Purifying Selection and Demographic Expansion Affect Sequence Diversity of the Ligand-Binding Domain of a Glutamate-Gated Chloride Channel Gene of Haemonchus placei

Ninety-five genomic sequences of the ligand-binding domain of glutamate-gated chloride channel genes of three populations of the parasitic nematode H. placei were evaluated for patterns of diversity, demography, and selection. These genes code for subunits of ion channels, which are involved in the mode of action of the most commonly used antiparasitic drugs, the macrocyclic lactones. An extremely high frequency of unique segregating sites in exons and introns was observed, with significantly negative neutrality tests in each population for noncoding, synonymous, and nonsynonymous sites. Several tests indicated that support for balancing selection, positive selection, and hitchhiking was lacking. McDonald–Kreitman tests using H. contortus or C. elegans as an outgroup revealed an extreme excess of replacement polymorphism, consistent with weak purifying selection. Although these tests agree that negative selection may explain the excess of replacement changes, an alternative interpretation is required for the significantly negative Fu and Lis D statistics based on silent and noncoding sites. These include homogeneous forces such as background selection and demographic expansion. The lack of population subdivision and the negative values of Tajimas D for this outbreeding parasitic nematode render background selection less likely than demographic expansion. Comparison of D statistics based on different site types using neutral coalescent simulations supported this interpretation. Although this statistic was more negative for nonsynonymous sites than for synonymous sites, most comparisons of the D statistic were not significantly different between mutation classes. A few significant site comparisons were also consistent with demographic expansion, because the observed test statistic (D_neutral – D_selected) were low relative to the neutral expectations. Finally, previous mitochondrial studies also identified a demographic expansion of this parasitic nematode species, which lends further support to a scenario involving both demographic and purifying forces in the ligand-binding domain of H. placei.     

Positive Selection on Nucleotide Substitutions and Indels in Accessory Gland Proteins of the Drosophila pseudoobscura Subgroup

Genes encoding reproductive proteins often diverge rapidly due to positive selection on nucleotide substitutions. While this general pattern is well established, the extent to which specific reproductive genes experience similar selection in different clades has been little explored, nor have possible targets of positive selection other than nucleotide substitutions, such as indels, received much attention. Here, we inspect for the signature of positive selection in the genes encoding five accessory gland proteins (Acps) (Acp26Aa, Acp32CD, Acp53Ea, Acp62F, and Acp70A) originally described from Drosophila melanogaster but with recognizable orthologues in the D. pseudoobscura subgroup. We compare patterns of selection within the D. psuedoobscura subgroup to those in the D. melanogaster subgroup. Similar patterns of positive selection were found in Acp26Aa and Acp62F in the two subgroups, while Acp53Ea and Acp70A experienced purifying selection in both subgroups. These proteins have thus remained targets for similar types of selection over long (>21-MY) periods of time. We also found several indel substitutions and polymorphisms in Acp26Aa and Acp32CD. These indels occur in the same regions as positively selected nucleotide substitutions for Acp26Aa in the D. pseudoobscura subgroup but not in the D. melanogaster subgroup. Rates of indel substitution within Acp26Aa in the D. pseudoobscura subgroup were up to several times those in noncoding regions of the Drosophila genome. This suggests that indel substitutions may be under positive selection and may play a key role in the divergence of some Acps. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Willis Swanson]     

Domesticated P Elements in the Drosophila montium Species Subgroup Have a New Function Related to a DNA Binding Property

Molecular domestication of a transposable element is defined as its functional recruitment by the host genome. To date, two independent events of molecular domestication of the P transposable element have been described: in the Drosophila obscura species group and in the Drosophila montium species subgroup. These P neogenes consist of stationary, nonrepeated sequences, potentially encoding 66-kDa repressor-like (RL) proteins. Here we investigate the function of the montium P neogenes. We provide evidence for the presence of RL proteins in two montium species (D. tsacasi and D. bocqueti) specifically expressed in adult and larval brain and gonads. We tested the hypothesis that the montium P neogenes’ function is related to the repression of the transposition of distantly related mobile P elements which coexist in the genome. Our results strongly suggest that the montium P neogenes are not recruited to downregulate the P element transposition. Given that all the proteins encoded by mobile or stationary P homologous sequences show a strong conservation of the DNA binding domain, we tested the capacity of the RL proteins to bind DNA in vivo. Immunostaining of polytene chromosomes in D. melanogaster transgenic lines strongly suggests that montium P neogenes encode proteins that bind DNA in vivo. RL proteins show multiple binding to the chromosomes. We suggest that the property recruited in the case of the montium P neoproteins is their DNA binding property. The possible functions of these neogenes are discussed. [Reviewing Editor: Dr. Dmitri Petrov     

Identification of One Intron Loss and Phylogenetic Evolution of Dfak Gene in the Drosophila melanogaster Species Group

Intron loss and its evolutionary significance have been noted in Drosophila. The current study provides another example of intron loss within a single-copy Dfak gene in Drosophila. By using polymerase chain reaction (PCR), we amplified about 1.3 kb fragment spanning intron 5–10, located in the position of Tyr kinase (TyK) domain of Dfak gene from Drosophila melanogaster species group, and observed size difference among the amplified DNA fragments from different species. Further sequencing analysis revealed that D. melanogaster and D. simulans deleted an about 60 bp of DNA fragment relative to other 7 Drosophila species, such as D. elegans, D. ficusphila, D. biarmipes, D. takahashii, D. jambulina, D. prostipennis and D. pseudoobscura, and the deleted fragment located precisely in the position of one intron. The data suggested that intron loss might have occurred in the Dfak gene evolutionary process of D. melanogaster and D. simulans of Drosophila melanogaster species group. In addition, the constructed phylogenetic tree based on the Dfak TyK domains clearly revealed the evolutionary relationships between subgroups of Drosophila melanogaster species group, and the intron loss identified from D. melanogaster and D. simulans provides a unique diagnostic tool for taxonomic classification of the melanogaster subgroup from other group of genus Drosophila.     

Further Study on the Esterase Patterns of Sibling Species in the Drosophila saltans Subgroup (saltans Group): Intraspecific and Interspecific Variations in the Development

Twenty of the 32 esterase bands previously detected in the adults of D. prosaltans, D. saltans and D.␣austrosaltans were found in larvae and pupae studied in this work. The results showed that, in addition to expressing the highest number of esterase bands, the adult stage of the three species exhibited the highest degree of expression (amount of synthesis) for most of the bands. Differences between larval and pupal stages were detected in the degree of expression (amount of synthesis) of the bands and in the frequency of samples expressing them. The frequencies of expression of the bands corresponding to genes in loci 1–3 were greater in pupae than in larvae while the frequencies of expression of the bands corresponding to genes in loci 4–9 were predominantly expressed in larvae or were equal in both developmental stages. Like the adults, larvae, pupae and empty pupal cases (which were also studied in this work) showed specific esterases. Taken together, the observations showed that, in the species studied, every developmental stage is characterized by specific bands and by specific frequency and degree of expression of the bands shared with other stages.     

Esterase Patterns and Phylogenetic Relationships of Drosophila Species in the Saltans Subgroup (Saltans Group)

The esterase patterns of sixteen strains from four species in the saltans subgroup were analyzed using polyacrylamide gel electrophoresis. Thirty-four esterase bands were detected. By using and naphthyl acetates as substrates, they were classified in 18 -esterases (they hydrolyse the -naphtyl substrate), 15 -esterases (they hydrolyse the -naphtyl substrate) and 1 /-esterase (it hydrolyses the and -naphtyl substrates). Among the -esterases, three were detected exclusively in males. Malathion, Eserine and pCMB were used as inhibitors in order to characterize biochemically the esterases. The results indicated the presence of cholinesterases, carboxylesterases and acetylesterases. The degree of mobility of the bands in the gels, their specificity to and naphthyl acetates and the results of the inhibition tests allowed us to recognize tentatively nine genetic loci. Phylogenetic relationships among species inferred on the basis of the esterase patterns by PAUP 4.0b8, with neighbor-joining search and a bootstrap analysis showed that, although the four species are closely related, D. septentriosaltans, D. saltans and D. austrosaltans are closer to each other than to D. prosaltans. These results showed to be consistent with phylogenetic relationships previously inferred from inversion polymorphism.     

Identification of Two Subfamilies of micropia Transposable Element in Species of the repleta Group of Drosophila

The occurrence, number of insertion sites and antisense RNA expression of micropia transposable element were studied in 26 species that belong to three subgroups (mercatorum, mulleri and hydei) of repleta group of Drosophila. Under high specific PCR, micropia sequences were detected in 11 species, but under less stringent condition, this retrotransposon was detected in all species. The widespread distribution of micropia suggests that this element was already present at the common ancestor of the repleta group of Drosophila. Southern blot analysis showed a variation from 0 to 17 different insertion sites and the occurrence of male-specific sequences. We found that the expression of the 1.0 kb micropia antisense RNA is variable among the species and tissues (soma and testis), which suggests that more than one mechanism regulates transposition in these species. Variation of amplification by PCR and of antisense RNA expression, as well as divergence of nucleotide sequences among the species allow us to suggest that at least two subfamilies of micropia transposable element are harbored by the genome of this species group.     

Molecular Phylogeny of the <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> Species Subgroup

Although molecular and phenotypic evolution have been studied extensively in Drosophila melanogaster and its close relatives, phylogenetic relationships within the D. melanogaster species subgroup remain unresolved. In particular, recent molecular studies have not converged on the branching orders of the D. yakuba–D. teissieri and D. erecta–D. orena species pairs relative to the D. melanogaster–D. simulans–D. mauritiana–D. sechellia species complex. Here, we reconstruct the phylogeny of the melanogaster species subgroup using DNA sequence data from four nuclear genes. We have employed vectorette PCR to obtain sequence data for orthologous regions of the Alcohol dehydrogenase (Adh), Alcohol dehydrogenase related (Adhr), Glucose dehydrogenase (Gld), and rosy (ry) genes (totaling 7164 bp) from six melanogaster subgroup species (D. melanogaster, D. simulans, D. teissieri, D. yakuba, D. erecta, and D. orena) and three species from subgroups outside the melanogaster species subgroup [D. eugracilis (eugracilis subgroup), D. mimetica (suzukii subgroup), and D. lutescens (takahashii subgroup)]. Relationships within the D. simulans complex are not addressed. Phylogenetic analyses employing maximum parsimony, neighbor-joining, and maximum likelihood methods strongly support a D. yakuba–D. teissieri and D. erecta–D. orena clade within the melanogaster species subgroup. D. eugracilis is grouped closer to the melanogaster subgroup than a D. mimetica–D. lutescens clade. This tree topology is supported by reconstructions employing simple (single parameter) and more complex (nonreversible) substitution models. Present address (Ryan M. David): University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284, USA     

A New Test for Selection Applied to Codon Usage in Drosophila simulans and D. mauritiana

In many organisms, synonymous codon usage is biased by a history of natural selection. However, codon bias, itself, does not indicate that selection is ongoing; it may be a vestige of past selection. Simple statistical tests have been devised to infer ongoing selection on codon usage by comparing the derived state frequency spectra at polymorphic sites segregating either derived preferred codons or derived unpreferred codons; if selection is effective, the frequency of derived states should be higher in the former. We propose a new test that uses the inferred degree of preference, essentially calculating the correlation of derived state frequency and the difference in preference between the derived and the ancestral states; the correlation should be positive if selection is effective. When implementing the test, derived and ancestral states can be assigned by parsimony or on the basis of relative probability. In either case, statistical significance is estimated by a simple permutation test. We explored the statistical power of the test by sampling polymorphism data from 14 loci in 16 strains of D. simulans, finding that the test retains 80% power even when quite a few of the data are discarded. The power of the test likely reflects better use of multiple features of the data, combining population frequencies of polymorphic variants and quantitative estimates of codon preferences. We also applied this novel test to 14 newly sequenced loci in five strains of D. mauritiana, showing for the first time ongoing selection on codon usage in this species.     

Variability on the dot chromosome in the Drosophila simulans clade

A recent study suggested that recent nuclear gene introgression between Drosophila simulans and D. mauritiana may have obscured efforts to estimate the phylogeny of the species of the D. simulans clade, which includes these two species and D. sechellia. Here, we report sequence variation of an intron of the eyeless gene in this species group. This gene should introgress freely between these species because it is not linked to any known barriers to gene exchange. We have also reevaluated levels of sequence divergence among species in this clade, noting differences between loci in regions of low recombination (as in all chromosome 4 loci) relative to other loci. Overall, none of the data analyzed were consistent with recent introgression exclusively between D. simulans and D. mauritiana.     

Evolutionary experiments on mate recognition in the Drosophila serrata species complex

It is becoming increasingly apparent that at least some aspects of the evolution of mate recognition may be amenable to manipulation in evolutionary experiments. Quantitative genetic analyses that focus on the genetic consequences of evolutionary processes that result in mate recognition evolution may eventually provide an understanding of the genetic basis of the process of speciation. We review a series of experiments that have attempted to determine the genetic basis of the response to natural and sexual selection on mate recognition in the Drosophila serrata species complex. The genetic basis of mate recognition has been investigated at three levels: (1) between the species of D. serrata and D. birchii using interspecific hybrids, (2) between populations of D. serrata that are sympatric and allopatric with respect to D. birchii, and (3) within populations of D. serrata. These experiments suggest that it may be possible to use evolutionary experiments to observe important events such as the reinforcement of mate recognition, or the generation of the genetic associations that are central to many sexual selection models.     

Reduced Selection for Codon Usage Bias in <Emphasis Type="Italic">Drosophila miranda</Emphasis>

Biased codon usage in many species results from a balance among mutation, weak selection, and genetic drift. Here I show that selection to maintain biased codon usage is reduced in Drosophila miranda relative to its ancestor. Analyses of mutation patterns in noncoding DNA suggest that the extent of this reduction cannot be explained by changes in mutation bias or by biased gene conversion. Low levels of variability in D. miranda relative to its sibling species, D. pseudoobscura, suggest that it has a much smaller effective population size. Reduced codon usage bias in D. miranda may thus result from the reduced efficacy of selection against newly arising mutations to unpreferred codons. [Reviewing Editor: Dr. Richard Kliman]     

A Genomic Comparison of Faster-Sex, Faster-X, and Faster-Male Evolution Between Drosophila melanogaster and Drosophila pseudoobscura

A genomic comparison of Drosophila melanogaster and Drosophila pseudoobscura provides a unique opportunity to investigate factors involved in sequence divergence. The chromosomal arrangements of these species include an autosomal segment in D. melanogaster which is homologous to part of the X chromosome in D. pseudoobscura. Using orthologues to calculate rates of nonsynonymous (d_N) substitutions, we found genes on the X chromosome to be significantly more diverged than those on the autosomes, but it is not true for segment 3L-XR which is autosomal in D. melanogaster (3L) and X-linked in D. pseudoobscura (XR). We also found that the median d_N values for genes having reproductive functions in either the male, the female, or both sexes are higher than those for sequences without reproductive function and even higher for sequences involved in male-specific function. These estimates of divergence for male sex-related sequences are most likely underestimates, as the very rapidly evolving reproductive genes would tend to lose homology sooner and thus not be included in the comparison of orthologues. We also noticed a high proportion of male reproductive genes among the othologous genes with the highest rates of d_N. Reproductive genes with and without an orthologue in D. pseudoobscura were compared among D. melanogaster, D. simulans, and D. yakuba and it was found that there were in fact higher rates of divergence in the group without a D. pseudoobscura orthologue. These results, from widely separated taxa, bolster the thesis that sexual system genes experience accelerated rates of change in comparison to nonsexual genes in evolution and speciation. [Reviewing Editor: Dr. Willie J. Swanson]     

In Situ Hybridisation Analysis of the X-Linked Genes in the Species of the Virilis Group of Drosophila

When estimating the level of DNA sequence variation within and between populations or when planning QTL analysis, it is essential to know the location of the genes under study. In the present work, five X chromosomal genes, earlier localised in Drosophila virilis and D. littoralis, were mapped by in situ hybridisation on the larval polytene chromosomes of four other virilis group species, D. a. americana, D. flavomontana, D. lacicola and D. montana. Conjugation of X chromosomes of the most interesting species pairs was studied in interspecific hybrids. Three of the marker genes were used as RFLP markers to examine the occurrence of recombination in D. flavomontana and D. montana hybrid females. The gene arrangement of all species studied, appeared to be different at the proximal end of the X chromosome, which prevented normal conjugation along the most part of the X chromosome. The data illustrating the locations of five X chromosomal marker genes are presented for D. a. americana, D. flavomontana, D. lacicola and D. montana.     

Selection against homozygotes in the ADH polymorphic system of Drosophila melanogaster associated with the lethal factor l(2) Stm

In the natural populations ⁺Tüb, ⁺Prov, and ⁺Rov, similar Adh ^F allele frequencies occur (q _F=0.11, 0.18, and 0.08, respectively). However, there is a discrepancy in that the Adh ^F allele in ⁺Tüb is closely linked to the lethal factor 1(2)Stm, which reduces relative fitness of the F phenotype to zero. In spite of this, polymorphism is maintained also in ⁺Tüb, because the heterozygotes are superior to the homozygous S type (relative fitness=0.88). Under laboratory culture conditions, in ⁺Tüb the relative fitness of the S genotype further decreases to 0.6. After outcrossing the lethal factor, relative fitnesses for S, FS, and F become 0.6, 1, and 0.48, respectively, implying that fitness for S remains the same. Relative values for S, FS, and F in ⁺Prov, not affected by the lethal factor, are calculated by the maximum average fitness method to be 1, 1.2, and 0.2 under the assumption that heterozygous FS are similarly superior to S as in the natural ⁺Tüb population and all allele frequencies found are stable equilibrium values.     

Effects on ADH activity and distribution,following selection for tolerance to ethanol in Drosophila melanogaster

Strains of Drosophila melanogaster homozygous for either the Adh^F or the Adh^S allele were kept on food supplemented with ethanol for 20 generations. These strains (FE and SE) were tested for tolerance to ethanol and compared with control strains (FN and SN). The E strains showed increased tolerance to ethanol both in the adult and in the juvenile life stages. In adults the increase in tolerance was not accompanied by an increase in overall ADH activity. However, there were changes in the distribution of ADH over the body parts. Flies of the FE strain possessed significantly more ADH in the abdomen, compared with FN. Another set of FN and SN populations were started both on standard food and on ethanol food with reduced yeast concentrations. After 9 months ADH activities were determined in flies from these populations which had been placed on three different media: the food the populations had been kept on, regular food and regular food supplemented with ethanol. The phenotypic effects of yeast reduction on ADH activity were considerably, but longterm genetic effects were limited.     

Copulatory Courtship in Drosophila birchii and D. serrata,Species Recognition and Sexual Selection

Two endemic Australian Drosophila species, D. birchii and D. serrata, have a copulatory courtship, i.e., the males court the female mainly during copulation. In the present study we found the males of both species to mount their prospective mating partners selectively, exhibiting both sex and species recognition. The males began to sing after mounting the female, and they often exhibited also postcopulatory displays typical to copulatory courtship. D. birchii and D. serrata females discriminated against males which did not sing during mounting/copulation, which suggests that the females utilize cryptic female choice. Our findings raise the question of how widespread a phenomenon cryptic female choice is in Drosophila species.     

Selection at the alcohol dehydrogenase locus of Drosophila melanogaster: Effects of ethanol and temperature

Drosophila melanogaster larvae were subjected to 10 generations of selection on 6% ethanol at 17, 25, and 30°C. For each temperature there was a significant (P<0.01) increase in the frequency of the Adh isoallele. Controls with no ethanol showed no change in the frequency of the Adh^F isoallele. Larvae subjected to stronger selection on 8% ethanol confirmed the results. When adults of various ages were subjected to 16 and 32°C, the ADH^F isoenzyme retained its twofold advantage in activity over ADH^S regardless of the temperature. The same result was obtained with larvae at 16 and 35°C. Although some effect of temperature was demonstrated, it was concluded that the effect was not strong enough for temperature to be a selective factor under the conditions studied. However, ethanol is a strong selective factor for laboratory populations.     

Molecular Evolution and Positive Selection of the Symbiotic Gene NORK in Medicago truncatula

Understanding the selective constraints of partner specificity in mutually beneficial symbiosis is a significant, yet largely unexplored, prospect of evolutionary biology. These selective constraints can be explored through the study of nucleotide polymorphism at loci controlling specificity. The membrane-anchored receptor NORK (nodulation receptor kinase) of the legume Medicago truncatula controls early steps of root infection by two symbiotic microorganisms: nitrogen-fixing bacteria (rhizobia) and endomycorrhizal fungi (Glomales). We analyzed the diversity of the gene NORK by sequencing 4 kilobases in 28 inbred lines sampled from natural populations. We detected 33 polymorphic sites with only one nonsynonymous change. Analysis based on Tajima’s D and Fay and Wu’s H summary statistics revealed no departure from the neutral model. We analyzed divergence using sequences from the closely related species M. coerulea. The McDonald-Kreitman test indicated a significant excess of nonsynonymous changes contributing to this divergence. Furthermore, maximum-likelihood analysis of a molecular phylogeny of a few legume species indicated that a number of amino acid sites, likely located in the receptor domain of the protein, evolved under the regime of positive selection. Further research should focus on the rate and direction of molecular coevolution between microorganisms’ signaling molecules and legumes’ receptors. [Reviewing Editor: Dr. Deborah Charlesworth] Sequence data were deposited in the GenBank database under accession nos. AY676428 to AY676457 and AJ884582.