MBE Advance Access originally published online on December 3, 2007
Molecular Biology and Evolution 2008 25(2):402-408; doi:10.1093/molbev/msm268
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Research Articles |
Large-Scale Appearance of Ultraconserved Elements in Tetrapod Genomes and Slowdown of the Molecular Clock
ARC Special Research Centre for Functional and Applied Genomics and ARC Centre of Excellence in Bioinformatics, Institute for Molecular Bioscience, University of Queensland, St Lucia, Australia
E-mail: j.mattick{at}imb.uq.edu.au.
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Abstract |
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Mammalian genomes contain millions of highly conserved noncoding sequences, many of which are regulatory. The most extreme examples are the 481 ultraconserved elements (UCEs) that are identical over at least 200 bp in human, mouse, and rat and show 96% identity with chicken, which diverged approximately 310 MYA. If the substitution rate in UCEs remained constant, these elements should also be present with a high level of identity in fish (
450 Myr), but this is not the case, suggesting that many appeared in the amniotes or tetrapods or that the molecular clock has slowed down in these lineages, or both. Taking advantage of the availability of multiple genomes, we identified 13,736 UCEs in the human genome that are identical over at least 100 bp in at least 3 of 5 placental mammals, including 2,189 sequences over at least 200 bp, thereby greatly expanding the repertoire of known UCEs, and investigated the evolution of these sequences in opossum, chicken, frog, and fish. We conclude that there was a massive genome-wide acquisition and expansion of UCEs during tetrapod and then amniote evolution, accompanied by a slowdown of the molecular clock, particularly in the amniotes, a process consistent with their functional exaptation in these lineages. The majority of tetrapod-specific UCEs are noncoding and associated with genes involved in regulation of transcription and development. In contrast, fish genomes contain relatively few UCEs, the majority of which are common to all bony vertebrates. These elements are different from other conserved noncoding elements and appear to be important regulatory innovations that became fixed following the emergence of vertebrates from the sea to the land.
Key Words: ultraconserved elements • exaptation • molecular clock
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionSupplementary MaterialAcknowledgementsReferences |
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It is clear that non–protein-coding sequences make up a larger fraction of the most highly conserved regions of the mammalian genome than protein-coding sequences (Waterston et al. 2002
; Siepel et al. 2005). Because all vertebrates and, indeed, all metazoans have a similar number of protein-coding genes and a similar extent of protein-coding sequences and that the relative proportion of noncoding sequences scales broadly with developmental complexity, it has been suggested that non–protein-coding regions of the genome play key roles in the developmental programming and phenotypic diversity of higher organisms (Taft et al. 2007). There are millions of highly conserved sequences presumably under selection for biological function (Dermitzakis et al. 2002; Boffelli et al. 2003; Margulies et al. 2003; Siepel et al. 2005), and subsequent studies have proven that hundreds of these are regulatory (de la Calle-Mustienes et al. 2005; Poulin et al. 2005; Pennacchio et al. 2006; Lareau et al. 2007; Ni et al. 2007).
In 2004, 481 ultraconserved elements (UCEs) that are identical between human, mouse, and rat over 200 bp were described (Bejerano et al. 2004). These sequences show a very low substitution rate over considerable evolutionary distances—the identity between human and chicken sequences is on average approximately 96% (Bejerano et al. 2004), indicating that the substitution rate within UCEs is less than 1% per site per 100 Myr. If such a low substitution rate remained unchanged throughout vertebrate evolution, the majority of UCEs should be present in fish genomes (common ancestor 450 Myr) with identity close to 90%. However, this is not observed (Bejerano et al. 2004), which strongly suggests that either the substitution rate within UCEs has changed in the course of evolution or that the UCEs were exapted into functional roles in the amniote or tetrapod lineages, or both. It is also evident that there are also many more shorter elements with similar properties (Bejerano et al. 2004), although these sequences have not been studied.
The subsequent publication of other mammalian and vertebrate genome sequences allowed us to undertake a more detailed analysis of both the incidence and the evolution of UCEs from fish to mammals. We identified almost 14,000 UCEs 
100 bp that are identical in at least 3 placental mammals and found that these elements largely appeared during tetrapod evolution, concomitant with a massive slowdown in their molecular clock, particularly in the amniotes, indicative of genome-wide functional exaptation followed by strong purifying selection.
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Materials and Methods |
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Identification of UCEs
Human-centric and mouse-centric multiple alignments of 17 species were downloaded from the University of California Santa Cruz (UCSC) genome browser (Kuhn et al. 2007
) (genome assembly versions human hg18, mouse mm8, rat rn4, dog canFam2, cow bosTau2, opossum monDom4, chicken galGal2, frog xenTro1, puffer fish fr1) and scanned for identical regions in 3 placental mammals excluding unassembled ("random" or unknown), haplotype and mitochondrial chromosomes. We identified 11,110 elements identical for at least 100 bp between human, dog, and cow (HDC); 5,505 between human, mouse, and rat (HMR); and 5,546 between mouse, rat, and dog (MRD). These elements identical in 3 mammals’ comparisons often have substitutions in other species, and so the elements do not always overlap. The MRD elements were mapped to human coordinates using the multiple alignments and requiring at least 95% identity with human and no more than 5% indels (removing 66 elements). We then made a union set of the HDC, HMR, and mapped MRD elements yielding 13,736 nonoverlapping elements (EU100+, table 1) covering a total of 2.131 Mb. Every base of these elements are identical in one of these sets (at least 3 of 5 mammals), but may contain some substitutions in the other species as shown in supplementary figure S1 (Supplementary Material online).
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Zebrafish-centric multiple alignments of 7 species were downloaded from the UCSC genome browser (Kuhn et al. 2007
) (genome assembly versions zebrafish danRer4, tetraodon tetNig1, and puffer fish fr1) and scanned for identical regions at least 100 bp long between the 3 fish, finding 43 unique elements after discarding 7 which were duplicated in the zebrafish genome assembly (table 1). Stickleback-centric multiple alignment of 8 species were downloaded from the same site and contain the following genome assembly versions: stickleback (February 2006, gasAcu1), medaka (October 2005, oryLat1), puffer fish (October 2004, fr2), tetraodon (February 2004, tetNig1), zebrafish (March 2006, danRer4), chicken (May 2006, galGal3), mouse (February 2006, mm8), and human (March 2006, hg18).
Orthologous UCEs
To determine the presence of orthologous elements in different species, we required elements to have at least 20 alignable nucleotides. We defined ancient elements as those with detectable orthologs in puffer fish. We defined elements appearing in the tetrapod ancestor as those with orthologs in frog but not in puffer fish; elements appearing in amniotes as those with orthologs in chicken but not frog or puffer fish; elements appearing in theria as those with orthologs in opossum but not in chicken, frog, or puffer fish; and elements appearing in eutheria as those without orthologs in opossum, chicken, frog, or puffer fish.
Gene Ontology Enrichments
Enrichments and P values were calculated essentially as described in Simons et al. (2006) counting genes once (not their multiple isoforms), requiring at least 2-fold enrichment and Fisher's exact P value 
10–10 and only considering terms with at least 5 associated genes. Intergenic UCEs were associated with the nearest gene to the "left" and the nearest to the "right" within 100 kb (if any). We did not directly correct for multiple hypothesis testing because, in practice, we performed less than 104 individual tests.
Phylogenetic Trees
The phylogenetic trees and standard errors for branch length were calculated in MEGA3 (Kumar et al. 2004) using Neighbor-Joining method with Kimura 2 parameter and uniform rates among sites.
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Results and Discussion |
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionSupplementary MaterialAcknowledgementsReferences |
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Expansion of the Set of Eutherian UCEs
The initial 3-way mouse–human–rat comparisons defined the original set of UCEs as syntenically equivalent sequences (i.e., those that are located in orthologous regions) that are identical among these species over at least 200 bp. However, examination of other genomes whose sequences have subsequently been published showed that these UCEs are not always identical (although very highly conserved) in other placental mammals and also that there are much larger numbers of shorter UCEs of length 100–199 bp which have similar very low substitution rates (see below). We therefore undertook other 3-way genomic comparisons using multiple alignments (Kuhn et al. 2007
) creating 3 sets of UCEs now defined as being syntenically equivalent sequences 100 bp that are identical in human–dog–cow (11,110 UCEs), human–mouse–rat (5,505 UCEs), and mouse–rat–dog (5,546 UCEs), respectively. We then formed a union of elements from all 3 sets (in human coordinates, see Materials and Methods) resulting in a set of 13,736 eutherian UCEs 100 bp covering 2.1 Mb (EU100+), which have similar properties as the original UCEs (see below) and became the reference set for subsequent analyses. This set contains 2,189 elements 200 bp (EU200+), over 4-fold more than originally reported from human–mouse–rat comparisons alone (Bejerano et al. 2004).
Incidence of Sequences Orthologous to Eutherian UCEs in Other Vertebrates
We then examined the distribution and conservation of these elements in other key vertebrate species whose genomes have been published, namely opossum, chicken, frog, and puffer fish, based on the human-centric alignment of 17 vertebrate genomes. We found that these elements are highly conserved in amniotes—over 83% are present in chicken and on average 92% identical with human—but that both the number of UCEs and their level of identity decrease sharply with more distant lineages. Two-thirds have recognizable orthologous sequences in amphibia (albeit tending to shorter lengths, see below), but only 39% have recognizable orthologous sequences in puffer fish (Fugu rubripes), where the average identity drops to 74% (table 1). Other bony fish species whose genome sequences have recently become available show very similar proportions of detectable orthologs and degree of divergence of UCEs, with stickleback and medaka having 38% (5,799) and 42% (5,251) recognizable orthologous sequences, respectively, both at 73% average identity.
We divided UCEs into several groups based on when they are first detectable in evolutionary history and found that the major increase in elements appeared before tetrapod speciation. There are approximately 5,500 (40%) "ancient" elements that are evident in fish, 4,160 (30%) that are first detected in frog, and 2,540 (18%) that first appeared in the amniotes. Another 1,388 (10%) arose in the therian ancestor (180 MYA) and just 244 (2%) appeared during the evolution of placental mammals. A large proportion of UCEs that are located within protein-coding exons or overlapping splice sites are ancient and preserved throughout vertebrate evolution, whereas the emergence of newer elements has mainly occurred in intronic and intergenic regions (fig. 1). We refer to the 8,332 elements that are first detected in terrestrial vertebrates (i.e., first detected in frog, chicken, or mammals) as "new" elements to contrast them with those that are ancient and (still) detectable in puffer fish.
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Genomic Distribution of UCEs
The EU100+ elements are overwhelmingly noncoding—based on human RefSeq annotations (Kuhn et al. 2007
; Pruitt et al. 2007), 46% of UCEs are intergenic, 30% are intronic, and although 24% overlap exons, half of these overlap splice sites (suggesting that their presence is related mainly to splicing regulation rather than structure–function constraints in the encoded protein), and only 391 (3%) lie entirely within protein-coding exons (fig. 1). The vast majority of UCEs (12,917) are unique within the well-assembled fraction of the human genome, as determined by BLAT searches. The remaining 819 UCEs have more than one BLAT hit (ranging from 2 to 83 hits) within the well-assembled fraction of the human genome with different levels of similarity. Among these, 555 (two-thirds) UCEs overlap exons of RefSeq genes, and the corresponding repeated sequences appear to be located in homologous pseudogenes. In addition, some UCEs have very similar sequences present in the vicinity of paralogous genes, and it appears that the selection pressures that maintain these sequences have been continued after duplication (Bejerano et al. 2004; Sandelin et al. 2004).
UCEs are preferentially located near or within genes involved in regulation of transcription and of development (as has been noted for highly conserved noncoding sequences by others, e.g., Woolfe et al. 2005) according to Gene Ontology (GO) annotations (Camon et al. 2004; Consortium 2006; Hsu et al. 2006) (April 2007) (supplementary tables S1–S5, Supplementary Material online). Ancient UCEs are 3-fold enriched in or around genes annotated with the "biological process" GO terms of regulating cell differentiation (P < 10–10) and specifically brain (P < 10–11) and central nervous system (CNS) development (P < 10–17). Additionally, of the 57 RNA splicing genes that contain ancient UCEs, 51 have UCEs overlapping exons (over 3-fold enrichment, P < 10–12), consistent with earlier observations (Bejerano et al. 2004) and suggesting an ancient origin of homeostatic autoregulation of splicing regulators (Lareau et al. 2007; Ni et al. 2007). Elements first detected before tetrapod speciation are 6-fold enriched in or around genes involved in brain development (P < 10–25) and cell fate commitment (P < 10–17) and have a total of 26 terms with over 3-fold enrichment (5 times as many as for ancient elements), including 7 relating to neurogenesis. Elements first detected before amniote speciation are 4-fold enriched for CNS development (P < 10–13). In contrast, the term "chromatin modification" shows the highest enrichment in elements first detected in the mammalian lineage (4-fold enrichment, P < 10–12). Curiously, some of these genes such as histone deacetylases HDAC5 and HDAC8, retinoblastoma-binding protein RBBP7, Wolf-Hirschhorn syndrome candidate 1 protein WHSC1, tousled-like kinase TLK1, and chromodomain helicase DNA-binding protein 3 CHD3 contain only mammalian-specific elements. Calculating the enrichment in terms of base-pair overlap by UCEs, we found that for the terms mentioned above UCEs are at least 2-fold and up to 6.4-fold enriched for sequence overlap except for ancient exonic elements which are 30-fold enriched for coverage of RNA splicing genes (supplementary table S6, Supplementary Material online).
New elements also tend to appear in the vicinity of genes that already had ancient elements. Of the 3,329 genes associated with UCEs, 2,018 genes (61%) have ancient elements and 1,010 (50%) of these have acquired novel ones in subsequent lineages. An example of the clustering of these elements during evolution is shown for chromosome 2 in supplementary figure S2 (Supplementary Material online). UCEs are also highly associated with transposon-free regions (TFRs) of the human genome (Simons et al. 2006)—51% of all TFRs 10 kb contain a UCE—and this is particularly pronounced in ancient elements (35-fold enriched for UCEs by sequence coverage) and elements exapted in tetrapods (28-fold enriched). These results suggest that the evolution of the tetrapod lineage coincides with increasing complexity of the regulatory system in and around key developmental genes.
There are also interesting distributions of UCEs within intergenic regions (outside protein-coding genes). There are 3,918 annotated "intergenic" noncoding RNA genes, and 195 (5.0%) of these contain 542 (3.9%) UCEs (coding and noncoding annotations from UCSC genes, April 2007, Hsu et al. 2006). There are no functional annotations for these noncoding RNAs (which including introns cover 71 Mb or 4.1% of intergenic regions), but they are 2.2-fold enriched for intergenic UCEs by base-pair coverage (1.6-fold enriched for all UCEs), compared with just 1.4-fold enrichment for UCEs in protein-coding genes (covering 40% of the genome). Intergenic UCEs are also abundant in gene deserts (Ovcharenko et al. 2005)—2,527 (18%) are located at least 100 kb from the nearest coding or noncoding gene, and 1,205 (8.8%) are at least 250 kb from the nearest gene (see example in supplementary fig. S2, Supplementary Material online).
Substitution Rates in UCEs and Slowdown of the Molecular Clock
Ancient UCEs show a significant decrease in the substitution rate within amniotes compared with the orthologous sequences in ancestral lineages (fig. 2A). A similar decline in the substitution rate in amniotes is also observed in UCEs first detected before tetrapod speciation (fig. 2B). The amniotic branches of the phylogenetic trees are disproportionately short compared with the amphibian and especially the fish branches, placing chicken very close to mammals. This contrasts markedly with the uniform substitution rate and constancy of the molecular clock observed in protein-coding sequences (CDS, over 16 Mb aligned in all investigated species with all human coding exons at least 100 bp long), over the entire span of vertebrate evolution, resulting in a phylogenetic tree that is congruent with the accepted divergence times (Hedges and Kumar 2003) (fig. 2C).
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Although the phylogenetic trees provide a summary of the data, the distribution of identities of individual elements show that this property holds for the majority of elements and indicate that the elements that appeared in the amniote ancestor also show a similar decline in substitution rate in mammals (supplementary fig. S3, Supplementary Material online).
The slowdown in substitution rate is even more pronounced in the subset of elements at least 200 bp long but is less pronounced in sequences identical over only 75–99 bp and even less so in shorter sequences identical over only 50–74 bp, suggesting that the latter comprise a mixture of different types (supplementary fig. S4, Supplementary Material online). Furthermore, the extremely reduced substitution rate in amniotes is not a general property of highly conserved sequences. We built a tree using the most conserved approximately 5,000 PhastCons (Siepel et al. 2005) elements (highest scoring elements from 17-way alignments, totaling 1.6 Mb aligned in all investigated species). Even though 33% of these sequences overlap with ancient UCEs and are only 13% protein coding, the phylogenetic tree shows a longer distance between human and chicken (and frog), similar to the CDS tree, indicating that the reduction in substitution rate is a feature unique to the UCEs (see supplementary fig. S4, Supplementary Material online). This data show that the substitution rate in the majority of UCEs that are ancient or first detected in the tetrapod ancestor is significantly reduced in the amniote lineage. These sequences have evolved in a non–clock-like manner over vertebrate evolution, in contrast to coding sequences (fig. 2D).
It is also clear that UCEs are not uniformly conserved across their length, which gives insight into their emergence. Figure 3 shows that the central sequences of UCEs have been more highly conserved over evolutionary time than those at the periphery, which suggests that the UCEs have not only increased in number during tetrapod evolution but also expanded in size from a core whose boundaries spread laterally as these elements evolved, presumably by acquiring more complex functions and interactions.
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The Origins of UCEs
There are 2 explanations for the observed slowing of the substitution rates within UCEs in tetrapods and particularly amniotes. The first is that the decrease in substitution rate is due to the functional exaptation (Gould and Vrba 1982
) of these sequences de novo in these lineages. This is supported by recent analyses of the derived allele frequency spectrum which indicates that, rather than being mutational cold spots, UCEs are under strong negative selection (Katzman et al. 2007), which is universally taken as prima facie evidence for biological function. There is evidence that at least some UCEs act as "enhancers" to control developmental expression of neighboring genes. A recent study documented the exaptation of a UCE derived from a Short INterspersed Element (SINE) transposable element active in the Sarcopterygii (lobe-finned fishes and tetrapods), which is absent in bony fish such as zebrafish or Fugu (Bejerano et al. 2006). The conclusion drawn was that acquisition of new function led to a decrease in substitution rate resulting in the appearance of the UCE. The exaptation of a SINE element into the highly conserved neuronal enhancer of the proopiomelanocortin gene has also been described (Santangelo et al. 2007). Moreover, we have found that, out of 169 validated enhancers in human (Visel et al. 2007), 101 overlap ancient UCEs (conserved in Fugu) and 51 enhancers overlap new UCEs first detected during terrestrial vertebrate evolution.
The alternative hypothesis is that these elements were present in the ancestral vertebrate genome but either evolved at a higher rate or were deleted in fish, wherein the whole-genome duplication might have relaxed the selection constraints acting on UCEs. However, this explanation is less parsimonious. It assumes the existence of the UCEs in the antecedent species, for which there is no evidence and for which one would have to posit the existence of important genetic capacity (assuming the conservation of the UCEs reflects purifying selection) that was both present in the ancestor and maintained in all vertebrate descendants except fish. There is evidence that noncoding sequences in HoxA loci are more highly conserved between shark and human than between shark and zebrafish, indicating that the duplication that occurred in the bony fishes may have relaxed the constraints on these noncoding sequences (Chiu et al. 2002), which, in general, could account for a higher divergence rate in the bony fishes. However, if we assume that UCEs are indeed ancient and apply the substitution rate observed in amniotes retrospectively to the antecedents of fish, we would expect to find (given the estimated divergence time) that some of those extant in amniotes would be detectable in the sea squirt Ciona intestinalis, which is not the case, except for a limited number of exonic sequences (Bejerano et al. 2004). In this respect, analysis of the conservation of EU100+ elements in the shark genome will be of interest.
Thus, although the origin of eutherian UCEs is ancient (in that they arose early in vertebrate evolution), it appears unlikely that they have been lost in fish, but rather that these particular sequences have not further expanded and become fixed in fish, as they did in tetrapods. However, this begs the question whether there may be other sequences that are unique to the fish lineage that may become ultraconserved independently in the ensuing 450 Myr. This appears not to be the case. There are only 43 sequences identical over at least 100 bp between 3 aligned fish genomes—Fugu, Tetraodon, and zebrafish (table 1) are estimated to have diverged between 110–300 MYA (Volff 2005; Hashiguchi and Nishida 2006)—and only 50 sequences identical over at least 100 bp between stickleback, medaka, and zebrafish, 21 of which are homologous to those found in the Fugu, Tetraodon, and zebrafish set. In both cases, this is 2 orders of magnitude less than the number of UCEs in amniotes with similar divergence times: 11,110 in human–dog–cow (90–98 Myr, Hedges and Kumar 2003) and 2,343 in human–dog–chicken (310 Myr, Hedges and Kumar 2003). We identified 445 sequences identical over at least 100 bp between the genomes of stickleback, medaka, and Fugu, but these species diverged more recently, between 60 and 80 and approximately 190 Myr (Volff 2005; Hashiguchi and Nishida 2006). Even if we accept the upper estimate, the number of identified UCEs in stickleback, medaka, and Fugu would be nearly one order of magnitude smaller than in the human–dog–opossum set of species with a comparable divergence time (3,941 UCEs at least 100 bp long).
Moreover, most of the UCEs in fish are present in, and have very high identity with, other tetrapod genomes. Thirty-eight (88%) of Fugu, Tetraodon, and zebrafish UCEs have identifiable orthologs in the human genome at an average of 93% identity, with similar numbers in other tetrapods (table 1), and 80% of stickleback–medaka–Fugu UCEs have identifiable orthologs in the human genome at an average of 86% identity, indicating that most fish UCEs have evolved at extremely low rates that have remained relatively uniform in all vertebrate lineages including fish, amphibia, and amniotes. Hence, the majority of fish UCEs represent sequences common to all Osteichthyes (bony vertebrates), with very few being fish specific.
Recently, thousands of fragments of mobile elements were shown to have been exapted in the human genome and under strong purifying selection since the boroeutherian ancestor (100 MYA) (Lowe et al. 2007). We found that only 63 UCEs (0.5%) covering 4.8 kb (0.2%) of sequence are annotated in human as derived from transposable elements, but there are UCEs derived from mobile elements that are no longer recognizable (Bejerano et al. 2006). Comparison of the opossum and eutherian genomes revealed that around 20% of eutherian conserved noncoding elements are recent innovations (Mikkelsen et al. 2007). In contrast to both these relatively young classes of conserved elements, many UCEs have their origins in fish and underwent both a massive expansion during the evolution of the tetrapods and a remarkable subsequent slowdown in their substitution rate during the evolution of the amniotes, with as few as 244 (2%) being unique to placental mammals.
Although there are a few reports of protein-coding or ribosomal RNA genes that have shown a change in their substitution rate (Ayala 1997; Friedrich and Tautz 1997), this is one of the first demonstrations of a massive genome-wide appearance and then extreme slowdown in the evolutionary clock of a large class of genetic elements, presumably as a result of their exaptation (Gould and Vrba 1982). Consistent with our observations, Kim and Pritchard (2007) have recently reported that 32% of 98,910 eutherian conserved noncoding elements show variation of substitution rate in different branches, suggesting that evolution of many regulatory regions occurs in a nonlinear manner, presumably due to adaptation. Likewise, we suggest that UCEs, most of which arose in noncoding regions near developmental genes during tetrapod evolution, comprise important regulatory innovations that were required to meet the considerable developmental and physiological challenges associated with the colonization of the land. However, the mechanism of action and the structure–function constraints on UCEs are still unknown. The only other sequences that show a similarly slow evolutionary rate are ribosomal RNA sequences, which have strict structure–function constraints and multilateral interactions with other RNAs and proteins. There is some evidence that UCEs may be transcribed (Feng et al. 2006). The mystery is that given their collective appearance and then change in evolutionary rate, which implies that UCEs arose by and operate through similar mechanisms, few, if any, show any detectable sequence homology, other than those associated with duplications, despite the fact that their sequences have become virtually frozen after their presumed exaptation.
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Supplementary Material |
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionSupplementary MaterialAcknowledgementsReferences |
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Supplementary figures S1–S4 and tables S1–S6 are available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/).
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Acknowledgements |
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TOPAbstractIntroductionMaterials and MethodsResults and DiscussionSupplementary MaterialAcknowledgementsReferences |
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We thank present and past members of the Mattick laboratory for discussions and comments on the manuscript. This work was supported by a Federation Fellowship (FF0561986) awarded to J.S.M. by the Australian Research Council and cofunded by the University of Queensland.
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Footnotes |
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1 These authors contributed equally to this work.
Naruya Saitou, Associate Editor
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