Evolution of Single-Domain Globins in Hydrothermal Vent Scale-Worms.

Hypoxia at deep-sea hydrothermal vents represents one of the most basic challenges for metazoans, which then requires specific adaptations to acquire oxygen to meet their metabolic needs. Hydrothermal vent scale-worms (Polychaeta; Polynoidae) express large amounts of extracellular single- and multi-domain hemoglobins, in contrast with their shallow-water relatives that only possess intracellular globins in their nervous system (neuroglobins). We sequenced the gene encoding the single-domain (SD) globin from nine species of polynoids found in various vent and deep-sea reduced microhabitats (and associated constraints) to determine if the Polynoidae SD globins have been the targets of diversifying selection. Although extracellular, all the SD globins (and multi-domain ones) form a monophyletic clade that clusters within the intracellular globin group of other annelids, indicating that these hemoglobins have evolved from an intracellular myoglobin-like form. Positive selection could not be detected at the major ecological changes that the colonization of the deep-sea and hydrothermal vents represents. This suggests that no major structural modification was necessary to allow the globins to function under these conditions. The mere expression of these globins extracellularly may have been sufficiently advantageous for the polynoids living in hypoxic hydrothermal vents. Among hydrothermal vent species, positively selected amino acids were only detected in the phylogenetic lineage leading to the two mussel-commensal species (Branchipolynoe). In this lineage, the multiplicity of hemoglobins could have lessened the selective pressure on the SD hemoglobin, allowing the acquisition of novel functions by positive Darwinian selection. Conversely, the colonization of hotter environments (species of Branchinotogluma) does not seem to have required additional modifications.

Origin and evolution of the unique tetra-domain hemoglobin from the hydrothermal vent scale worm Branchipolynoe.

Hemoglobin is the most common respiratory pigment in annelids. It can be intra or extracellular, and this latter type can form large multimeric complexes. The hydrothermal vent scale worms Branchipolynoe symmytilida and Branchipolynoe seepensis express an extracellular tetra-domain hemoglobin (Hb) that is unique in annelids. We sequenced the gene for the single-domain and tetra-domain globins in these two species. The single-domain gene codes for a mature protein of 137 amino acids, and the tetra-domain gene codes for a mature protein of 552 amino acids. The single-domain gene has a typical three exon/two intron structure, with introns located at their typical positions (B12.2 and G7.0). This structure is repeated four times in the tetra-domain gene, with no bridge introns or linker sequences between domains. The phylogenetic position of Branchipolynoe globins among known annelid globins revealed that, although extracellular, they cluster within the annelid intracellular globins clade, suggesting that the extracellular state of these Hbs is the result of convergent evolution. The tetra-domain structure likely resulted from two tandem duplications, domain 1 giving rise to domain 2 and after this the two-domain gene duplicated to produce domains 3 and 4. The high O(2) affinity of Branchipolynoe extracellular globins may be explained by the two key residues (B10Y and E7Q) in the heme pocket in each of the domains of the single and tetra-domain globins, which have been shown to be essential in the oxygen-avid Hb from the nematode Ascaris suum. This peculiar globin evolutionary path seems to be very different from other annelid extracellular globins and is most likely the product of evolutionary tinkering associated with the strong selective pressure to adapt to chronic hypoxia that characterizes hydrothermal vents.

Molecular population genetics of sex determination genes: the transformer gene of Drosophila melanogaster.

The transformer locus (tra) produces an RNA processing protein that alternatively splices the doublesex pre-mRNA in the sex determination hierarchy of Drosophila melanogaster. Comparisons of the tra coding region among Drosophila species have revealed an unusually high degree of divergence in synonymous and nonsynonymous sites. In this study, we tested the hypothesis that the tra gene will be polymorphic in synonymous and nonsynonymous sites within species by investigating nucleotide sequence variation in eleven tra alleles within D. melanogaster. Of the 1063 nucleotides examined, two synonymous sites were polymorphic and no amino acid variation was detected. Three statistical tests were used to detect departures from an equilibrium neutral model. Two tests failed to reject a neutral model of molecular evolution because of low statistical power associated with low levels of genetic variation (Tajima/Fu and Li). The Hudson, Kreitman, and Aguade test rejected a neutral model when the tra region was compared to the 5'-flanking region of alcohol dehydrogenase (Adh). The lack of variability in the tra gene is consistent with a recent selective sweep of a beneficial allele in or near the tra locus.

Nucleotide sequence of the Adh gene region of Drosophila pseudoobscura: evolutionary change and evidence for an ancient gene duplication.

The alcohol dehydrogenase (Adh) locus (ADH; alcohol: NAD+ oxidoreductase, EC 1.1.1.1) of Drosophila pseudoobscura was cloned and sequenced. Forty-five percent of the "effectively silent sites" have changed between Adh in D. pseudoobscura of the obscura species group and the homologous DNA sequence in D. mauritiana, the latter representing the melanogaster species group. The untranslated leader sequence of the adult transcript of D. pseudoobscura has two deletions relative to the D. mauritiana message. The ADH protein sequences of D. pseudoobscura is missing the third and fourth amino acids at the N-terminus relative to the D. mauritiana enzyme. Of the remaining 254 amino acid positions, 27 (10.64%) differ between the two species. Amino acid replacements are randomly distributed into hydrophilic and hydrophobic domains of ADH. However, replacement substitutions are distributed nonrandomly across the three exons among D. pseudoobscura and members of the melanogaster subgroup, suggesting that functional constraints across the exons are different. Surprisingly, silent substitutions are also nonrandomly distributed with the third exon being the most divergent. This pattern suggests possible selective constraints on supposedly neutral silent substitutions and/or variation in underlying mutation rates across the gene. The presence of transcriptional and translational signals at the beginning and end of conserved sequences 3' to Adh implies the existence of a previously undescribed gene. Codon usage and patterns of nucleotide divergence are consistent with a protein coding function for this gene. In addition, conservation of nucleotide and amino acid sequence and similarity in hydropathy plots suggests that the gene 3' to Adh represents an ancient duplication of the Adh gene.

Estimates of linkage disequilibrium and the recombination parameter determined from segregating nucleotide sites in the alcohol dehydrogenase region of Drosophila pseudoobscura.

The alcohol dehydrogenase (Adh) region of Drosophila pseudoobscura, which includes the two genes Adh and Adh-Dup, was used to examine the pattern and organization of linkage disequilibrium among pairs of segregating nucleotide sites. A collection of 99 strains from the geographic range of D. pseudoobscura were nucleotide-sequenced with polymerase chain reaction-mediated techniques. All pairs of the 359 polymorphic sites in the 3.5-kb Adh region were tested for significant linkage disequilibrium with Fisher's exact test. Of the 74,278 pairwise comparisons of segregating sites, 127 were in significant linkage disequilibrium at the 5% level. The distribution of five linkage disequilibrium estimators D(ij), D2, r(ij), r2 and D(ij) were compared to theoretical distributions. The observed distributions of D(ij), D2, r(ij) and r2 were consistent with the theoretical distribution given an infinite sites model. The observed distribution of D(ij) differed from the theoretical distribution because of an excess of values at -1 and 1. No spatial pattern was observed in the linkage disequilibrium pattern in the Adh region except for two clusters of sites nonrandomly associated in the adult intron and intron 2 of Adh. The magnitude of linkage disequilibrium decreases significantly as nucleotide distance increases, or a distance effect. Adh-Dup had a larger estimate of the recombination parameter, 4Nc, than Adh, where N is the effective population size and c is the recombination rate. A comparison of the mutation and recombination parameters shows that 7-17 recombination events occur for each mutation event. The heterogeneous estimates of the recombination parameter and the inverse relationship between linkage disequilibrium and nucleotide distance are no longer significant when the two clusters of Adh intron sites are excluded from analyses. The most likely explanation for the two clusters of linkage disequilibria is epistatic selection between sites in the cluster to maintain pre-mRNA secondary structure.

Molecular population genetics of an electrophoretically monomorphic protein in the alcohol dehydrogenase region of Drosophila pseudoobscura.

Nucleotide sequence data from the alcohol dehydrogenase (Adh) region of 18 isochromosomal strains of Drosophila pseudoobscura were used to determine whether the lack of amino acid polymorphism in ADH results from a low neutral mutation rate or a recent directional selection event. We estimated the neutral mutation parameter, 4Nmu, in synonymous sites for 17 subregions of Adh. The nucleotide diversity data were tested for departures from an equilibrium neutral model with two statistical tests. The Tajima test and the Hudson, Kreitman and Aguade test each failed to reject a neutral model. These results suggest that the ADH enzyme of D. pseudoobscura lacks amino acid polymorphisms because the neutral mutation rate of nonsynonymous sites is low. The neutral mutation parameter for synonymous sites is heterogeneous between domains of the Adh region. These data indicate that selective constrains on synonymous sites can vary between functional domains.

Estimates of gene flow in Drosophila pseudoobscura determined from nucleotide sequence analysis of the alcohol dehydrogenase region.

The genetic structure of Drosophila pseudoobscura populations was inferred from a nucleotide sequence analysis of a 3.4-kb segment of the alcohol dehydrogenase (Adh) region. A total of 99 isochromosomal strains collected from 13 populations in North and South America were used to determine if any population departed from a neutral model and to estimate levels of gene flow between populations. This study also included the nucleotide sequences from two sibling species, D. persimilis and D. miranda. We estimated the neutral mutation parameter, 4N mu, in synonymous and noncoding sites for 17 subregions of Adh in each of nine populations with sample sizes greater than three. The nucleotide diversity data in the nine populations was tested for departures from an equilibrium neutral model with two statistical tests. The Tajima and the Hudson, Kreitman, Aguade tests showed that each population fails to reject a neutral model. Tests for genetic differentiation between populations fail to show any population substructure among the North American populations of D. pseudoobscura. The nucleotide diversity data is consistent with direct and indirect measures of gene flow that show extensive dispersal between populations of D. pseudoobscura.

Polymorphism and divergence at a Drosophila pseudogene locus.

The larval cuticle protein (Lcp) cluster in Drosophila melanogaster contains four functional genes and a closely related pseudogene. A 630-bp fragment including the larval cuticle pseudogene locus (Lcp psi) was nucleotide sequenced in 10 strains of D. melanogaster and a 458-bp Lcp psi fragment from D. simulans was also sequenced. We used these data to test the hypotheses that the rates of synonymous and nonsynonymous substitution are equal, that the absolute levels of variation are higher than in functional genes, and that intraspecific polymorphism is correlated with interspecific divergence. As predicted, synonymous and nonsynonymous substitution rates were equivalent, and overall nucleotide divergence between D. melanogaster and D. simulans (Jukes-Cantor distance = 0.149 +/- 0.150) was extremely high. However, within-species DNA sequence comparisons at Lcp psi revealed lower levels of polymorphism (theta = 0.001 +/- 0.001) than at many functional loci in D. melanogaster. Using the HUDSON, KREITMAN, and AGUADE (HKA) test, we show that the level of polymorphism in Lcp psi within D. melanogaster is lower than expected given the amount of divergence between D. melanogaster and D. simulans when the pseudogene data are compared to the Adh 5' flanking region. Because the Lcp psi lies in a region of relatively infrequent recombination, we suggest that the low level of within-species polymorphism is the result of background selection.

Molecular population genetics of X-linked genes in Drosophila pseudoobscura.

This article presents a nucleotide sequence analysis of 500 bp determined in each of five X-linked genes, runt, sisterlessA, period, esterase 5, and Heat-shock protein 83, in 40 Drosophila pseudoobscura strains collected from two populations. Estimates of the neutral migration parameter for the five loci show that gene flow among D. pseudoobscura populations is sufficient to homogenize inversion frequencies across the range of the species. Nucleotide diversity at each locus fails to reject a neutral model of molecular evolution. The sample of 40 chromosomes included six Sex-ratio inversions, a series of three nonoverlapping inversions that are associated with a strong meiotic drive phenotype. The selection driven by the Sex-ratio meiotic drive element has not fixed variation across the X chromosome of D. pseudoobscura because, while significant linkage disequilibrium was observed within the sisterlessA, period, and esterase 5 genes, we did not find evidence for nonrandom association among loci. The Sex-ratio chromosome was estimated to be 25,000 years old based on the decomposition of linkage disequilibrium between esterase 5 and Heat-shock protein 83 or 1 million years old based on the net divergence of esterase 5 between Standard and Sex-ratio chromosomes. Genetic diversity was depressed within esterase 5 within Sex-ratio chromosomes, while the four other genes failed to show a reduction in heterozygosity in the Sex-ratio background. The reduced heterogeneity in esterase 5 is due either to its location near one of the Sex-ratio inversion breakpoints or that it is closely linked to a gene or genes responsible for the Sex-ratio meiotic drive system.

Natural selection and the frequency distributions of "silent" DNA polymorphism in Drosophila.

In Escherichia coli, Saccharomyces cerevisiae, and Drosophila melanogaster, codon bias may be maintained by a balance among mutation pressure, genetic drift, and natural selection favoring translationally superior codons. Under such an evolutionary model, silent mutations fall into two fitness categories: preferred mutations that increase codon bias and unpreferred changes in the opposite direction. This prediction can be tested by comparing the frequency spectra of synonymous changes segregating within populations; natural selection will elevate the frequencies of advantageous mutations relative to that of deleterious changes. The frequency distributions of preferred and unpreferred mutations differ in the predicted direction among 99 alleles of two D. pseudoobscura genes and five alleles of eight D. simulans genes. This result confirms the existence of fitness classes of silent mutations. Maximum likelihood estimates suggest that selection intensity at silent sites is, on average, very weak in both D. pseudoobscura and D. simulans (magnitude of NS approximately 1). Inference of evolutionary processes from within-species sequence variation is often hindered by the assumption of a stationary frequency distribution. This assumption can be avoided when identifying the action of selection and tested when estimating selection intensity.

Protein variation in Adh and Adh-related in Drosophila pseudoobscura. Linkage disequilibrium between single nucleotide polymorphisms and protein alleles.

A 3.5-kb segment of the alcohol dehydrogenase (Adh) region that includes the Adh and Adh-related genes was sequenced in 139 Drosophila pseudoobscura strains collected from 13 populations. The Adh gene encodes four protein alleles and rejects a neutral model of protein evolution with the McDonald-Kreitman test, although the number of segregating synonymous sites is too high to conclude that adaptive selection has operated. The Adh-related gene encodes 18 protein haplotypes and fails to reject an equilibrium neutral model. The populations fail to show significant geographic differentiation of the Adh-related haplotypes. Eight of 404 single nucleotide polymorphisms (SNPs) in the Adh region were in significant linkage disequilibrium with three ADHR protein alleles. Coalescent simulations with and without recombination were used to derive the expected levels of significant linkage disequilibrium between SNPs and 18 protein haplotypes. Maximum levels of linkage disequilibrium are expected for protein alleles at moderate frequencies. In coalescent models without recombination, linkage disequilibrium decays between SNPs and high frequency haplotypes because common alleles mutate to haplotypes that are rare or that reach moderate frequency. The implication of this study is that linkage disequilibrium mapping has the highest probability of success with disease-causing alleles at frequencies of 10%.

Tubeworm succession at hydrothermal vents: use of biogenic cues to reduce habitat selection error?

Species colonizing new deep-sea hydrothermal vents along the East Pacific Rise show a distinct successional sequence: pioneer assemblages dominated by the vestimentiferan tubeworm Tevnia jerichonana being subsequently invaded by another vestimentiferan Riftia pachyptila, and eventually the mussel Bathymodiolus thermophilus. Using a manipulative approach modified from shallow-water ecological studies, we test three alternative hypotheses to explain the initial colonization by T. jerichonana and its subsequent replacement by R. pachyptila. We show that R. pachyptila and another vestimentiferan, Oasisia alvinae, colonized new surfaces only if the surfaces also were colonized by T. jerichonana. This pattern does not appear to be due to restricted habitat tolerances or inferior dispersal capabilities of R. pachyptila and O. alvinae, and we argue the alternative explanation that T. jerichonana facilitates the settlement of the other two species and is eventually outcompeted by R. pachyptila. Unlike the classic model of community succession, in which facilitating species promote their own demise by modifying the environment to make it more hospitable for competitors, we suggest that T. jerichonana may produce a chemical substance that induces settlement of these competitors. This process of selecting habitat based on biogenic cues may be especially adaptive and widespread among later-successional species that occupy a physically variable and unpredictable environment. In these cases, the presence of weedy species implies some integrated period of environmental suitability, whereas an instantaneous assessment of physical habitat conditions, such as water temperature for vent tubeworms, provides a poorer predictor of long-term habitat suitability.

Nucleotide sequence analysis of Adh genes estimates the time of geographic isolation of the Bogota population of Drosophila pseudoobscura.

The population of Drosophila pseudoobscura at Bogota, Columbia, is geographically and partially reproductively isolated from populations in the main body of the species in North America. The degree of genetic differentiation and time of divergence between populations at Bogota and Apple Hill, CA, were estimated by comparison of 3388 nucleotides in the alcohol dehydrogenase region (Adh and Adh-Dup genes) of 18 strains. Of the 146 polymorphic nucleotide sites detected, 68 and 31 were unique to the Apple Hill and Bogota samples, respectively, and 53 were shared. On the basis of an observed net divergence per nucleotide site of 0.264% between the two samples, the Bogota and North American populations were estimated to have been separated for at least 155,000 years. This divergence time suggests that D. pseudoobscura extended its range from North America to South America in a period of Pleistocene glaciation, when habitat suitable for the species presumably existed in lowland Central America.

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.

Restriction-map variation in the alcohol dehydrogenase region of Drosophila pseudoobscura.

A 32-kb region including the Adh structural gene was analyzed with six restriction endonucleases in 20 lines of Drosophila pseudoobscura, one line of D. persimilis, and one line of D. miranda. Nineteen lines of D. pseudoobscura from a single population were estimated to be polymorphic at one in every 15 nucleotides (p = 0.066). Any two homologous chromosomes chosen at random were heterozygous at one in 48 (H = 0.021) nucleotides. Two small insertions of 50 and 200 bp were found approximately 7 kb upstream from the Adh transcript. High haplotype diversity and low linkage disequilibrium suggest that the polymorphic restriction sites around Adh have segregated almost randomly during the history of this D. pseudoobscura population and that the effects of inbreeding and periodic reduction of population size have been negligible. The restriction-endonuclease analysis for the Adh region of D. pseudoobscura stands in sharp contrast to the strong linkage disequilibrium, high levels of insertion/deletion polymorphism, and lower estimates of nucleotide polymorphism found for this same region in D. melanogaster. A phylogeny for the Adh haplotypes is consistent with an early divergence of D. miranda, while D. persimilis falls within the cluster of D. pseudoobscura haplotypes.

Restriction-map variation in the Notch region of Drosophila melanogaster.

A worldwide sample of 37 X chromosomes of Drosophila melanogaster was analyzed with four restriction endonucleases for a 60-kb region of the Notch locus. Any two randomly chosen homologous chromosomes were heterozygous at one in 143 nucleotides (theta = 0.007). The chromosomes that were sampled contained no more than one insertion/deletion. The four insertions and one deletion observed in the 37 chromosomes sampled were located 3' to the Notch transcript; one insertion was represented twice in the sample. The amount of linkage disequilibrium in the Notch region appears to be lower than that of the alcohol dehydrogenase locus in D. melanogaster. The few instances of linkage disequilibrium observed could be due to geographic differentiation of African populations. The genetic variation estimates in the Notch region were comparable with those of the alcohol dehydrogenase region in D. melanogaster, suggesting that molecular genetic variation on the X chromosome is not dramatically reduced by selection against slightly deleterious alleles.

A comprehensive package for DNA sequence analysis in FORTRAN IV for the PDP-11.

A computer package written in Fortran-IV for the PDP-11 minicomputer is described. The package's novel features are: software for voice-entry of sequence data; a less memory intensive algorithm for optimal sequence alignment; and programs that fit statistical models to nucleic acid and protein sequences.

S-allele sequence diversity in natural populations of Solanum carolinense (Horsenettle).

S-allele diversity in Solanum carolinense was surveyed in two natural populations, located in Tennessee and North Carolina, with a molecular assay to determine the genotype of individual plants. A total of 13 different S-alleles were identified and sequenced. There is high overlap between the two populations sampled, with 10 alleles shared in common, one allele found only in Tennessee, and two found only in North Carolina. The number of alleles in this species appears to be extremely low compared with other species with gametophytic self-incompatibility. Sequence comparisons show that most alleles are extremely different one from another in their primary sequence and a phylogenetic analysis indicates extensive trans-specific evolution of S-lineages. In addition, some alleles appear to be derived much more recently. The implications of these observations are discussed in the light of recent theoretical results on S-allele population diversity and persistence.

Molecular evolution of inversions in Drosophila pseudoobscura: the amylase gene region.

The amylase region of the third chromosome of Drosophila pseudoobscura has been cloned and localized to cytological band 73A. It is contained within a series of highly polymorphic inversions and serves as a convenient tool for a molecular evolutionary analysis of the inverted gene arrangements. Amylase in D. pseudoobscura is a family of three genes, and some chromosomes have deletions for one or two of them. Two overlapping clones covering 26 kilobases were isolated and used as probes to survey DNA restriction map polymorphism among 28 lines, representing five of the major inversion types found in natural populations, as well as single chromosomes from the closely related species Drosophila persimilis and Drosophila miranda. Restriction-site differences are considerably greater among the various gene arrangements than among chromosomes with the same gene arrangement. Clustering the restriction map haplotypes yielded a dendrogram concordant with the phylogeny generated independently from cytogenetic considerations. The inversion polymorphism is estimated to be about 2 million years old.