Utility of the white gene in estimating phylogenetic relationships among mosquitoes (Diptera: Culicidae)

The utility of a nuclear protein-coding gene for reconstructing phylogenetic relationships within the family Culicidae was explored. Relationships among 13 species representing three subfamilies and nine genera of Culicidae were analyzed using a 762-bp fragment of coding sequence from the eye color gene, white. Outgroups for the study were two species from the sister group Chaoboridae. Sequences were determined from clone PCR products amplified from genomic DNA, and aligned following conceptual intron splicing and amino acid translation. Third codon positions were characterized by high levels of divergence and biased nucleotide composition, the intensity and direction of which varied among taxa. Equal weighting of all characters resulted in parsimony and neighboring-joining trees at odds with the generally accepted phylogenetic hypothesis based on morphology and rDNA sequences. The application of differential weighting schemes recovered the traditional hypothesis, in which the subfamily Anophelinae formed the basal clade. The subfamily Toxorhynchitinae occupied an intermediate position, and was a sister group to the subfamily Culicinae. Within Culicinae, the genera Sabethes and Tripteroides formed an ancestral clade, while the Culex-Deinocerites and Aedes- Haemagogus clades occupied increasingly derived positions in the molecular phylogeny. An intron present in the Culicinae- Toxorhynchitinae lineage and one outgroup taxon was absent in the basal Anophelinae lineage and the second outgroup taxon, suggesting that intron insertions or deletions may not always be reliable systematic characters.      

Rapid sequence turnover at an intergenic locus in Drosophila

Closely related species of Drosophila tend to have similar genome sizes. The strong imbalance in favor of small deletions relative to insertions implies that the unconstrained DNA in Drosophila is unlikely to be passively inherited from even closely related ancestors, and yet most DNA in Drosophila genomes is intergenic and potentially unconstrained. In an attempt to investigate the maintenance of this intergenic DNA, we studied the evolution of an intergenic locus on the fourth chromosome of the Drosophila melanogaster genome. This 1.2-kb locus is marked by two distinct, large insertion events: a nuclear transposition of a mitochondrial sequence and a transposition of a nonautonomous DNA transposon DNAREP1_DM. Because we could trace the evolutionary histories of these sequences, we were able to reconstruct the length evolution of this region in some detail. We sequenced this locus in all four species of the D. melanogaster species complex: D. melanogaster, D. simulans, D. sechellia, and D. mauritiana. Although this locus is similar in size in these four species, less than 10% of the sequence from the most recent common ancestor remains in D. melanogaster and all of its sister species. This region appears to have increased in size through several distinct insertions in the ancestor of the D. melanogaster species complex and has been shrinking since the split of these lineages. In addition, we found no evidence suggesting that the size of this locus has been maintained over evolutionary time; these results are consistent with the model of a dynamic equilibrium between persistent DNA loss through small deletions and more sporadic DNA gain through less frequent but longer insertions. The apparent stability of genome size in Drosophila may belie very rapid sequence turnover at intergenic loci.     

Selective constraints on intron evolution in Drosophila

Intron sizes show an asymmetrical distribution in a number of organisms, with a large number of "short" introns clustered around a minimal intron length and a much broader distribution of longer introns. In Drosophila melanogaster, the short intron class is centered around 61 bp. The narrow length distribution suggests that natural selection may play a role in maintaining intron size. A comparison of 15 orthologous introns among species of the D. melanogaster subgroup indicates that, in general, short introns are not under greater DNA sequence or length constraints than long introns. There is a bias toward deletions in all introns (deletion/insertion ratio is 1.66), and the vast majority of indels are of short length (<10 bp). Indels occurring on the internal branches of the phylogenetic tree are significantly longer than those occurring on the terminal branches. These results are consistent with a compensatory model of intron length evolution in which slightly deleterious short deletions are frequently fixed within species by genetic drift, and relatively rare larger insertions that restore intron length are fixed by positive selection. A comparison of paralogous introns shared among duplicated genes suggests that length constraints differ between introns within the same gene. The janusA, janusB, and ocnus genes share two short introns derived from a common ancestor. The first of these introns shows significantly fewer indels than the second intron, although the two introns show a comparable number of substitutions. This indicates that intron-specific selective constraints have been maintained following gene duplication, which preceded the divergence of the D. melanogaster species subgroup.     

Indel evolution of mammalian introns and the utility of non-coding nuclear markers in eutherian phylogenetics

Nuclear DNA intron sequences are increasingly used to investigate evolutionary relationships among closely related organisms. The phylogenetic usefulness of intron sequences at higher taxonomic levels has, however, not been firmly established and very few studies have used these markers to address evolutionary questions above the family level. In addition, the mechanisms driving intron evolution are not well understood. We compared DNA sequence data derived from three presumably independently segregating introns (THY, PRKC I and MGF) across 158 mammalian species. All currently recognized extant eutherian mammalian orders were included with the exception of Cingulata, Dermoptera and Scandentia. The total aligned length of the data was 6366 base pairs (bp); after the exclusion of autapomorphic insertions, 1511 bp were analyzed. In many instances the Bayesian and parsimony analyses were complementary and gave significant posterior probability and bootstrap support (>80) for the monophyly of Afrotheria, Euarchontoglires, Laurasiatheria and Boreoeutheria. Apart from finding congruent support when using these methods, the intron data also provided several indels longer than 3 bp that support, among others, the monophyly of Afrotheria, Paenungulata, Ferae and Boreoeutheria. A quantitative analysis of insertions and deletions suggested that there was a 75% bias towards deletions. The average insertion size in the mammalian data set was 16.49 bp +/- 57.70 while the average deletion was much smaller (4.47 bp +/- 14.17). The tendency towards large insertions and small deletions is highlighted by the observation that out of a total of 17 indels larger than 100 bp, 15 were insertions. The majority of indels (>60% of all events) were 1 or 2 bp changes. Although the average overall indel substitution rate of 0.00559 per site is comparable to that previously reported for rodents and primates, individual analyses among different evolutionary lineages provide evidence for differences in the formation rate of indels among the different mammalian groups.     

General-use polymerase chain reaction primers for amplification and direct sequencing of enolase, a single-copy nuclear gene, from different animal phyla

In contrast to mitochondrial DNA, remarkably few general-use primer sets are available for single-copy nuclear genes across animal phyla. Here, we present a primer set that yields a c. 364-bp coding fragment of the metabolic gene enolase, which includes an intron in some taxa. In species where introns are absent or have few insertions/deletions, the amplified fragment can be sequenced directly for phylogenetic or population analysis. Between-species variation in the coding region occurs widely at third codon positions, even between closely related taxa, making the fragment useful for species-level systematics. In low gene-flow species, the primers may also be of use for population genetics, as intraspecific polymorphisms occur at several silent positions in the taxa examined.     

Species-specific double-strand break repair and genome evolution in plants

Even closely related eukaryotic species may differ drastically in genome size. While insertion of retroelements represents a major source of genome enlargement, the mechanism mediating species- specific deletions is fairly obscure. We analyzed the formation of deletions during double-strand break (DSB) repair in Arabidopsis thaliana and tobacco, two dicotyledonous plant species differing >20-fold in genome size. DSBs were induced by the rare cutting restriction endonuclease I-SCE:I and deletions were identified by loss of function of a negative selectable marker gene containing an I-SCE:I site. Whereas the partial use of micro-homologies in junction formation was similar in both species, in tobacco 40% of the deletions were accompanied by insertions. No insertions could be detected in Arabidopsis , where larger deletions were more frequent, indicating a putative inverse correlation between genome size and the average length of deletions. Such a correlation has been postulated before by a theoretical study on the evolution of related insect genomes and our study now identifies a possible molecular cause for the phenomenon, indicating that species-specific differences in DSB repair might indeed influence genome evolution.     

Recombination drives vertebrate genome contraction

Selective and/or neutral processes may govern variation in DNA content and, ultimately, genome size. The observation in several organisms of a negative correlation between recombination rate and intron size could be compatible with a neutral model in which recombination is mutagenic for length changes. We used whole-genome data on small insertions and deletions within transposable elements from chicken and zebra finch to demonstrate clear links between recombination rate and a number of attributes of reduced DNA content. Recombination rate was negatively correlated with the length of introns, transposable elements, and intergenic spacer and with the rate of short insertions. Importantly, it was positively correlated with gene density, the rate of short deletions, the deletion bias, and the net change in sequence length. All these observations point at a pattern of more condensed genome structure in regions of high recombination. Based on the observed rates of small insertions and deletions and assuming that these rates are representative for the whole genome, we estimate that the genome of the most recent common ancestor of birds and lizards has lost nearly 20% of its DNA content up until the present. Expansion of transposable elements can counteract the effect of deletions in an equilibrium mutation model; however, since the activity of transposable elements has been low in the avian lineage, the deletion bias is likely to have had a significant effect on genome size evolution in dinosaurs and birds, contributing to the maintenance of a small genome. We also demonstrate that most of the observed correlations between recombination rate and genome contraction parameters are seen in the human genome, including for segregating indel polymorphisms. Our data are compatible with a neutral model in which recombination drives vertebrate genome size evolution and gives no direct support for a role of natural selection in this process.     

Growth and decline of introns

To gauge the processes that might direct the length of introns, I studied the balance of indels (insertions or deletions, determined using Alu and LINE1 retroposon repeats) and the density of these repeats in the introns of the human genome. The indel balance is biased in favour of deletions and correlated with the divergence of repeats. At fixed repeat divergence, the indel bias correlated with the intron size: the shorter the intron, the more deletions were favoured over insertions. This correlation with the intron size was stronger than with the gene-wide or isochore-wide parameters. The density of repeats (the number of repeats in a unit of intron length) correlated positively with the intron size. Thus, quite different mechanisms, the indel bias and the integration and/or persistence of retroposons, act in the same direction in regards to intron size, which suggests selection for the size of individual introns.     

Chloroplast microsatellites and mitochondrial nad1 intron 2 sequences indicate congruent phylogenetic relationships among Swiss stone pine (Pinus cembra), Siberian stone pine (Pinus sibirica), and Siberian dwarf pine (Pinus pumila)

We studied the phylogenetic relationships among the three stone pine species, Pinus cembra, P. sibirica, and P. pumila, using chloroplast microsatellites and mitochondrial nad1 intron 2 sequences. The three chloroplast microsatellite loci combined into a total of 18 haplotypes. Fourteen haplotypes were detected in 15 populations of P. cembra and one population of P. sibirica, five of which were shared between the two species, and the two populations of P. pumila comprised four species-specific haplotypes. Mitochondrial intron sequences confirmed this grouping of species. Sequences of P. cembra and P. sibirica were identical, but P. pumila differed by several nucleotide substitutions and insertions/deletions. A repeat region found in the former two species showed no intraspecific variation. These results indicate a relatively recent evolutionary separation of P. cembra and P. sibirica, despite their currently disjunct distributions. The species-specific chloroplast and mitochondrial markers of P. sibirica and P. pumila should help to trace the hybridization in their overlapping distribution area and to identify fossil remains with respect to the still unresolved postglacial re-colonization history of these two species.     

A multi-gene phylogeny of aquiline eagles (Aves: Accipitriformes) reveals extensive paraphyly at the genus level

The phylogeny of the tribe Aquilini (eagles with fully feathered tarsi) was investigated using 4.2 kb of DNA sequence of one mitochondrial (cyt b) and three nuclear loci (RAG-1 coding region, LDH intron 3, and adenylate-kinase intron 5). Phylogenetic signal was highly congruent and complementary between mtDNA and nuclear genes. In addition to single-nucleotide variation, shared deletions in nuclear introns supported one basal and two peripheral clades within the Aquilini. Monophyly of the Aquilini relative to other birds of prey was confirmed. However, all polytypic genera within the tribe, Spizaetus, Aquila, Hieraaetus, turned out to be non-monophyletic. Old World Spizaetus and Stephanoaetus together appear to be the sister group of the rest of the Aquilini. Spizastur melanoleucus and Oroaetus isidori are nested among the New World Spizaetus species and should be merged with that genus. The Old World 'Spizaetus' species should be assigned to the genus Nisaetus (Hodgson, 1836). The sister species of the two spotted eagles (Aquila clanga and Aquila pomarina) is the African Long-crested Eagle (Lophaetus occipitalis). Hieraaetus fasciatus/spilogaster are closest to Aquila verreauxii and should be merged with that genus. Wahlberg's Eagle H. wahlbergi, formerly placed in Aquila, is part of a clade including three small Hieraaetus species (pennatus, ayresii, and morphnoides). The Martial Eagle (Polemaetus bellicosus) is the sister species of the Aquila/Hieraaetus/Lophaetus clade. Basal relationships within this clade remained unresolved. Parsimony reconstruction of the evolution of plumage pattern within Aquilini suggests that: (1) transverse barring of parts of the body plumage was lost in the Palearctic Aquila-Hieraaetus clade, (2) pale underparts in adult plumage evolved three times independently, and (3) dimorphic adult plumage is a derived character of the small-bodied Hieraaetus clade.     

Structure and sequence of the Cu,Zn Sod gene in the Mediterranean fruit fly, Ceratitis capitata: intron insertion/deletion and evolution of the gene.

We have cloned a 4-kb region encompassing the Cu,Zn superoxide dismutase (Sod) gene from a genomic library of the Mediterranean fruit fly, Ceratitis capitata, using a cDNA probe from Drosophila melanogaster. The coding sequence of 462 bases is equally as long as that in Drosophila species. The rate of amino acid replacement over the past 100 million years is approximately the same in the Diptera and in mammals, thus excluding the hypothesis (proposed to account for an apparent acceleration in rate of evolution of Sod over geological time) that the evolution of the SOD protein is much higher in the mammals than in other organisms. The coding region is interrupted by two introns in Ceratitis, whereas only one occurs in Drosophila. Phylogenetic comparisons indicate that the second intron was present in the common dipteran ancestor, but was lost shortly after the divergence of the Drosophila lineage from other Diptera. Analysis of the exon/intron structure of Sod in various animal phyla, plants, and fungi indicates that intron insertions as well as deletions have occurred in the evolution of the Sod gene.     

Phylogenetic distribution of intron positions in alpha-amylase genes of bilateria suggests numerous gains and losses

Most eukaryotes have at least some genes interrupted by introns. While it is well accepted that introns were already present at moderate density in the last eukaryote common ancestor, the conspicuous diversity of intron density among genomes suggests a complex evolutionary history, with marked differences between phyla. The question of the rates of intron gains and loss in the course of evolution and factors influencing them remains controversial. We have investigated a single gene family, alpha-amylase, in 55 species covering a variety of animal phyla. Comparison of intron positions across phyla suggests a complex history, with a likely ancestral intronless gene undergoing frequent intron loss and gain, leading to extant intron/exon structures that are highly variable, even among species from the same phylum. Because introns are known to play no regulatory role in this gene and there is no alternative splicing, the structural differences may be interpreted more easily: intron positions, sizes, losses or gains may be more likely related to factors linked to splicing mechanisms and requirements, and to recognition of introns and exons, or to more extrinsic factors, such as life cycle and population size. We have shown that intron losses outnumbered gains in recent periods, but that "resets" of intron positions occurred at the origin of several phyla, including vertebrates. Rates of gain and loss appear to be positively correlated. No phase preference was found. We also found evidence for parallel gains and for intron sliding. Presence of introns at given positions was correlated to a strong protosplice consensus sequence AG/G, which was much weaker in the absence of intron. In contrast, recent intron insertions were not associated with a specific sequence. In animal Amy genes, population size and generation time seem to have played only minor roles in shaping gene structures.     

How intron splicing affects the deletion and insertion profile in Drosophila melanogaster

Studies of "dead-on-arrival" transposable elements in Drosophila melanogaster found that deletions outnumber insertions approximately 8:1 with a median size for deletions of approximately 10 bp. These results are consistent with the deletion and insertion profiles found in most other Drosophila pseudogenes. In contrast, a recent study of D. melanogaster introns found a deletion/insertion ratio of 1.35:1, with 84% of deletions being shorter than 10 bp. This discrepancy could be explained if deletions, especially long deletions, are more frequently strongly deleterious than insertions and are eliminated disproportionately from intron sequences. To test this possibility, we use analysis and simulations to examine how deletions and insertions of different lengths affect different components of splicing and determine the distribution of deletions and insertions that preserve the original exons. We find that, consistent with our predictions, longer deletions affect splicing at a much higher rate compared to insertions and short deletions. We also explore other potential constraints in introns and show that most of these also disproportionately affect large deletions. Altogether we demonstrate that constraints in introns may explain much of the difference in the pattern of deletions and insertions observed in Drosophila introns and pseudogenes.     

Studies of the subtilin leader peptide as a translocation signal in Escherichia coli K12

In the protozoan Stylonychia lemnae 10 different histone H3 genes were discovered by polymerase chain reaction (PCR) amplification and sequence analysis. One of them is interrupted by a short intron sequence. These genes code for nine divergent histone H3 proteins. The genetic distances between some of these variants are very high. Most of the substitutions, as well as insertions/deletions, were found in the amino-terminal region. One variant shows an extremely elongated and altered N-terminus, which did not allow an unambiguous alignment with other histone H3 variants in this region. Hybridization experiments using the different H3 genes as probes indicate that even more histone H3 variants must exist in this species.     

The correlation between intron length and recombination in drosophila. Dynamic equilibrium between mutational and selective forces

Intron length is negatively correlated with recombination in both Drosophila melanogaster and humans. This correlation is not likely to be the result of mutational processes alone: evolutionary analysis of intron length polymorphism in D. melanogaster reveals equivalent ratios of deletion to insertion in regions of high and low recombination. The polymorphism data do reveal, however, an excess of deletions relative to insertions (i.e., a deletion bias), with an overall deletion-to-insertion events ratio of 1.35. We propose two types of selection favoring longer intron lengths. First, the natural mutational bias toward deletion must be opposed by strong selection in very short introns to maintain the minimum intron length needed for the intron splicing reaction. Second, selection will favor insertions in introns that increase recombination between mutations under the influence of selection in adjacent exons. Mutations that increase recombination, even slightly, will be selectively favored because they reduce interference among selected mutations. Interference selection acting on intron length mutations must be very weak, as indicated by frequency spectrum analysis of Drosophila intron length polymorphism, making the equilibrium for intron length sensitive to changes in the recombinational environment and population size. One consequence of this sensitivity is that the advantage of longer introns is expected to decrease inversely with the rate of recombination, thus leading to a negative correlation between intron length and recombination rate. Also in accord with this model, intron length differs between closely related Drosophila species, with the longest variant present more often in D. melanogaster than in D. simulans. We suggest that the study of the proposed dynamic model, taking into account interference among selected sites, might shed light on many aspects of the comparative biology of genome sizes including the C value paradox.     

Evolution of Piperales--matK gene and trnK intron sequence data reveal lineage specific resolution contrast

Piperales represent the largest basal angiosperm order with a nearly worldwide distribution. The order includes three species rich genera, Piper (ca. 2000 species), Peperomia (ca. 1500-1700 species), and Aristolochia s. l. (ca. 500 species). Sequences of the matK gene and the non-coding trnK group II intron are analysed for a dense set of 105 taxa representing all families (except Hydnoraceae) and all generic segregates (except Euglypha within Aristolochiaceae) of Piperales. A large number of highly informative indels are found in the Piperales trnK/matK dataset. Within a narrow region approximately 500 nt downstream in the matK coding region (CDS), a length variable simple sequence repeat (SSR) expansion segment occurs, in which insertions and deletions have led to short frame-shifts. These are corrected shortly afterwards, resulting in a maximum of six amino acids being affected. Furthermore, additional non-functional matK copies were found in Zippelia begoniifolia, which can easily be discriminated from the functional open reading frame (ORF). The trnK/matK sequence data fully resolve relationships within Peperomia, whereas they are not effective within Piper. The resolution contrast is correlated with the rate heterogeneity between those lineages. Parsimony, Bayesian and likelihood analyses result in virtually the same topology, and converge on the monophyly of Piperaceae and Saururaceae. Lactoris gains high support as sister to Aristolochiaceae subf. Aristolochioideae, but the different tree inference methods yield conflicting results with respect to the relationships of subfam. Asaroideae. In Piperaceae, a clade formed by the monotypic genus Zippelia and the small genus Manekia (=Sarcorhachis) is sister to the two large genera Piper and Peperomia.     

Intron length evolution in Drosophila

I present data on the evolution of intron lengths among 3 closely related Drosophila species, D. melanogaster, Drosophila simulans, and Drosophila yakuba. Using D. yakuba as an outgroup, I mapped insertion and deletion mutations in 148 introns (spanning approximately 30 kb) to the D. melanogaster and D. simulans lineages. Intron length evolution in the 2 sister species has been different: in D. melanogaster, X-linked introns have increased slightly in size, whereas autosomal ones have decreased slightly in size; in D. simulans, both X-linked and autosomal introns have decreased in size. To understand the possible evolutionary causes of these lineage- and chromosome-specific patterns of intron evolution, I studied insertion-deletion (indel) polymorphism and divergence in D. melanogaster. Small insertion mutations segregate at elevated frequencies and enjoy elevated probabilities of fixation, particularly on the X chromosome. In contrast, there is no detectable X chromosome effect on fixations in D. simulans. These findings suggest X chromosome-specific selection or biased gene conversion-gap repair favoring insertions in D. melanogaster but not in D. simulans. These chromosome- and lineage-specific patterns of indel substitution are not easily explained by existing general population genetic models of intron length evolution. Genomic data from D. melanogaster further suggest that the forces described here affect introns and intergenic regions similarly.     

Mind the gaps: evidence of bias in estimates of multiple sequence alignments

Multiple sequence alignment (MSA) is a crucial first step in the analysis of genomic and proteomic data. Commonly occurring sequence features, such as deletions and insertions, are known to affect the accuracy of MSA programs, but the extent to which alignment accuracy is affected by the positions of insertions and deletions has not been examined independently of other sources of sequence variation. We assessed the performance of 6 popular MSA programs (ClustalW, DIALIGN-T, MAFFT, MUSCLE, PROBCONS, and T-COFFEE) and one experimental program, PRANK, on amino acid sequences that differed only by short regions of deleted residues. The analysis showed that the absence of residues often led to an incorrect placement of gaps in the alignments, even though the sequences were otherwise identical. In data sets containing sequences with partially overlapping deletions, most MSA programs preferentially aligned the gaps vertically at the expense of incorrectly aligning residues in the flanking regions. Of the programs assessed, only DIALIGN-T was able to place overlapping gaps correctly relative to one another, but this was usually context dependent and was observed only in some of the data sets. In data sets containing sequences with non-overlapping deletions, both DIALIGN-T and MAFFT (G-INS-I) were able to align gaps with near-perfect accuracy, but only MAFFT produced the correct alignment consistently. The same was true for data sets that comprised isoforms of alternatively spliced gene products: both DIALIGN-T and MAFFT produced highly accurate alignments, with MAFFT being the more consistent of the 2 programs. Other programs, notably T-COFFEE and ClustalW, were less accurate. For all data sets, alignments produced by different MSA programs differed markedly, indicating that reliance on a single MSA program may give misleading results. It is therefore advisable to use more than one MSA program when dealing with sequences that may contain deletions or insertions, particularly for high-throughput and pipeline applications where manual refinement of each alignment is not practicable.     

Evolution of Mhc–DRB Introns: Implications for the Origin of Primates

Introns are generally believed to evolve too rapidly and too erratically to be of much use in phylogenetic reconstructions. Few phylogenetically informative intron sequences are available, however, to ascertain the validity of this supposition. In the present study the supposition was tested on the example of the mammalian class II major histocompatibility complex (Mhc) genes of the DRB family. Since the Mhc genes evolve under balancing selection and are believed to recombine or rearrange frequently, the evolution of their introns could be expected to be particularly rapid and subject to scrambling. Sequences of intron 4 and 5 DRB genes were obtained from polymerase chain reaction-amplified fragments of genomic DNA from representatives of six eutherian orders—Primates, Scandentia, Chiroptera, Dermoptera, Lagomorpha, and Insectivora. Although short stretches of the introns have indeed proved to be unalignable, the bulk of the intron sequences from all six orders, spanning >85 million years (my) of evolution, could be aligned and used in a study of the tempo and mode of intron evolution. The analysis has revealed the Mhc introns to evolve at a rate similar to that of other genes and of synonymous sites of non-Mhc genes. No evidence of homogenization or large-scale scrambling of the intron sequences could be found. The Mhc introns apparently evolve largely by point mutations and insertions/deletions. The phylogenetic signals contained in the intron sequences could be used to identify Scandentia as the sister group of Primates, to support the existence of the Archonta superorder, and to confirm the monophyly of the Chiroptera. Received: 26 October 1998 / Accepted: 21 December 1998