Direct Evidence of Genic Differentiation between Sex Ratio and Standard Gene Arrangements of X Chromosome in DROSOPHILA PSEUDOOBSCURA

Evidence of strong association of alleles at the Esterase-5 and adult Acid phosphatase-6 loci with the Sex Ratio and the Standard X chromosome arrangements in D. pseudoobscura is presented. There is no differentiation at these two loci, either in the Sex Ratio or the Standard arrangement from two widely separated geographic populations. At the Esterase-5 locus, alleles characteristic of one arrangement are found in low frequencies in the other arrangement. It is concluded, from these and other observations that the differentiation between the Sex Ratio and the Standard arrangement, at these two loci, is a result of natural selection favoring different coadapted gene blocks in these arrangements. The average heterozygosity at these two loci in different karyotypes is in the following order: ST/SR > ST/ST >> SR/SR which agrees very well with the fitness estimates of these karyotypes.     

Associations of Alleles of the Esterase-1 Locus with Gene Arrangements of the Left Arm of the Second Chromosome in DROSOPHILA ROBUSTA

Evidence of strong associations of Est-1 alleles with the 2L, 2L1 and 2L3 gene arrangements of the left arm of the second chromosome in D. robusta is presented. Each gene arrangement is polymorphic for three to four Est-1 alleles. The allele frequencies differ in the 2L3 and 2L arrangements; the allele Est-1^.92 is 8% in the 2L3 arrangement (n=203)—this allele is 82% in the 2L arrangement (n=203); the allele Est-1^1.0 is 66% and 14.8% in the 2L3 and 2L arrangements, respectively. There are no differences in allele frequencies in 2L3 arrangements from any of the widely separated seven different populations; similarly the allele frequencies in the 2L arrangement are alike in all five widely separated populations studied. The allele frequencies in the 2L1 arrangement are intermediate to those observed in the 2L3 and the 2L arrangements and show north-south clinal change. These associations between Est-1 alleles and gene arrangements of the left arm of the second chromosome are due to natural selection favoring different allele frequencies in different gene arrangements, as a result of epistatic interactions between the Est-1 locus and the loci on the gene arrangements. As expected, we observe that the proportion of heterozygotes is greater in the inversion heterokaryotypes than in the homokaryotypes.     

A Comparative Study of the Esterase-5 Locus in DROSOPHILA PSEUDOOBSCURA,D. PERSIMILIS and D. MIRANDA

Electrophoretic phenotypes of the esterase-5 locus were examined in the sibling species D. pseudoobscura, D. persimilis and D. miranda. D. persimilis alleles were found to have uniformly higher charge on monomers than corresponding alleles of either D. pseudoobscura or D. miranda. Consequently, D. persimilis shares no alleles in common with either D. pseudoobscura or D. miranda, while the latter two species share a number of alleles. It was discovered that by increasing the concentration of acrylamide gel and increasing the length of migration, more allelic differences could be distinguished. Also more alleles were discovered by examining monomer mobility in addition to dimer mobility. In D. persimilis and D. miranda it was found that the previously known high frequency allelic classes broke down into several allelic classes. A test of goodness-of-fit to the infinite alleles model was done and a rough agreement with the model was found.     

Gene Differences between Third-Chromosome Inversions of DROSOPHILA PSEUDOOBSCURA

Associations of alleles of the acid phosphatase-3 locus with the different third-chromosome inversions from different populations of D. pseudoobscura are described. We observe only the allele AP-3 ^1.0 in the Standard and Arrow-head inversions and the allele AP-3^.98 in the Santa Cruz, Treeline, Cuernavaca and the Pikes Peak arrangements. The Chiricahua gene arrangement is polymorphic.     

Evidence for Extensive Genetic Differentiation between the Sex-Ratio and the Standard Arrangement of DROSOPHILA PSEUDOOBSCURA and D. PERSIMILIS and Identification of Hybrid Sterility Factors

This study deals with sex-ratio genes tightly linked within the Sex-Ratio inversion. By taking advantage of the fact that the Sex-Ratio chromosome of Drosophila persimilis [SR(B)] is homosequential to the Standard chromosome of D. pseudoobscura [ST(A)], we carried out two reciprocal introgression experiments. Individual segments of SR(B) or ST(A) were introgressed into the genome of D. pseudoobscura or D. persimilis, respectively. Males possessing a hybrid SR(B)-ST(A) X chromosome and a genetic background derived from either of the two species were tested for fertility and sex-ratio expression.—It was found that, in terms of the meiotic drive genes, the Sex-Ratio chromosome differs extensively from the Standard chromosome. Because recombinations of these genes result in a complete loss of sex-ratio expression, this finding lends strong support to the hypothesis of gene coadaptation. Coadaptation, in this context, is the advantage of being transmitted preferentially. In light of this finding, the evolution of the sex-ratio system in these two sibling species is discussed.—Introgression experiments also yielded information about hybrid sterility. With reciprocal introgression, sterility interactions were found to be "asymmetric." The asymmetry is fully expected from the viewpoint of evolution of postmating reproductive isolation.     

Gene Differences between the Sex Ratio and Standard Gene Arrangements of the X Chromosome and Linkage Disequilibrium between Loci in the Standard Gene Arrangement of the X Chromosome in DROSOPHILA PSEUDOOBSCURA

The Standard and Sex Ratio gene arrangements of the X chromosome of D. pseudobscura differ from each other in allele frequencies at the four X chromosome loci, esterase-5, adult acid phosphatase-6, phosphoglucomutase-1 and octanol dehydrogenase-3. The Standard arrangement which is the common arrangement in all populations is polymorphic at these loci in varying degrees, the geographically less widespread Sex Ratio arrangement has little polymorphism and is genically predominantly E-5(1.04) AP-6(-) Pgm1(1.0) ODH-3(1.0). The Sex Ratio arrangement from different populations is alike at all of the four loci, the Standard arrangement shows some gene frequency differences among populations. The Standard and Sex Ratio arrangements differ from each other by three inversions which suggests that the two arrangements are "old". Gene differences between these two chromosome arrangements can be explained due to differential natural selection of alleles in the Standard and Sex Ratio arrangments.-The order and percent recombination among these four loci in the Standard arrangement are: E-5-.294-AP-6-.335-Pgm-1-.024-ODH-3. The Standard X chromosomes from four different wild populations were analyzed for evidence of linkage disequilibrium between pairs of loci at these four loci. No evidence of linkage disequilibrium between pairs of loci was obtained. However, when linkages involving simultaneously three loci, E-5, AP-6 and Pgm-1 are considered, then significant departure from linkage equilibrium is observed.     

Molecular and chromosomal polymorphism in continental and insular populations from the southwestern range of Drosophila subobscura

Eight insular and continental populations from the south-western range of Drosophila subobscura have been studied with regard to molecular and inversion polymorphisms. Heterogeneity between populations with respect to allele frequencies of 4 gene loci (Amy, Est-8, Est-9 and Pep-1) is the highest known for natural populations of the species. Moreover, the most common allele for these loci is not the same in all populations. Cladograms based on UPGMA clustering of the genetic distances based on allele frequency do not coincide with those constructed with inversion data. The allele-frequency differences between the populations may be due to non-random associations between enzyme alleles and gene arrangements and to founder effects appearing in the insular populations.     

Genomics of natural populations: Evolutionary forces that establish and maintain gene arrangements in Drosophila pseudoobscura

The evolution of complex traits in heterogeneous environments may shape the order of genes within chromosomes. Drosophila pseudoobscura has a rich gene arrangement polymorphism that allows one to test evolutionary genetic hypotheses about how chromosomal inversions are established in populations. D. pseudoobscura has >30 gene arrangements on a single chromosome that were generated through a series of overlapping inversion mutations with >10 inversions with appreciable frequencies and wide geographic distributions. This study analyses the genomic sequences of 54 strains of Drosophila pseudoobscura that carry one of six different chromosomal arrangements to test whether (i) genetic drift, (ii) hitchhiking with an adaptive allele, (iii) direct effects of inversions to create gene disruptions caused by breakpoints, or (iv) indirect effects of inversions in limiting the formation of recombinant gametes are responsible for the establishment of new gene arrangements. We found that the inversion events do not disrupt the structure of protein coding genes at the breakpoints. Population genetic analyses of 2,669 protein coding genes identified 277 outlier loci harbouring elevated frequencies of arrangement‐specific derived alleles. Significant linkage disequilibrium occurs among distant loci interspersed between regions with low levels of association indicating that distant allelic combinations are held together despite shared polymorphism among arrangements. Outlier genes showing evidence of genetic differentiation between arrangements are enriched for sensory perception and detoxification genes. The data presented here support the indirect effect of inversion hypothesis where chromosomal inversions are favoured because they maintain linked associations among multilocus allelic combinations among different arrangements.     

Serum esterase genetics in rabbits. II. Genetic analysis of the prealbumin esterase system,including atropinesterase and cocainesterase polymorphism

The zymotypic variation of rabbit prealbumin esterases is controlled by three autosomal loci, each with two alleles: Est-1 ^S and Est-1 ^s, Est-2^F and Est-2 ^f′, Est-3^D and Est-3 ^d. Est-1^S gives rise to the three S zones possessing the cocainesterase activity, Est-2 ^F to the three F zones with atropinesterase activity. Presence of the latter allele is never manifested without the Est-1 ^S allele. Est-3 ^D codes for the D zone. This D esterase reacts with the currently used substrate α-naphthylacetate only in the presence of the F zones. Est-1 and Est-2 loci are closely linked (<0.5% recombination); Est-3 shows no coupling with Est-1 and Est-2. The Est-1 ^S and Est-3 ^D alleles have a complete dominant expression, whereas the Est-2 alleles are codominant. Gene frequencies of the Est-1 and Est-2 loci vary between the examined breeds. A Hardy-Weinberg equilibrium is found in two populations (Cpb:ALU and Cpb:VW). A significant surplus of heterozygotes is demonstrated in a third population (Cpb:CH).     

Gene Polymorphism in Natural Populations of DROSOPHILA PERSIMILIS

Genetic variation at 43 loci has been studied in six different populations of Drosophila persimilis by electrophoresis of enzymes and proteins. In D. persimilis the mean proportion of polymorphic loci is 0.362, the mean proportion of heterozygous loci per individual is 0.100 and the average number of alleles per locus is 1.651. In all populations, the loci coding for the hydrolytic and other nonspecific enzymes are much more variable than the loci coding for the enzymes of the glycolytic pathway, Kreb's cycle, other specific enzymes and larval proteins. Most loci have similar allele frequency in all populations except the two loci, Amylase and Pt-12, which show a pattern of associations of different alleles with different third chromosome inversions.     

Genic Heterogeneity at Two Alcohol Dehydrogenase Loci in DROSOPHILA PSEUDOOBSCURA and DROSOPHILA PERSIMILIS

A sequential electrophoretic survey of the second chromosome loci, alcohol dehydrogenase-6 (Adh-6) and octanol dehydrogenase ( Odh), was performed on 147 isochromosomal lines of Drosophila pseudoobscura and 60 lines of its sibling species, D. persimilis. Gels run with a variety of acrylamide concentrations and buffer pH's revealed the presence of 18 alleles of Adh-6 in the two species, where only eight had been previously detected by conventional electrophoretic methods. Only two alleles were added with our techniques to the previous total of nine in both species at the largely monomorphic Odh locus. Both enzymes show a predominance of one allele, with the other variants being fairly rare. There was no evidence of increased genetic divergence between the two species, but we found a striking increase in differentiation of Adh-6 alleles between the main body of D. pseudoobscura populations and the conspecific isolate from Bogotá, Colombia. These results are compared with our previous surveys of xanthine dehydrogenase in these species and discussed in reference to theories of genic polymorphism.     

An evolutionary model for the duplication and divergence of esterase genes in Drosophila

Summary The esterase 5 (Est-5 = gene, EST 5 = protein) enzyme in Drosophila pseudoobscura is encoded by one of three paralogous genes, Est-5A, Est5B, and Est-5C, that are tightly clustered on the right arm of the X chromosome. The homologous Est-6 locus in Drosophila melanogaster has only one paralogous neighbor, Est-P. Comparisons of coding and flanking DNA sequences among the three D. pseudoobscura and two D. melanogaster genes suggest that two paralogous genes were present before the divergence of D. pseudoobscura from D. melanogaster and that, later, a second duplication occurred in D. pseudoobscura. Nucleotide sequences of the coding regions of the three D. pseudoobscura genes showed 78–85% similarity in pairwise comparisons, whereas the relatedness between Est-6 and Est-P was only 67%. The higher degree of conservation in D. pseudoobscura likely results from the comparatively recent divergence of Est-5B and Est-5C and from possible gene conversion events between Est-5A and Est-5B. Analyses of silent and replacement site differences in the two exons of the paralogous and orthologous genes in each species indicate that common selective forces are acting on all five loci. Further evidence for common purifying selective constraints comes from the conservation of hydropathy profiles and proposed catalytic residues. However, different levels of amino acid substitution between the paralogous genes in D. melanogaster relative to those in D. pseudoobscura suggest that interspecific differences in selection also exist.Offprint requests to: R.C. Richmond     

Serum esterase genetics in rabbits. IV. The prealbumin and β-globulin systems

Discontinuous starch gel electrophoresis revealed a fourth allele of rabbit prealbumin serum esterase at locus Est-2. This allele is designated Est-2 ^f and appears to be silent. In addition to the prealbumin serum esterases, another serum esterase system was studied in rabbits. This system is localized in the β-globulin region. Genetic analysis indicated that one locus with two codominant alleles controls the variation in this region. Linkage of this system with Est-1 and Est-2 of the prealbumin serum esterases was demonstrated. Comparison of the arrangement of these esterase loci on linkage group VI with the esterase loci on chromosome 8 of the mouse gives additional support for the theory of evolutionary conservation of chromosomal segments coding for mammalian esterases.     

Frequency-Dependent Selection at the PGM-1 Locus of DROSOPHILA PSEUDOOBSCURA

Frequency-dependent fitness was studied at the Pgm-1 locus of Drosophila pseudoobscura with respect to two fitness components: rate of development and larva-to-adult survival. The Pgm-1 locus is very polymorphic with only two alleles, Pgm-1¹⁰⁰ and Pgm-1¹⁰⁴, occurring at high frequencies. For each of these two alleles, 20 homozygous strains were obtained from a sample of 1,140 wild-inseminated females. First-instar larvae of the two genotypes were combined in a set of eight different frequencies: 0.0, 0.10, 0.25, 0.40, 0.60, 0.75, 0.90, and 1.0. Frequency-dependent fitness effects were observed for the two survival-related fitness components examined: larvae of the less common genotype develop faster and have a higher probability of survival than larvae of the more common genotype. The rate of survival at intermediate genotypic frequencies is similar to that in pure cultures. If selection acted solely as frequency-dependent effects on survival-related components of fitness, the equilibrium frequency of the Pgm-1¹⁰⁰ allele would be 0.615 for a two-genotype system, which fits an observed frequency range for this allele in nature between 0.55 and 0.71. Experimentally created linkage disequilibrium was excluded from the experiment by using a large number of independent strains. It is nevertheless possible that the frequency-dependent selection may not affect the Pgm-1 locus per se, but may reflect a linkage disequilibrium present in the natural population. Even if this were the case, the frequency-dependent selection could affect the frequency of the Pgm-1 alleles in nature.     

The Genomics of Speciation in Drosophila: Diversity, Divergence, and Introgression Estimated Using Low-Coverage Genome Sequencing

In nature, closely related species may hybridize while still retaining their distinctive identities. Chromosomal regions that experience reduced recombination in hybrids, such as within inversions, have been hypothesized to contribute to the maintenance of species integrity. Here, we examine genomic sequences from closely related fruit fly taxa of the Drosophila pseudoobscura subgroup to reconstruct their evolutionary histories and past patterns of genic exchange. Partial genomic assemblies were generated from two subspecies of Drosophila pseudoobscura (D. ps.) and an outgroup species, D. miranda. These new assemblies were compared to available assemblies of D. ps. pseudoobscura and D. persimilis, two species with overlapping ranges in western North America. Within inverted regions, nucleotide divergence among each pair of the three species is comparable, whereas divergence between D. ps. pseudoobscura and D. persimilis in non-inverted regions is much lower and closer to levels of intraspecific variation. Using molecular markers flanking each of the major chromosomal inversions, we identify strong crossover suppression in F₁ hybrids extending over 2 megabase pairs (Mbp) beyond the inversion breakpoints. These regions of crossover suppression also exhibit the high nucleotide divergence associated with inverted regions. Finally, by comparison to a geographically isolated subspecies, D. ps. bogotana, our results suggest that autosomal gene exchange between the North American species, D. ps. pseudoobscura and D. persimilis, occurred since the split of the subspecies, likely within the last 200,000 years. We conclude that chromosomal rearrangements have been vital to the ongoing persistence of these species despite recent hybridization. Our study serves as a proof-of-principle on how whole genome sequencing can be applied to formulate and test hypotheses about species formation in lesser-known non-model systems.     

Patterns of Gene Variation in Central and Marginal Populations of DROSOPHILA ROBUSTA

The central and marginal populations of D. robusta differ greatly in the level of inversion polymorphism; the marginal populations are monomorphic or nearly so and the central populations are highly polymorphic. This paper presents the frequencies of alleles at forty gene loci in various populations of D. robusta, studied by electrophoresis of proteins and enzymes. Population samples were obtained from eight widely separated populations of D. robusta which included the central, the extreme marginal and the intervening populations between the center and the margins. We find that the proportion of polymorphic loci and average heterozygosity per individual is slightly higher in the marginal populations than the central populations. In D. robusta on an average, 39% of the loci are polymorphic and the average proportion of loci heterozygous per individual is 11%. A breakdown of loci in three categories, viz, hydrolytic enzymes and some other enzymes, larval proteins and glycolytic and Kreb's cycle enzymes, shows that in all populations the level of polymorphism is highest in the hydrolytic enzymes, intermediate in larval proteins and least in the glycolytic and Kreb's cycle enzymes. On the average, the proportion of loci heterozygous per individual for three groups of loci is: hydrolytic enzymes and others (.164), larval proteins (.115) and glycolytic and Kreb's cycle enzymes (.037). We also observe that in all populations the level of polymorphism on the X chromosome is far less than the expected 38%; in salivary gland cells the euchromatic length of the X chromosome is 38% of the entire genome. Lower levels of polymorphism for the X chromosome loci are explained due to low probability of balanced polymorphisms for the X-linked loci since the conditions for establishment of balanced polymorphism for X-linked loci are more restrictive than for the autosomal loci.-The polymorphic loci can be grouped according to pattern of allele frequencies in different populations as follows: (1) The allele frequencies are similar in all populations at the XDH, Pep-1 and Hex-1 loci. (2) The alleles at the Est-1, Est-2, Amy loci and the AP-4(1.0) and the LAP-1(.90) alleles show north south clinal change in frequency. (3) There is north south and east west differentiation at the Pt-5, Pt-8 and Pt-9 loci and the allele AP-4(.81). (4) Polymorphism at loci such as Fum, B.Ox, Hex-8, Pep-2 and Pep-3 are restricted to only one or two of the populations. (5) Allele frequencies at the MDH and ODH loci fluctuate between populations. (6) Allele frequencies at many polymorphic loci such as Est-1, Est-2, LAP-1, AP-4, Pt-5, Pt-8, Pt-9, Pt-16, MDH, Fum change clinally within a gene arrangement. The pattern of gene variation in D. robusta is very complex and cannot be easily explained due to migration of neutral alleles between once-isolated populations or to semi-isolation of neutral alleles. The observations of the pattern of allele variation in different populations, high levels of polymorphism in the marginal populations which have small population size and low levels of polymorphism of the X chromosome loci all support the argument in favor of balancing selection as the main mechanism for the maintenance of these polymorphisms. Environmental factors must play a role in the maintenance of a great deal of these polymorphisms, since we observe clinal allele frequency changes even within a given inversion type.     

An esterase duplication in Drosophila: Differences in expression of duplicate loci within and among related species

An esterase duplication is described in the sibling species pair Drosophila mojavensis and Drosophila arizonensis. We present evidence for two separate structural loci mapping at a distance of less than 0.16 recombination units from each other. Alleles at the two loci have the same substrate specificities and form small amounts of interlocus heterodimers. One locus (Est-5) is functioning throughout the insect's life cycle and appears at high concentrations in the hemolymph and the fat body. Its duplicate (Est-4) functions only during the late larval stage and is concentrated mainly in the carcass. No null alleles at either locus were observed in population surveys. An examination of 12 other species from the repleta group, to which D. mojavensis and D. arizonesis belong, suggests that Est-5 is universally present, but the activity levels of Est-4 vary among species and may be totally absent in some species. Variation in the level of Est-4 activity does not closely follow the phylogenetic relationship.     

The effect of relative humidity on photoresponse in sympatric species of Drosophila: Short term exposure to desiccating environments

Photoresponses of isofemale strains of sympatric sibling species, Drosophila pseudoobscura and D. persimilis from MacDonald Ranch, California and D. pseudoobscura and D. miranda from Mather, California were compared at relative humidities of 95, 50 and 5% for a period of 30 min. The sympatric species could be distinguished behaviourally by their responses at the three humidities. Although the average photoresponses of all four populations were negative, D. pseudoobscura was significantly more photonegative than its sibling sympatric species in each habitat. There was no effect of humidity on light response over the short term duration of exposure to desiccating environments. A hypothesis is presented to account for this phenomenon.     

Linkage relationships and chromosomal locations of enzyme-coding genes in pepper,Capsicum annuum

The linkage relationships and chromosomal locations of 14 enzyme-coding genes were investigated in Capsicum annuum L. (garden pepper) by monitoring segregations in backcross and F₂ progeny of an interspecific cross between C. annuum cv. NM6-4 and C. chinense CA4 and by studying allele dosage effects in five hybrid primary trisomics. These conclusions can be reached: 6Pgdh-i is on the metacentric chromosome corresponding to the noir trisomie; Idh-1 and Est-3 are on chromosome 12; an aerocentric chromosome which corresponds to the pourple trisomie; Idh-1 is near the centromere and Est-3 is distal on the long arm of that chromosome. The other loci can be arranged in the following linkage groups and are apparently not located on the trisome corresponding to any of the trisomics tested: Est-4-18cM-(Pgi-1-3cM-Pgm-1); Prx-7-2cM-Tk-1; Pgm-2-2cM-Skdh-1; Est-1-OcM-Est-7. Linkage and dosage data combined with karyotype and meiotic analyses of the two species and F₁ hybrids suggest that Idh-1 and Skdh-1/Pgm-2 are near the breakpoints on the two chromosomes involved in a reciprocal translocation for which the two species differ. One locus, Pgm-3, was detected only in C. annuum cultivars and is apparently the result of a duplication of Pgm-2 which codes for cytosolic phosphoglucomutase activity. Pgm-2 and Pgm-3 are not tightly linked (approximately 20% recombination) which supports the proposal that Pgm-3 originated from a mechanism other than unequal crossing over. A comparison of the linkage relationships of enzyme-coding genes in pepper with those of putative orthologous loci in tomato reveals that two linkage blocks, Est-1-Est-7 and Pgi-1-Est-4, may have remained intact since the divergence of Capsicum and Lycopersicon.     

Gene Flow and the Geographical Distribution of a Molecular Polymorphism in DROSOPHILA PSEUDOOBSCURA

This paper discusses the relation between the geographical distribution of an enzyme polymorphism and population structure in Drosophila pseudoobscura. California populations of this species living in very different montane and lowland habitats separated by several kilometers are similar to each other in the frequency of an esterase allele. Previous estimates suggest that gene flow is too limited to account for this homogeneity of genetic structure, so that it must reflect some balancing force of natural selection. We show, however, that dispersal over unfavorable habitats is much greater than earlier supposed. Isolated populations of D. pseudoobscura separated by 15 km from other populations are subject to large amounts of immigration. This is shown by changes in the seasonal abundance of this species and in the annual pattern of lethal alleles in such populations. The genetic structure of an experimentally perturbed isolated population in an oasis returned to normal within a single year, suggesting that such populations are ephemeral and that the oasis is subject to annual recolonization by distant migrants. Direct assessment of marked flies shows that they can move at least 10 km in 24 hours over a desert. Such extensive gene flow may help explain the distribution of the esterase allele, and is relevant to the high level of molecular polymorphism and its general lack of geographic differentiation throughout the range of D. pseudoobscura.