The Patterns of Codon Usage between Chordates and Arthropods are Different but Co-evolving with Mutational Biases.

Different frequencies amongst codons that encode the same amino acid (i.e. synonymous codons) have been observed in multiple species. Studies focused on uncovering the forces that drive such codon usage showed that a combined effect of mutational biases and translational selection works to produce different frequencies of synonymous codons. However, only few have been able to measure and distinguish between these forces that may leave similar traces on the coding regions. Here, we have developed a codon model that allows the disentangling of mutation, selection on amino acids and synonymous codons, and GC-biased gene conversion (gBGC) which we employed on an extensive dataset of 415 chordates and 191 arthropods. We found that chordates need 15 more synonymous codon categories than arthropods to explain the empirical codon frequencies, which suggests that the extent of codon usage can vary greatly between animal phyla. Moreover, methylation at CpG sites seems to partially explain these patterns of codon usage in chordates but not in arthropods. Despite the differences between the two phyla, our findings demonstrate that in both, GC-rich codons are disfavored when mutations are GC-biased, and the opposite is true when mutations are AT-biased. This indicates that selection on the genomic coding regions might act primarily to stabilize its GC/AT content on a genome-wide level. Our study shows that the degree of synonymous codon usage varies considerably among animals, but is likely governed by a common underlying dynamic.

Polymorphism-Aware Models in RevBayes: Species Trees, Disentangling Balancing Selection, and GC-Biased Gene Conversion.

The role of balancing selection is a long-standing evolutionary puzzle. Balancing selection is a crucial evolutionary process that maintains genetic variation (polymorphism) over extended periods of time; however, detecting it poses a significant challenge. Building upon the Polymorphism-aware phylogenetic Models (PoMos) framework rooted in the Moran model, we introduce a PoMoBalance model. This novel approach is designed to disentangle the interplay of mutation, genetic drift, and directional selection (GC-biased gene conversion), along with the previously unexplored balancing selection pressures on ultra-long timescales comparable with species divergence times by analyzing multi-individual genomic and phylogenetic divergence data. Implemented in the open-source RevBayes Bayesian framework, PoMoBalance offers a versatile tool for inferring phylogenetic trees as well as quantifying various selective pressures. The novel aspect of our approach in studying balancing selection lies in polymorphism-aware phylogenetic models' ability to account for ancestral polymorphisms and incorporate parameters that measure frequency-dependent selection, allowing us to determine the strength of the effect and exact frequencies under selection. We implemented validation tests and assessed the model on the data simulated with SLiM and a custom Moran model simulator. Real sequence analysis of Drosophila populations reveals insights into the evolutionary dynamics of regions subject to frequency-dependent balancing selection, particularly in the context of sex-limited color dimorphism in Drosophila erecta.

Increased Positive Selection in Highly Recombining Genes Does not Necessarily Reflect an Evolutionary Advantage of Recombination.

It is commonly thought that the long-term advantage of meiotic recombination is to dissipate genetic linkage, allowing natural selection to act independently on different loci. It is thus theoretically expected that genes with higher recombination rates evolve under more effective selection. On the other hand, recombination is often associated with GC-biased gene conversion (gBGC), which theoretically interferes with selection by promoting the fixation of deleterious GC alleles. To test these predictions, several studies assessed whether selection was more effective in highly recombining genes (due to dissipation of genetic linkage) or less effective (due to gBGC), assuming a fixed distribution of fitness effects (DFE) for all genes. In this study, I directly derive the DFE from a gene's evolutionary history (shaped by mutation, selection, drift, and gBGC) under empirical fitness landscapes. I show that genes that have experienced high levels of gBGC are less fit and thus have more opportunities for beneficial mutations. Only a small decrease in the genome-wide intensity of gBGC leads to the fixation of these beneficial mutations, particularly in highly recombining genes. This results in increased positive selection in highly recombining genes that is not caused by more effective selection. Additionally, I show that the death of a recombination hotspot can lead to a higher dN/dS than its birth, but with substitution patterns biased towards AT, and only at selected positions. This shows that controlling for a substitution bias towards GC is therefore not sufficient to rule out the contribution of gBGC to signatures of accelerated evolution. Finally, although gBGC does not affect the fixation probability of GC-conservative mutations, I show that by altering the DFE, gBGC can also significantly affect nonsynonymous GC-conservative substitution patterns.

GC Content Across Insect Genomes: Phylogenetic Patterns, Causes and Consequences.

The proportions of A:T and G:C nucleotide pairs are often unequal and can vary greatly between animal species and along chromosomes. The causes and consequences of this variation are incompletely understood. The recent release of high-quality genome sequences from the Darwin Tree of Life and other large-scale genome projects provides an opportunity for GC heterogeneity to be compared across a large number of insect species. Here we analyse GC content along chromosomes, and within protein-coding genes and codons, of 150 insect species from four holometabolous orders: Coleoptera, Diptera, Hymenoptera, and Lepidoptera. We find that protein-coding sequences have higher GC content than the genome average, and that Lepidoptera generally have higher GC content than the other three insect orders examined. GC content is higher in small chromosomes in most Lepidoptera species, but this pattern is less consistent in other orders. GC content also increases towards subtelomeric regions within protein-coding genes in Diptera, Coleoptera and Lepidoptera. Two species of Diptera, Bombylius major and B. discolor, have very atypical genomes with ubiquitous increase in AT content, especially at third codon positions. Despite dramatic AT-biased codon usage, we find no evidence that this has driven divergent protein evolution. We argue that the GC landscape of Lepidoptera, Diptera and Coleoptera genomes is influenced by GC-biased gene conversion, strongest in Lepidoptera, with some outlier taxa affected drastically by counteracting processes.

The influence of GC-biased gene conversion on non-adaptive sequence evolution in short introns of Drosophila melanogaster.

Population genetic inference of selection on the nucleotide sequence level often proceeds by comparison to a reference sequence evolving only under mutation and population demography. Among the few candidates for such a reference sequence is the 5' part of short introns (5SI) in Drosophila. In addition to mutation and population demography, however, there is evidence for a weak force favouring GC bases, likely due to GC-biased gene conversion (gBGC), and for the effect of linked selection. Here, we use polymorphism and divergence data of Drosophila melanogaster to detect and describe the forces affecting the evolution of the 5SI. We separately analyse mutation classes, compare them between chromosomes, and relate them to recombination rate frequencies. GC-conservative mutations seem to be mainly influenced by mutation and drift, with linked selection mostly causing differences between the central and the peripheral (i.e., telomeric and centromeric) regions of the chromosome arms. Comparing GC-conservative mutation patterns between autosomes and the X chromosome showed differences in mutation rates, rather than linked selection, in the central chromosomal regions after accounting for differences in effective population sizes. On the other hand, GC-changing mutations show asymmetric site frequency spectra, indicating the presence of gBGC, varying among mutation classes and in intensity along chromosomes, but approximately equal in strength in autosomes and the X chromosome.

GC heterogeneity reveals sequence-structures evolution of angiosperm ITS2.

BACKGROUND: Despite GC variation constitutes a fundamental element of genome and species diversity, the precise mechanisms driving it remain unclear. The abundant sequence data available for the ITS2, a commonly employed phylogenetic marker in plants, offers an exceptional resource for exploring the GC variation across angiosperms. RESULTS: A comprehensive selection of 8666 species, comprising 165 genera, 63 families, and 30 orders were used for the analyses. The alignment of ITS2 sequence-structures and partitioning of secondary structures into paired and unpaired regions were performed using 4SALE. Substitution rates and frequencies among GC base-pairs in the paired regions of ITS2 were calculated using RNA-specific models in the PHASE package. The results showed that the distribution of ITS2 GC contents on the angiosperm phylogeny was heterogeneous, but their increase was generally associated with ITS2 sequence homogenization, thereby supporting the occurrence of GC-biased gene conversion (gBGC) during the concerted evolution of ITS2. Additionally, the GC content in the paired regions of the ITS2 secondary structure was significantly higher than that of the unpaired regions, indicating the selection of GC for thermodynamic stability. Furthermore, the RNA substitution models demonstrated that base-pair transformations favored both the elevation and fixation of GC in the paired regions, providing further support for gBGC. CONCLUSIONS: Our findings highlight the significance of secondary structure in GC investigation, which demonstrate that both gBGC and structure-based selection are influential factors driving angiosperm ITS2 GC content.

Revised time estimation of the ancestral human chromosome 2 fusion.

BACKGROUND: The reduction of the chromosome number from 48 in the Great Apes to 46 in modern humans is thought to result from the end-to-end fusion of two ancestral non-human primate chromosomes forming the human chromosome 2 (HSA2). Genomic signatures of this event are the presence of inverted telomeric repeats at the HSA2 fusion site and a block of degenerate satellite sequences that mark the remnants of the ancestral centromere. It has been estimated that this fusion arose up to 4.5 million years ago (Mya). RESULTS: We have developed an enhanced algorithm for the detection and efficient counting of the locally over-represented weak-to-strong (AT to GC) substitutions. By analyzing the enrichment of these substitutions around the fusion site of HSA2 we estimated its formation time at 0.9 Mya with a 95% confidence interval of 0.4-1.5 Mya. Additionally, based on the statistics derived from our algorithm, we have reconstructed the evolutionary distances among the Great Apes (Hominoidea). CONCLUSIONS: Our results shed light on the HSA2 fusion formation and provide a novel computational alternative for the estimation of the speciation chronology.

Inferring Adaptive Codon Preference to Understand Sources of Selection Shaping Codon Usage Bias.

Alternative synonymous codons are often used at unequal frequencies. Classically, studies of such codon usage bias (CUB) attempted to separate the impact of neutral from selective forces by assuming that deviations from a predicted neutral equilibrium capture selection. However, GC-biased gene conversion (gBGC) can also cause deviation from a neutral null. Alternatively, selection has been inferred from CUB in highly expressed genes, but the accuracy of this approach has not been extensively tested, and gBGC can interfere with such extrapolations (e.g., if expression and gene conversion rates covary). It is therefore critical to examine deviations from a mutational null in a species with no gBGC. To achieve this goal, we implement such an analysis in the highly AT rich genome of Dictyostelium discoideum, where we find no evidence of gBGC. We infer neutral CUB under mutational equilibrium to quantify "adaptive codon preference," a nontautologous genome wide quantitative measure of the relative selection strength driving CUB. We observe signatures of purifying selection consistent with selection favoring adaptive codon preference. Preferred codons are not GC rich, underscoring the independence from gBGC. Expression-associated "preference" largely matches adaptive codon preference but does not wholly capture the influence of selection shaping patterns across all genes, suggesting selective constraints associated specifically with high expression. We observe patterns consistent with effects on mRNA translation and stability shaping adaptive codon preference. Thus, our approach to quantifying adaptive codon preference provides a framework for inferring the sources of selection that shape CUB across different contexts within the genome.

Using GC Content to Compare Recombination Patterns on the Sex Chromosomes and Autosomes of the Guppy, Poecilia reticulata, and Its Close Outgroup Species.

Genetic and physical mapping of the guppy (Poecilia reticulata) have shown that recombination patterns differ greatly between males and females. Crossover events occur evenly across the chromosomes in females, but in male meiosis they are restricted to the tip furthest from the centromere of each chromosome, creating very high recombination rates per megabase, as in pseudoautosomal regions of mammalian sex chromosomes. We used GC content to indirectly infer recombination patterns on guppy chromosomes, based on evidence that recombination is associated with GC-biased gene conversion, so that genome regions with high recombination rates should be detectable by high GC content. We used intron sequences and third positions of codons to make comparisons between sequences that are matched, as far as possible, and are all probably under weak selection. Almost all guppy chromosomes, including the sex chromosome (LG12), have very high GC values near their assembly ends, suggesting high recombination rates due to strong crossover localization in male meiosis. Our test does not suggest that the guppy XY pair has stronger crossover localization than the autosomes, or than the homologous chromosome in the close relative, the platyfish (Xiphophorus maculatus). We therefore conclude that the guppy XY pair has not recently undergone an evolutionary change to a different recombination pattern, or reduced its crossover rate, but that the guppy evolved Y-linkage due to acquiring a male-determining factor that also conferred the male crossover pattern. We also identify the centromere ends of guppy chromosomes, which were not determined in the genome assembly.

Runaway GC Evolution in Gerbil Genomes.

Recombination increases the local GC-content in genomic regions through GC-biased gene conversion (gBGC). The recent discovery of a large genomic region with extreme GC-content in the fat sand rat Psammomys obesus provides a model to study the effects of gBGC on chromosome evolution. Here, we compare the GC-content and GC-to-AT substitution patterns across protein-coding genes of four gerbil species and two murine rodents (mouse and rat). We find that the known high-GC region is present in all the gerbils, and is characterized by high substitution rates for all mutational categories (AT-to-GC, GC-to-AT, and GC-conservative) both at synonymous and nonsynonymous sites. A higher AT-to-GC than GC-to-AT rate is consistent with the high GC-content. Additionally, we find more than 300 genes outside the known region with outlying values of AT-to-GC synonymous substitution rates in gerbils. Of these, over 30% are organized into at least 17 large clusters observable at the megabase-scale. The unusual GC-skewed substitution pattern suggests the evolution of genomic regions with very high recombination rates in the gerbil lineage, which can lead to a runaway increase in GC-content. Our results imply that rapid evolution of GC-content is possible in mammals, with gerbil species providing a powerful model to study the mechanisms of gBGC.

The Interaction of Natural Selection and GC Skew May Drive the Fast Evolution of a Sand Rat Homeobox Gene.

Several processes can lead to strong GC skew in localized genomic regions. In most cases, GC skew should not affect conserved amino acids because natural selection will purge deleterious alleles. However, in the gerbil subfamily of rodents, several conserved genes have undergone radical alteration in association with strong GC skew. An extreme example concerns the highly conserved homeobox gene Pdx1, which is uniquely divergent and GC rich in the sand rat Psammomys obesus and close relatives. Here, we investigate the antagonistic interplay between very rare amino acid changes driven by GC skew and the force of natural selection. Using ectopic protein expression in cell culture, pulse-chase labeling, in vitro mutagenesis, and drug treatment, we compare properties of mouse and sand rat Pdx1 proteins. We find that amino acid change driven by GC skew resulted in altered protein stability, with a significantly longer protein half-life for sand rat Pdx1. Using a reversible inhibitor of the 26S proteasome, MG132, we find that sand rat and mouse Pdx1 are both degraded through the ubiquitin proteasome pathway. However, in vitro mutagenesis reveals this pathway operates through different amino acid residues. We propose that GC skew caused loss of a key ubiquitination site, conserved through vertebrate evolution, and that sand rat Pdx1 evolved or fixed a new ubiquitination site to compensate. Our results give molecular insight into the power of natural selection in the face of maladaptive changes driven by strong GC skew.

Influence of Recombination and GC-biased Gene Conversion on the Adaptive and Nonadaptive Substitution Rate in Mammals versus Birds.

Recombination is expected to affect functional sequence evolution in several ways. On the one hand, recombination is thought to improve the efficiency of multilocus selection by dissipating linkage disequilibrium. On the other hand, natural selection can be counteracted by recombination-associated transmission distorters such as GC-biased gene conversion (gBGC), which tends to promote G and C alleles irrespective of their fitness effect in high-recombining regions. It has been suggested that gBGC might impact coding sequence evolution in vertebrates, and particularly the ratio of nonsynonymous to synonymous substitution rates (dN/dS). However, distinctive gBGC patterns have been reported in mammals and birds, maybe reflecting the documented contrasts in evolutionary dynamics of recombination rate between these two taxa. Here, we explore how recombination and gBGC affect coding sequence evolution in mammals and birds by analyzing proteome-wide data in six species of Galloanserae (fowls) and six species of catarrhine primates. We estimated the dN/dS ratio and rates of adaptive and nonadaptive evolution in bins of genes of increasing recombination rate, separately analyzing AT → GC, GC → AT, and G ↔ C/A ↔ T mutations. We show that in both taxa, recombination and gBGC entail a decrease in dN/dS. Our analysis indicates that recombination enhances the efficiency of purifying selection by lowering Hill-Robertson effects, whereas gBGC leads to an overestimation of the adaptive rate of AT → GC mutations. Finally, we report a mutagenic effect of recombination, which is independent of gBGC.

Episodic and guanine-cytosine-biased bursts of intragenomic and interspecific synonymous divergence in Ajugoideae (Lamiaceae) mitogenomes.

Synonymous substitution rates in plant mitochondrial genomes vary by orders of magnitude among species, whereas synonymous rates among genes within a genome are generally consistent. Exceptionally, genes within the Ajuga reptans (Lamiaceae) mitochondrial genome exhibit unprecedented intragenomic heterogeneity in synonymous sequence divergence, but the biological mechanisms underlying this rate variation remain unclear. We tracked the origin and evolutionary trajectory of mitochondrial rate variations by dense sampling in Ajugoideae and found differences in the timing and magnitude of rate acceleration for particular genes. The most divergent genes accelerated earlier, retained a high rate across Ajugoideae, and are generally devoid of RNA editing, whereas moderately diverged genes accelerated later and retained relatively higher RNA editing frequency. The acceleration of mutation rates correlates with increased guanine-cytosine (GC) content, suggesting a key role for GC-biased gene conversion and/or repair after the breakage of ancestral gene clusters.

GC content of vertebrate exome landscapes reveal areas of accelerated protein evolution.

BACKGROUND: Rapid accumulation of vertebrate genome sequences render comparative genomics a powerful approach to study macro-evolutionary events. The assessment of phylogenic relationships between species routinely depends on the analysis of sequence homology at the nucleotide or protein level. RESULTS: We analyzed mRNA GC content, codon usage and divergence of orthologous proteins in 55 vertebrate genomes. Data were visualized in genome-wide landscapes using a sliding window approach. Landscapes of GC content reveal both evolutionary conservation of clustered genes, and lineage-specific changes, so that it was possible to construct a phylogenetic tree that closely matched the classic "tree of life". Landscapes of GC content also strongly correlated to landscapes of amino acid usage: positive correlation with glycine, alanine, arginine and proline and negative correlation with phenylalanine, tyrosine, methionine, isoleucine, asparagine and lysine. Peaks of GC content correlated strongly with increased protein divergence. CONCLUSIONS: Landscapes of base- and amino acid composition of the coding genome opens a new approach in comparative genomics, allowing identification of discrete regions in which protein evolution accelerated over deep evolutionary time. Insight in the evolution of genome structure may spur novel studies assessing the evolutionary benefit of genes in particular genomic regions.

Population size influences the type of nucleotide variations in humans.

BACKGROUND: It is well known that the effective size of a population (Ne) is one of the major determinants of the amount of genetic variation within the population. However, it is unclear whether the types of genetic variations are also dictated by the effective population size. To examine this, we obtained whole genome data from over 100 populations of the world and investigated the patterns of mutational changes. RESULTS: Our results revealed that for low frequency variants, the ratio of AT→GC to GC→AT variants (ß) was similar across populations, suggesting the similarity of the pattern of mutation in various populations. However, for high frequency variants, ß showed a positive correlation with the effective population size of the populations. This suggests a much higher proportion of high frequency AT→GC variants in large populations (e.g. Africans) compared to those with small population sizes (e.g. Asians). These results imply that the substitution patterns vary significantly between populations. These findings could be explained by the effect of GC-biased gene conversion (gBGC), which favors the fixation of G/C over A/T variants in populations. In large population, gBGC causes high ß. However, in small populations, genetic drift reduces the effect of gBGC resulting in reduced ß. This was further confirmed by a positive relationship between Ne and ß for homozygous variants. CONCLUSIONS: Our results highlight the huge variation in the types of homozygous and high frequency polymorphisms between world populations. We observed the same pattern for deleterious variants, implying that the homozygous polymorphisms associated with recessive genetic diseases will be more enriched with G or C in populations with large Ne (e.g. Africans) than in populations with small Ne (e.g. Europeans).

Open chromatin reveals the functional maize genome.

Cellular processes mediated through nuclear DNA must contend with chromatin. Chromatin structural assays can efficiently integrate information across diverse regulatory elements, revealing the functional noncoding genome. In this study, we use a differential nuclease sensitivity assay based on micrococcal nuclease (MNase) digestion to discover open chromatin regions in the maize genome. We find that maize MNase-hypersensitive (MNase HS) regions localize around active genes and within recombination hotspots, focusing biased gene conversion at their flanks. Although MNase HS regions map to less than 1% of the genome, they consistently explain a remarkably large amount (∼40%) of heritable phenotypic variance in diverse complex traits. MNase HS regions are therefore on par with coding sequences as annotations that demarcate the functional parts of the maize genome. These results imply that less than 3% of the maize genome (coding and MNase HS regions) may give rise to the overwhelming majority of phenotypic variation, greatly narrowing the scope of the functional genome.

Direct Determination of the Mutation Rate in the Bumblebee Reveals Evidence for Weak Recombination-Associated Mutation and an Approximate Rate Constancy in Insects.

Accurate knowledge of the mutation rate provides a base line for inferring expected rates of evolution, for testing evolutionary hypotheses and for estimation of key parameters. Advances in sequencing technology now permit direct estimates of the mutation rate from sequencing of close relatives. Within insects there have been three prior such estimates, two in nonsocial insects (Drosophila: 2.8 × 10-9 per bp per haploid genome per generation; Heliconius: 2.9 × 10-9) and one in a social species, the honeybee (3.4 × 10-9). Might the honeybee's rate be ∼20% higher because it has an exceptionally high recombination rate and recombination may be directly or indirectly mutagenic? To address this possibility, we provide a direct estimate of the mutation rate in the bumblebee (Bombus terrestris), this being a close relative of the honeybee but with a much lower recombination rate. We confirm that the crossover rate of the bumblebee is indeed much lower than honeybees (8.7 cM/Mb vs. 37 cM/Mb). Importantly, we find no significant difference in the mutation rates: we estimate for bumblebees a rate of 3.6 × 10-9 per haploid genome per generation (95% confidence intervals 2.38 × 10-9 and 5.37 × 10-9) which is just 5% higher than the estimate that of honeybees. Both genomes have approximately one new mutation per haploid genome per generation. While we find evidence for a direct coupling between recombination and mutation (also seen in honeybees), the effect is so weak as to leave almost no footprint on any between-species differences. The similarity in mutation rates suggests an approximate constancy of the mutation rate in insects.

Impact of Recombination on the Base Composition of Bacteria and Archaea.

The mutational process in bacteria is biased toward A and T, and most species are GC-rich relative to the mutational input to their genome. It has been proposed that the shift in base composition is an adaptive process-that natural selection operates to increase GC-contents-and there is experimental evidence that bacterial strains with GC-rich versions of genes have higher growth rates than those strains with AT-rich versions expressing identical proteins. Alternatively, a nonadaptive process, GC-biased gene conversion (gBGC), could also increase the GC-content of DNA due to the mechanistic bias of gene conversion events during recombination. To determine what role recombination plays in the base composition of bacterial genomes, we compared the spectrum of nucleotide polymorphisms introduced by recombination in all microbial species represented by large numbers of sequenced strains. We found that recombinant alleles are consistently biased toward A and T, and that the magnitude of AT-bias introduced by recombination is similar to that of mutations. These results indicate that recombination alone, without the intervention of selection, is unlikely to counteract the AT-enrichment of bacterial genomes.

Avian Genomes Revisited: Hidden Genes Uncovered and the Rates versus Traits Paradox in Birds.

According to current assemblies, avian genomes differ from those of the other lineages of amniotes in 1) containing a lower number of genes; 2) displaying a high stability of karyotype and recombination map; and 3) lacking any correlation between evolutionary rates (dN/dS) and life-history traits, unlike mammals and nonavian reptiles. We question the reality of the bird missing genes and investigate whether insufficient representation of bird gene content might have biased previous evolutionary analyses. Mining RNAseq data, we show that the vast majority of the genes missing from avian genome assemblies are actually present in most species of birds. These mainly correspond to the GC-rich fraction of the bird genome, which is the most difficult to sequence, assemble and annotate. With the inclusion of these genes in a phylogenomic analysis of high-quality alignments, we uncover a positive and significant correlation between the ratio of nonsynonymous to synonymous substitution rate (dN/dS) and life-history traits in Neoaves. We report a strong effect of GC-biased gene conversion on the dN/dS ratio in birds and a peculiar behavior of Palaeognathae (ostrich and allies) and Galloanserae (chickens, ducks and allies). Avian genomes do not contain fewer genes than mammals or nonavian reptiles. Previous analyses have overlooked ∼15% of the bird gene complement. GC-rich regions, which are the most difficult to access, are a key component of amniote genomes. They experience peculiar molecular processes and must be included for unbiased functional and comparative genomic analyses in birds.

Conserved Nonexonic Elements: A Novel Class of Marker for Phylogenomics.

Noncoding markers have a particular appeal as tools for phylogenomic analysis because, at least in vertebrates, they appear less subject to strong variation in GC content among lineages. Thus far, ultraconserved elements (UCEs) and introns have been the most widely used noncoding markers. Here we analyze and study the evolutionary properties of a new type of noncoding marker, conserved nonexonic elements (CNEEs), which consists of noncoding elements that are estimated to evolve slower than the neutral rate across a set of species. Although they often include UCEs, CNEEs are distinct from UCEs because they are not ultraconserved, and, most importantly, the core region alone is analyzed, rather than both the core and its flanking regions. Using a data set of 16 birds plus an alligator outgroup, and ∼3600-∼3800 loci per marker type, we found that although CNEEs were less variable than bioinformatically derived UCEs or introns and in some cases exhibited a slower approach to branch resolution as determined by phylogenomic subsampling, the quality of CNEE alignments was superior to those of the other markers, with fewer gaps and missing species. Phylogenetic resolution using coalescent approaches was comparable among the three marker types, with most nodes being fully and congruently resolved. Comparison of phylogenetic results across the three marker types indicated that one branch, the sister group to the passerine + falcon clade, was resolved differently and with moderate (>70%) bootstrap support between CNEEs and UCEs or introns. Overall, CNEEs appear to be promising as phylogenomic markers, yielding phylogenetic resolution as high as for UCEs and introns but with fewer gaps, less ambiguity in alignments and with patterns of nucleotide substitution more consistent with the assumptions of commonly used methods of phylogenetic analysis.