N, Jones SJ, Clark LN, Corton CH, Barrell BG, Blaxter ML. (2002) Conservation of long-range synteny and microsynteny between the genomes of two distantly related nematodes. Genome Biol 3:RESEARCH0057.[Medline]

Hill AA, Hunter CP, Tsung BT, Tucker-Kellogg G, Brown EL. (2000) Genomic analysis of gene expression in C. elegans. Science 290:809–812.[Abstract/Free Full Text]

Iwamoto M, Maekawa M, Saito A, Higo H, Higo K. (1998) Evolutionary relationship of plant catalase genes inferred from exon-intron structures: isozyme divergence after the separation of monocots and dicots. Theor Appl Genet 97:9–19.

Jeffares DC, Mourier T, Penny D. (2006) The biology of intron gain and loss. Trends Genet 22:16–22.[CrossRef][ISI][Medline]

Kent WJ and Zahler AM. (2000) Conservation, regulation, synteny, and introns in a large-scale C. briggsae-C. elegans genomic alignment. Genome Res 10:1115–1125.[Abstract/Free Full Text]

Kiontke K, Gavin NP, Raynes Y, Rehrig C, Piano F, Fitch DH. (2004) Caenorhabditis phylogeny predicts convergence of hermaphroditism and extensive intron loss. Proc Natl Acad Sci USA 101:9003–9008.[Abstract/Free Full Text]

Lin H, Zhu W, Silva JC, Gu X, Buell CR. (2006) Intron gain and loss in segmentally duplicated genes in rice. Genome Biol 7:R41.[CrossRef][Medline]

Llopart A, Comeron JM, Brunet FG, Lachaise D, Long M. (2002) Intron presence-absence polymorphism in Drosophila driven by positive Darwinian selection. Proc Natl Acad Sci USA 99:8121–8126.[Abstract/Free Full Text]

Jr Logsdon JM. (2004) Worm genomes hold the smoking guns of intron gain. Proc Natl Acad Sci USA 101:11195–11196.[Free Full Text]

Logsdon JM Jr, Stoltzfus A, Doolittle WF. (1998) Molecular evolution: recent cases of spliceosomal intron gain? . Curr Biol 8:R560–R563.[CrossRef][ISI][Medline]

Martin W and Koonin EV. (2006) Introns and the origin of nucleus-cytosol compartmentalization. Nature 440:41–45.[CrossRef][Medline]

Mourier T and Jeffares DC. (2003) Eukaryotic intron loss. Science 300:1393.[Free Full Text]

Nielsen CB, Friedman B, Birren B, Burge CB, Galagan JE. (2004) Patterns of intron gain and loss in fungi. PLoS Biol 2:e422.[CrossRef][Medline]

Rodríguez-Trelles F, Tarrío R, Ayala FJ. (2006) Origins and evolution of spliceosomal introns. Annu Rev Genet 40:47–76.[Medline]

Rogozin IB, Wolf YI, Sorokin AV, Mirkin BG, Koonin EV. (2003) Remarkable interkingdom conservation of intron positions and massive, lineage-specific intron loss and gain in eukaryotic evolution. Curr Biol 13:1512–1517.[CrossRef][ISI][Medline]

Roy SW. (2004) The origin of recent introns: transposons? Genome Biol 5:251.[CrossRef][Medline]

Roy SW, Fedorov A, Gilbert W. (2003) Large-scale comparison of intron positions in mammalian genes shows intron loss but no gain. Proc Natl Acad Sci USA 100:7158–7162.[Abstract/Free Full Text]

Roy SW and Gilbert W. (2006) The evolution of spliceosomal introns: patterns, puzzles and progress. Nat Rev Genet 7:211–221.[CrossRef][ISI][Medline]

Roy SW and Hartl DL. (2006) Very little intron loss/gain in Plasmodium: intron loss/gain mutation rates and intron number. Genome Research 16:750–756.[Abstract/Free Full Text]

Roy SW, Irimia M, Penny D. (2006) Very little intron gain in Entamoeba hystolitica genes laterally transferred from prokaryotes. Mol Biol Evol 23:1824–1827.[Abstract/Free Full Text]

Sakharkar MK, Tan TW, de Souza SJ. (2001) Generation of a database containing discordant intron positions in eukaryotic genes (MIDB). Bioinformatics 17:671–675.[Abstract/Free Full Text]

Seo H-C, Kube M, Edvardsen RB, et al. (11 co-authors). (2001) Miniature genome in the marine chordate Oikopleura dioica. Science 294:2506.[Free Full Text]

Slamovits CH and Keeling PJ. (2006) A high density of ancient spliceosomal introns in oxymonad excavates. BMC Evol Biol 6:34.[CrossRef][Medline]

Stajich JE and Dietrich FS. (2006) Evidence of mRNA-mediated intron loss in the human-pathogenic fungus Cryptococcus neoformans. Eukaryotic Cell 5:789–793.[Abstract/Free Full Text]

Tarrío R, Rodríguez-Trelles F, Ayala FJ. (1998) New Drosophila introns originate by duplication. Proc Natl Acad Sci USA 95:1658–1662.[Abstract/Free Full Text]

Tarrío R, Rodríguez-Trelles F, Ayala FJ. (2003) A new Drosophila spliceosomal intron position is common in plants. Proc Natl Acad Sci USA 100:6580–6583.[Abstract/Free Full Text]

Venkatesh B, Ning Y, Brenner S. (1999) Late changes in spliceosomal introns define clades in vertebrate evolution. Proc Natl Acad Sci USA 96:10267–10271.[Abstract/Free Full Text]

Wada H, Kobayashi M, Sato R, Satoh N, Miyasaka H, Shirayama Y. (2002) Dynamic insertion-deletion of introns in deuterostome EF-1 alpha genes. J Mol Evol 54:118–128.[CrossRef][ISI][Medline]

Wolf YI, Kondrashov FA, Koonin EV. (2001) Footprints of primordial introns on the eukaryotic genome: still no clear traces. Trends Genet 17:499–450.[CrossRef][ISI][Medline]

Accepted for publication August 24, 2006.

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This Article Abstract FREE Full Text (PDF) All Versions of this Article: 23/12/2259    most recent msl098v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Request Permissions Google Scholar Articles by Roy, S. W. Articles by Penny, D. Search for Related Content PubMed PubMed Citation Articles by Roy, S. W. Articles by Penny, D. Social Bookmarking          What's this?

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