Patterns and relative rates of nucleotide and insertion/deletion evolution at six chloroplast intergenic regions in new world species of the Lecythidaceae

Insertions and deletions (indels) in chloroplast noncoding regions are common genetic markers to estimate population structure and gene flow, although relatively little is known about indel evolution among recently diverged lineages such as within plant families. Because indel events tend to occur nonrandomly along DNA sequences, recurrent mutations may generate homoplasy for indel haplotypes. This is a potential problem for population studies, because indel haplotypes may be shared among populations after recurrent mutation as well as gene flow. Furthermore, indel haplotypes may differ in fitness and therefore be subject to natural selection detectable as rate heterogeneity among lineages. Such selection could contribute to the spatial patterning of cpDNA haplotypes, greatly complicating the interpretation of cpDNA population structure. This study examined both nucleotide and indel cpDNA variation and divergence at six noncoding regions (psbB-psbH, atpB-rbcL, trnL-trnH, rpl20-5'rps12, trnS-trnG, and trnH-psbA) in 16 individuals from eight species in the Lecythidaceae and a Sapotaceae outgroup. We described patterns of cpDNA changes, assessed the level of indel homoplasy, and tested for rate heterogeneity among lineages and regions. Although regression analysis of branch lengths suggested some degree of indel homoplasy among the most divergent lineages, there was little evidence for indel homoplasy within the Lecythidaceae. Likelihood ratio tests applied to the entire phylogenetic tree revealed a consistent pattern rejecting a molecular clock. Tajima's 1D and 2D tests revealed two taxa with consistent rate heterogeneity, one showing relatively more and one relatively fewer changes than other taxa. In general, nucleotide changes showed more evidence of rate heterogeneity than did indel changes. The rate of evolution was highly variable among the six cpDNA regions examined, with the trnS-trnG and trnH-psbA regions showing as much as 10% and 15% divergence within the Lecythidaceae. Deviations from rate homogeneity in the two taxa were constant across cpDNA regions, consistent with lineage-specific rates of evolution rather than cpDNA region-specific natural selection. There is no evidence that indels are more likely than nucleotide changes to experience homoplasy within the Lecythidaceae. These results support a neutral interpretation of cpDNA indel and nucleotide variation in population studies within species such as Corythophora alta.     

Evolutionary rearrangement of the amylase genomic regions between Drosophila melanogaster and Drosophila pseudoobscura

Two Drosophila pseudoobscura genomic clones have sequence similarity to the Drosophila melanogaster amylase region that maps to the 53CD region on the D. melanogaster cytogenetic map. The two clones with similarity to amylase map to sections 73A and 78C of the D. pseudoobscura third chromosome cytogenetic map. The complete sequences of both the 73A and 78C regions were compared to the D. melanogaster genome to determine if the coding region for amylase is present in both regions and to determine the evolutionary mechanism responsible for the observed distribution of the amylase gene or genes. The D. pseudoobscura 73A and 78C linkage groups are conserved with the D. melanogaster 41E and 53CD regions, respectively. The amylase gene, however, has not maintained its conserved linkage between the two species. These data indicate that amylase has moved via a transposition event in the D. melanogaster or D. pseudoobscura lineage. The predicted genes within the 73A and 78C regions show patterns of molecular evolution in synonymous and nonsynonymous sites that are consistent with previous studies of these two species.     

Molecular evolution of X-linked accessory gland proteins in Drosophila pseudoobscura

In Drosophila melanogaster and Drosophila simulans, positive Darwinian selection drives high rates of evolution of male reproductive genes, and accessory gland proteins (Acps) in particular. Here, we tested whether 13 X-linked male-specific genes, 4 Acps and 9 non-Acps, are under selective forces in the Drosophila pseudoobscura species group, much as those in the D. melanogaster group. We observed a statistically significant correlation in relative rates of nonsynonymous evolution between the two species groups tested. One Acp examined had a higher rate of nonsynonymous substitution than predicted by a neutral model in both species groups, suggesting its divergence was driven by positive Darwinian selection. To further test for the signature of selection, we examined polymorphism of three Acps within D. pseudoobscura. From this test, no Acp individually bore the signature of positive selection, but the 3 Acps together possessed an excess of nonsynonymous differences between species, relative to polymorphism within species. We conclude that faster evolution of Acps in the D. pseudoobscura group appears to be driven by positive selection, as previously suggested in the D. melanogaster group.     

Evolution of the glucose dehydrogenase gene in Drosophila

The glucose dehydrogenase genes (Gld) of Drosophila melanogaster, of D. pseudoobscura, and of D. virilis have been isolated and compared with each other in order to identify conserved and divergent aspects of their structure and expression. The exon/intron structure of Gld is conserved. The Gld mRNAs are similar, with a range of 2.6-2.8 kb among the three species. All three species exhibit peaks of Gld expression during every major developmental stage, although considerable variation in the precise timing of these peaks exists between species. Interspecific gene transfer experiments demonstrate that the regulation and function of the D. pseudoobscura Gld is similar enough to the homologous gene in D. melanogaster to substitute for its essential role in the eclosion process. Comparison of the putative promoter sequences has identified both shared and divergent sequence elements which are likely responsible, respectively, for the conserved and divergent patterns of expression observed. The entire coding sequences of the pseudoobscura and melanogaster Gld genes are presented and shown to encode a 612-amino-acid pre-protein. The inferred amino acid sequences are 92% conserved between the two species. In general the intronic regions of Gld are unusually well conserved.     

The Speciation History of Drosophila Pseudoobscura and Close Relatives: Inferences from DNA Sequence Variation at the Period Locus

Thirty-five period locus sequences from Drosophila pseudoobscura and its siblings species, D. p. bogotana, D. persimilis, and D. miranda, were studied. A large amount of variation was found within D. pseudoobscura and D. persimilis, consistent with histories of large effective population sizes. D. p. bogotana, however, has a severe reduction in diversity. Combined analysis of per with two other loci, in both D. p. bogotana and D. pseudoobscura, strongly suggest this reduction is due to recent directional selection at or near per within D. p. bogotana. Since D. p. bogotana is highly variable and shares variation with D. pseudoobscura at other loci, the low level of variation at per within D. p. bogotana can not be explained by a small effective population size or by speciation via founder event. Both D. pseudoobscura and D. persimilis have considerable intraspecific gene flow. A large portion of one D. persimilis sequence appears to have arisen via introgression from D. pseudoobscura. The time of this event appears to be well after the initial separation of these two species. The estimated times since speciation are one mya for D. pseudoobscura and D. persimilis and 2 mya since the formation of D. miranda.     

Molecular population genetics of sequence length diversity in the Adh region of Drosophila pseudoobscura

Positive and negative selection on indel variation may explain the correlation between intron length and recombination levels in natural populations of Drosophila. A nucleotide sequence analysis of the 3.5 kilobase sequence of the alcohol dehydrogenase (Adh) region from 139 Drosophila pseudoobscura strains and one D. miranda strain was used to determine whether positive or negative selection acts on indel variation in a gene that experiences high levels of recombination. A total of 30 deletion and 36 insertion polymorphisms were segregating within D. pseudoobscura populations and no indels were fixed between D. pseudoobscura and its two sibling species D. miranda and D. persimilis. The ratio of Tajima's D to its theoretical minimum value (D(min)) was proposed as a metric to assess the heterogeneity in D among D. pseudoobscura loci when the number of segregating sites differs among loci. The magnitude of the D/D(min) ratio was found to increase as the rate of population expansion increases, allowing one to assess which loci have an excess of rare variants due to population expansion versus purifying selection. D. pseudoobscura populations appear to have had modest increases in size accounting for some of the observed excess of rare variants. The D/D(min) ratio rejected a neutral model for deletion polymorphisms. Linkage disequilibrium among pairs of indels was greater than between pairs of segregating nucleotides. These results suggest that purifying selection removes deletion variation from intron sequences, but not insertion polymorphisms. Genome rearrangement and size-dependent intron evolution are proposed as mechanisms that limit runaway intron expansion.     

Evaluation of the genomic extent of effects of fixed inversion differences on intraspecific variation and interspecific gene flow in Drosophila pseudoobscura and D. persimilis

There is increasing evidence that chromosomal inversions may facilitate the formation or persistence of new species by allowing genetic factors conferring species-specific adaptations or reproductive isolation to be inherited together and by reducing or eliminating introgression. However, the genomic domain of influence of the inverted regions on introgression has not been carefully studied. Here, we present a detailed study on the consequences that distance from inversion breakpoints has had on the inferred level of gene flow and divergence between Drosophila pseudoobscura and D. persimilis. We identified the locations of the inversion breakpoints distinguishing D. pseudoobscura and D. persimilis in chromosomes 2, XR, and XL. Population genetic data were collected at specific distances from the inversion breakpoints of the second chromosome and at two loci inside the XR and XL inverted regions. For loci outside the inverted regions, we found that distance from the nearest inversion breakpoint had a significant effect on several measures of divergence and gene flow between D. pseudoobscura and D. persimilis. The data fitted a logarithmic relationship, showing that the suppression of crossovers in inversion heterozygotes also extends to loci located outside the inversion but close to it (within 1-2 Mb). Further, we detected a significant reduction in nucleotide variation inside the inverted second chromosome region of D. persimilis and near one breakpoint, consistent with a scenario in which this inversion arose and was fixed in this species by natural selection.     

Genetics of hybrid male sterility among strains and species in the Drosophila pseudoobscura species group

Taxa in the early stages of speciation may bear intraspecific allelic variation at loci conferring barrier traits in hybrids such as hybrid sterility. Additionally, hybridization may spread alleles that confer barrier traits to other taxa. Historically, few studies examine within- and between-species variation at loci conferring reproductive isolation. Here, we test for allelic variation within Drosophila persimilis and within the Bogota subspecies of D. pseudoobscura at regions previously shown to contribute to hybrid male sterility. We also test whether D. persimilis and the USA subspecies of D. pseudoobscura share an allele conferring hybrid sterility in a D. pseudoobscura bogotana genetic background. All loci conferred similar hybrid sterility effects across all strains studied, although we detected some statistically significant quantitative effect variation among D. persimilis alleles of some hybrid incompatibility QTLs. We also detected allelism between D. persimilis and D. pseudoobscura USA at a second chromosome hybrid sterility QTL. We hypothesize that either the QTL is ancestral in D. persimilis and D. pseudoobscura USA and lost in D. pseudoobscura bogotana, or gene flow transferred the QTL from D. persimilis to D. pseudoobscura USA. We discuss our findings in the context of population features that may contribute to variation in hybrid incompatibilities.     

A test of the chromosomal rearrangement model of speciation in Drosophila pseudoobscura

Recent studies suggest that chromosomal rearrangements play a significant role in speciation by preventing recombination and maintaining species persistence despite interspecies gene flow. Factors conferring adaptation or reproductive isolation are maintained in rearranged regions in the face of hybridization, while such factors are eliminated from collinear regions. As a direct test of this rearrangement model, we evaluated the genetic basis of hybrid male sterility in a sympatric species pair, Drosophila pseudoobscura pseudoobscura and D. persimilis, and an allopatric species pair, D. pseudoobscura bogotana and D. persimilis. Our results are consistent with the proposed model: virtually all of the sterility factors in the former pair are associated with three inverted regions, whereas sterility factors are present in the collinear regions in the latter pair. These findings indicate recombination and selection may have eliminated sterility factors outside the inverted regions between D. p. pseudoobscura and D. persimilis, suggesting chromosomal rearrangements may facilitate species persistence despite hybridization.     

Molecular evolution of sex-biased genes in Drosophila

Studies of morphology, interspecific hybridization, protein/DNA sequences, and levels of gene expression have suggested that sex-related characters (particularly those involved in male reproduction) evolve rapidly relative to non-sex-related characters. Here we report a general comparison of evolutionary rates of sex-biased genes using data from cDNA microarray experiments and comparative genomic studies of Drosophila. Comparisons of nonsynonymous/synonymous substitution rates (d(N)/d(S)) between species of the D. melanogaster subgroup revealed that genes with male-biased expression had significantly faster rates of evolution than genes with female-biased or unbiased expression. The difference was caused primarily by a higher d(N) in the male-biased genes. The same pattern was observed for comparisons among more distantly related species. In comparisons between D. melanogaster and D. pseudoobscura, genes with highly biased male expression were significantly more divergent than genes with highly biased female expression. In many cases, orthologs of D. melanogaster male-biased genes could not be identified in D. pseudoobscura through a Blast search. In contrast to the male-biased genes, there was no clear evidence for accelerated rates of evolution in female-biased genes, and most comparisons indicated a reduced rate of evolution in female-biased genes relative to unbiased genes. Male-biased genes did not show an increased ratio of nonsynonymous/synonymous polymorphism within D. melanogaster, and comparisons of polymorphism/divergence ratios suggest that the rapid evolution of male-biased genes is caused by positive selection.     

Unusual pattern of single nucleotide polymorphism at the exuperantia2 locus of Drosophila pseudoobscura

We have investigated the pattern of DNA sequence variation at the exuperantia2 locus in Drosophila pseudoobscura. This adds to the increasing dataset of genetic variation in D. pseudoobscura, a useful model species for evolutionary genetic studies. The level of silent site nucleotide diversity and the divergence from an outgroup Drosophila miranda are comparable with those for other X-linked loci. One peculiar pattern at the exu2 locus of D. pseudoobscura is a complete linkage disequilibrium between two SNPs, one of which is a replacement site. As a result, there are two distinct haplotype groups in our dataset. Based upon the comparisons with the outgroup sequences from D. miranda and Drosophila persimilis, we show that the newly derived haplotype group has lower diversity than the ancestral haplotype group. The pattern of protein evolution at exu2 shows some deviation from the neutral model. Together, these and other characteristics of the exu2 locus suggest the action of selection on the pattern of SNP variation, consistent with a partial selective sweep associated with the newly derived haplotype.     

Evolution of lineage-specific functions in ancient cis-regulatory modules

Morphological evolution is driven both by coding sequence variation and by changes in regulatory sequences. However, how cis-regulatory modules (CRMs) evolve to generate entirely novel expression domains is largely unknown. Here, we reconstruct the evolutionary history of a lens enhancer located within a CRM that not only predates the lens, a vertebrate innovation, but bilaterian animals in general. Alignments of orthologous sequences from different deuterostomes sub-divide the CRM into a deeply conserved core and a more divergent flanking region. We demonstrate that all deuterostome flanking regions, including invertebrate sequences, activate gene expression in the zebrafish lens through the same ancient cluster of activator sites. However, levels of gene expression vary between species due to the presence of repressor motifs in flanking region and core. These repressor motifs are responsible for the relatively weak enhancer activity of tetrapod flanking regions. Ray-finned fish, however, have gained two additional lineage-specific activator motifs which in combination with the ancient cluster of activators and the core constitute a potent lens enhancer. The exploitation and modification of existing regulatory potential in flanking regions but not in the highly conserved core might represent a more general model for the emergence of novel regulatory functions in complex CRMs.     

Structure and evolution of teleost mitochondrial control regions

We amplified and sequenced the mitochondrial control region from 23 species representing six families of teleost fish. The length of this segment is highly variable among even closely related species due to the presence of tandemly repeated sequences and large insertions. The position of the repetitive sequences suggests that they arise during replication both near the origin of replication and at the site of termination of the D-loop strand. Many of the conserved sequence blocks (CSBs) observed in mammals are also found among fish. In particular, the mammalian CSB-D is present in all of the fish species studied. Study of potential secondary structures of RNAs from the conserved regions provides little insight into the functional constraints on these regions. The variable structure of these control regions suggests that particular care should be taken to identify the most appropriate segment for studies of intraspecific variation. Correspondence to: T.D. Kocher     

Direct and correlated responses to artificial selection on developmental time and wing length in Drosophila buzzatii

Abstract.— Developmental time and body size are two positively correlated traits closely related to fitness in many organisms including Drosophila . Previous work suggested that these two traits are involved in a trade-off that may result from a negative genetic correlation between their effects on pre-adult and adult fitness. Here, we examine the evolution of developmental time and body size (indexed by wing length) under artificial selection applied to one or both traits in replicated D. buzzatii populations. Directional changes in both developmental time and wing length indicate the presence of substantial additive genetic variance for both traits. The strongest response to selection for fast development was found in lines selected simultaneously to reduce both developmental time and wing length, probably as an expected consequence of a synergistic effect of indirect selection. When selection was applied in the direction opposite to the putative genetic correlation, that is, large wing length but fast development, no responses were observed for developmental time. Lines selected to reduce both wing length and developmental time diverged slightly faster from the control than lines selected to increase wing length and reduce developmental time. However, wing length did not diverge from the control in lines selected only for fast development. These results suggest a complex genetic basis of the correlation between developmental time and wing length, but are generally consistent with the hypothesis that both traits are related in a trade-off. However, we found that this trade-off may disappear under uncrowded conditions, with fast-developing lines exhibiting a higher pre-adult viability than other lines when tested at high larval density.     

Why the short face? Developmental disintegration of the neurocranium drives convergent evolution in neotropical electric fishes

Convergent evolution is widely viewed as strong evidence for the influence of natural selection on the origin of phenotypic design. However, the emerging evo‐devo synthesis has highlighted other processes that may bias and direct phenotypic evolution in the presence of environmental and genetic variation. Developmental biases on the production of phenotypic variation may channel the evolution of convergent forms by limiting the range of phenotypes produced during ontogeny. Here, we study the evolution and convergence of brachycephalic and dolichocephalic skull shapes among 133 species of Neotropical electric fishes (Gymnotiformes: Teleostei) and identify potential developmental biases on phenotypic evolution. We plot the ontogenetic trajectories of neurocranial phenotypes in 17 species and document developmental modularity between the face and braincase regions of the skull. We recover a significant relationship between developmental covariation and relative skull length and a significant relationship between developmental covariation and ontogenetic disparity. We demonstrate that modularity and integration bias the production of phenotypes along the brachycephalic and dolichocephalic skull axis and contribute to multiple, independent evolutionary transformations to highly brachycephalic and dolichocephalic skull morphologies.     

Microsatellite variation in populations of Drosophila pseudoobscura and Drosophila persimilis

We have isolated, characterized and mapped 33 dinucleotide, three trinucleotide and one tetranucleotide repeat loci from the four major chromosomes of Drosophila pseudoobscura. Average inferred repeat unit length of the dinucleotide repeats is 12 repeat units, similar to D. melanogaster. Assays of D. pseudoobscura and populations of its sibling species, D. persimilis, using 10 of these loci show extremely high levels of variation compared with similar studies of dinucleotide repeat variation in D. melanogaster populations. The high levels of variation are consistent with an average mutation rate of approximately 10(-6) per locus per generation and an effective population size of D. pseudoobscura approximately four times larger than that of D. melanogaster. Consistent with allozymes and nucleotide sequence polymorphism, the dinucleotide repeat loci reveal minimal structure across four populations of D. pseudoobscura. Finally, our preliminary recombinational mapping of 24 of these microsatellites suggests that the total recombinational genome size may be larger than previously inferred using morphological mutant markers.