HLA class II linkage disequilibrium and haplotype evolution in the Cayapa Indians of Ecuador.

DNA-based typing of the HLA class II loci in a sample of the Cayapa Indians of Ecuador reveals several lines of evidence that selection has operated to maintain and to diversify the existing level of polymorphism in the class II region. As has been noticed for other Native American groups, the overall level of polymorphism at the DRB1, DQA1, DQB1, and DPB1 loci is reduced relative to that found in other human populations. Nonetheless, the relative evenness in the distribution of allele frequencies at each of the four loci points to the role of balancing selection in the maintenance of the polymorphism. The DQA1 and DQB1 loci, in particular, have near-maximum departures from the neutrality model, which suggests that balancing selection has been especially strong in these cases. Several novel DQA1-DQB1 haplotypes and the discovery of a new DRB1 allele demonstrate an evolutionary tendency favoring the diversification of class II alleles and haplotypes. The recombination interval between the centromeric DPB1 locus and the other class II loci will, in the absence of other forces such as selection, reduce disequilibrium across this region. However, nearly all common alleles were found to be part of DR-DP haplotypes in strong disequilibrium, consistent with the recent action of selection acting on these haplotypes in the Cayapa.     

Polymorphism, recombination, and linkage disequilibrium within the HLA class II region.

Thirty-nine CEPH (Centre d'Etude du Polymorphisme Humain) families, comprised of 502 individuals, have been typed for the HLA class II genes DRB1, DQA1, DQB1, and DPB1 using nonradioactive sequence-specific oligonucleotide probes to analyze polymerase chain reaction amplified DNA. This population, which consists of 266 independent chromosomes, contains 27 DRB1, 7 DQA1, 12 DQB1, and 17 DPB1 alleles. Analysis of the distribution of allele frequencies using the homozygosity statistic, which gives an indication of past selection pressures, suggests that balancing selection has acted on the DRB1, DQA1, and DQB1 loci. The distribution of DPB1 alleles, however, suggests a different evolutionary past. Family data permits the estimation of recombination rates and the unambiguous assignment of haplotypes. No recombinants were found between DRB1, DQA1, and DQB1; however, recombinants were detected between DQB1 and DPB1, resulting in an estimated recombination fraction of greater than or equal to 0.008 +/- 0.004. Only 33 distinct DRB1-DQA1-DQB1 haplotypes were found in this population which illustrates the extreme nonrandom haplotypic association of alleles at these loci. A few of these haplotypes are unusual (previously unreported) for a Caucasian population and most likely result from past recombination events between the DR and DQ subregions. Examination of disequilibrium across the HLA region using these data and the available serologic HLA-A and HLA-B types of these samples shows that global disequilibrium between these loci declines with the recombination fraction, approaching statistic nonsignificance at the most distant interval, HLA-A to HLA-DP.DR-DQ haplotypes in linkage disequilibrium with DPB1 and B are noted and, finally, the evolutionary origin of certain class II haplotypes is addressed.     

PCR/oligonucleotide probe typing of HLA class II alleles in a Filipino population reveals an unusual distribution of HLA haplotypes.

We have analyzed the distribution of HLA class II alleles and haplotypes in a Filipino population by PCR amplification of the DRB1, DQB1, and DPB1 second-exon sequences from buccal swabs obtained from 124 family members and 53 unrelated individuals. The amplified DNA was typed by using nonradioactive sequence-specific oligonucleotide probes. Twenty-two different DRB1 alleles, including the novel Filipino *1105, and 46 different DRB1/DQB1 haplotypes, including the unusual DRB1*0405-DQB1*0503, were identified. An unusually high frequency (f = .383) of DPB1*0101, a rare allele in other Asian populations, was also observed. In addition, an unusual distribution of DRB1 alleles and haplotypes was seen in this population, with DR2 (f = .415) and DRB1*1502-DQB1*0502 (f = .233) present at high frequencies. This distribution of DRB1 alleles differs from the typical HLA population distribution, in which the allele frequencies are more evenly balanced. The distribution of HLA class II alleles and haplotypes in this Filipino population is different from that of other Asian and Pacific groups: of those populations studied to date; the Indonesian population is the most similar. DRB1*1502-DQB1*0502 was in strong linkage disequilibrium (D'' = .41) with DPB1*0101 (f = .126, for the extended haplotype), which is consistent with selection for this DR, DQ, DP haplotype being responsible for the high frequency of these three class II alleles in this population.     

Analysis of HLA class II haplotypes in the Cayapa Indians of Ecuador: a novel DRB1 allele reveals evidence for convergent evolution and balancing selection at position 86.

PCR amplification, oligonucleotide probe typing, and sequencing were used to analyze the HLA class II loci (DRB1, DQA1, DQB1, and DPB1) of an isolated South Amerindian tribe. Here we report HLA class II variation, including the identification of a new DRB1 allele, several novel DR/DQ haplotypes, and an unusual distribution of DPB1 alleles, among the Cayapa Indians (N = 100) of Ecuador. A general reduction of HLA class II allelic variation in the Cayapa is consistent with a population bottle-neck during the colonization of the Americas. The new Cayapa DRB1 allele, DRB1*08042, which arose by a G-->T point mutation in the parental DRB1*0802, contains a novel Val codon (GTT) at position 86. The generation of DRB1*08042 (Val-86) from DRB1*0802 (Gly-86) in the Cayapa, by a different mechanism than the (GT-->TG) change in the creation of DRB1*08041 (Val-86) from DRB1*0802 in Africa, implicates selection in the convergent evolution of position 86 DR beta variants. The DRB1*08042 allele has not been found in > 1,800 Amerindian haplotypes and thus presumably arose after the Cayapa separated from other South American Amerindians. Selection pressure for increased haplotype diversity can be inferred in the generation and maintenance of three new DRB1*08042 haplotypes and several novel DR/DQ haplotypes in this population. The DPB1 allelic distribution in the Cayapa is also extraordinary, with two alleles, DPB1*1401, a very rare allele in North American Amerindian populations, and DPB1*0402, the most common Amerindian DPB1 allele, constituting 89% of the Cayapa DPB1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Impact of historical founder effects and a recent bottleneck on MHC variability in Commander Arctic foxes (Vulpes lagopus)

Populations of Arctic foxes (Vulpes lagopus) have been isolated on two of the Commander Islands (Bering and Mednyi) from the circumpolar distributed mainland population since the Pleistocene. In 1970-1980, an epizootic outbreak of mange caused a severe population decline on Mednyi Island. Genes of the major histocompatibility complex (MHC) play a primary role in infectious disease resistance. The main objectives of our study were to compare contemporary variation of MHC class II in mainland and island Arctic foxes, and to document the effects of the isolation and the recent bottleneck on MHC polymorphism by analyzing samples from historical and contemporary Arctic foxes. In 184 individuals, we found 25 unique MHC class II DRB and DQB alleles, and identified evidence of balancing selection maintaining allelic lineages over time at both loci. Twenty different MHC alleles were observed in mainland foxes and eight in Bering Island foxes. The historical Mednyi population contained five alleles and all contemporary individuals were monomorphic at both DRB and DQB. Our data indicate that despite positive and diversifying selection leading to elevated rates of amino acid replacement in functionally important antigen-binding sites, below a certain population size, balancing selection may not be strong enough to maintain genetic diversity in functionally important genes. This may have important fitness consequences and might explain the high pathogen susceptibility in some island populations. This is the first study that compares MHC diversity before and after a bottleneck in a wild canid population using DNA from museum samples.     

Contrasting Roles of Interallelic Recombination at the Hla-a and Hla-B Loci

A statistical study of DNA sequences of alleles at the highly polymorphic class I MHC loci of humans, HLA-A and HLA-B, showed evidence of both large-scale recombination events (involving recombination of exons 1-2 of one allele with exons 3-8 of another) and small-scale recombination events (involving apparent exchange of short DNA segments). The latter events occurred disproportionately in the region of the gene encoding the antigen recognition site (ARS) of the class I molecule. Furthermore, they involved the ARS codons which are under the strongest selection favoring allelic diversity at the amino acid level. Thus, the frequency of recombinant alleles appears to have been increased by some form of balancing selection (such as overdominant selection) favoring heterozygosity in the ARS. These analyses also revealed a striking difference between the A and B loci. Recombination events appear to have occurred about twice as frequently at the B locus, and recombinants at the B locus were significantly more likely to affect polymorphic sites in the ARS. At the A locus, there are well-defined allelic lineages that have persisted since prior to the human-chimpanzee divergence; but at the B locus, there is no evidence for such long-lasting allelic lineages. Thus, relatively frequent interallelic recombination has apparently been a feature of the long-term evolution of the B locus but not of the A locus.     

Contrasting patterns of selection acting on MHC class I and class II DRB genes in the Alpine marmot (Marmota marmota)

The major histocompatibility complex (MHC) genes code for proteins that play a critical role in the immune system response. The MHC genes are among the most polymorphic genes in vertebrates, presumably due to balancing selection. The two MHC classes appear to differ in the rate of evolution, but the reasons for this variation are not well understood. Here, we investigate the level of polymorphism and the evolution of sequences that code for the peptide-binding regions of MHC class I and class II DRB genes in the Alpine marmot (Marmota marmota). We found evidence for four expressed MHC class I loci and two expressed MHC class II loci. MHC genes in marmots were characterized by low polymorphism, as one to eight alleles per putative locus were detected in 38 individuals from three French Alps populations. The generally limited degree of polymorphism, which was more pronounced in class I genes, is likely due to bottleneck the populations undergone. Additionally, gene duplication within each class might have compensated for the loss of polymorphism at particular loci. The two gene classes showed different patterns of evolution. The most polymorphic of the putative loci, Mama-DRB1, showed clear evidence of historical positive selection for amino acid replacements. However, no signal of positive selection was evident in the MHC class I genes. These contrasting patterns of sequence evolution may reflect differences in selection pressures acting on class I and class II genes.     

Balancing selection and heterozygote advantage in major histocompatibility complex loci of the bottlenecked Finnish wolf population

Maintaining effective immune response is an essential factor in the survival of small populations. One of the most important immune gene regions is the highly polymorphic major histocompatibility complex (MHC). We investigated how a population bottleneck and recovery have influenced the diversity and selection in three MHC class II loci, DLA‐DRB1, DLA‐DQA1 and DLA‐DQB1, in the Finnish wolf population. We studied the larger Russian Karelian wolf population for comparison and used 17 microsatellite markers as reference loci. The Finnish and Karelian wolf populations did not differ substantially in their MHC diversities ( = 0.047, P = 0.377), but differed in neutral microsatellite diversities ( = 0.148, P = 0.008). MHC allele frequency distributions in the Finnish population were more even than expected under neutrality, implying balancing selection. In addition, an excess of nonsynonymous compared to synonymous polymorphisms indicated historical balancing selection. We also studied association between helminth (Trichinella spp. and Echinococcus canadensis) prevalence and MHC diversity at allele and SNP level. MHC‐heterozygous wolves were less often infected by Trichinella spp. and carriers of specific MHC alleles, SNP haplotypes and SNP alleles had less helminth infections. The associated SNP haplotypes and alleles were shared by different MHC alleles, which emphasizes the necessity of single‐nucleotide‐level association studies also in MHC. Here, we show that strong balancing selection has had similar effect on MHC diversities in the Finnish and Russian Karelian wolf populations despite significant genetic differentiation at neutral markers and small population size in the Finnish population.     

HLA-DPB1 alleles in a population from South China

Using the polymerase chain reaction (PCR) and hybridization with oligonucleotide probes, we analyzed the distribution of DPB1 alleles in 99 healthy unrelated individuals from the city of Guangzhou (Canton), South China. Twelve different DPB1 alleles were found in this panel. The most common allele was DPB1*0501 (62.6%). Other major alleles detected included DPB1*02 (DPB1*0201 and DPB1*0202), DPB1*1301, DPB1*0401, and a recently described allele, designated DPB1*2101. The hybridization pattern of DPB1*2101 showed that this allele shared sequences with DPB1*0301 and DPB1*0601 in the A and F hypervariable regions, while the C, D, and E regions were identical to those of DPB1*0202. DPB1*2101 was observed in 11% of the subjects tested. It was found to be in strong linkage dis-equilibrium with DRB1*1202. In family studies, segregation of the haplotype DRB1*1202, DRB3*0301, DQA1*0601, DQB1*0301, DPB1*2101 was observed. The second exon of DPB1*2101 was sequenced from codon 8 to codon 90 and the sequence, inferred from the pattern of hybridization, was confirmed. DPB1*0301, DPB1*0402, DPB1*0101, DPB1*1401, DPB1*1901, and another recently recognized allele, now designated DPB1*2401, were detected with low frequencies. DPB1*2401 had the same hybridization pattern as DPB1*0501 except for a probe that matches codons 85–90. In this region, DPB1*2401 encoded the amino acid sequence GPMTLQ instead of EAVTLQ as in DPB1*0501.     

The Père David's deer MHC class I genes show unexpected diversity patterns, with monomorphic classical genes but polymorphic nonclassical genes and pseudogenes

Père David's deer (Elaphurus davidianus) is a highly inbred species that arose from 11 founders but now comprises a population of about 3,000 individuals, making it interesting to investigate the adaptive variation of this species from the major histocompatibility complex (MHC) perspective. In this study, we isolated Elda-MHC class I loci using magnetic bead-based cDNA hybridization, and examined the molecular variations of these loci using single-strand conformation polymorphism (SSCP) and sequence analysis. We obtained seven MHC class I genes, which we designated F1, F12, G2, I7, AF, I8, and C1. Our analyses of stop codons, phylogenetic trees, amino acid conservation, and G+C content revealed that F1, F12, G2, and I7 were classical genes, AF was a nonclassical gene, and I8 and C1 were pseudogenes. Our subsequent molecular examinations showed that the diversity pattern in the Père David's deer was unusual. Most mammals have more polymorphic classical class I loci vs. the nonclassical and neutral genes. In contrast, the Père David's deer was found to be monomorphic at classical genes F1, F12, G2, and I7, dimorphic at the nonclassical AF gene, dimorphic at pseudogene I8, and tetramorphic at pseudogene C1. The adverse polymorphism patterns of Elda-I genes might provide evidence for selection too faster deplete MHC variation than drift in the bottlenecked populations, while the postbottleneck tetramorphism of the C1 pseudogene appears to be evidence of strong historical balancing selection.     

Discordant patterns of linkage disequilibrium of the peptide-transporter loci within the HLA class II region.

Disequilibrium between genetic markers is expected to decline monotonically with recombinational map distance. We present evidence from the HLA class II region that seems to violate this principle. Pairwise disequilibrium values were calculated from six loci ranging in physical separation from 15 kb to 550 kb. The histocompatibility loci DRB1, DQA1, and DQB1, located on the distal end of the class II region, behave as a single evolutionary unit within which extremely high linkage disequilibrium exists. Lower but still significant levels of disequilibrium are present between these loci and DPB1, located at the proximal edge of the HLA complex. The peptide-transporter loci TAP1 and TAP2, located in the intervening region, reveal no disequilibrium with each other and low or negligible disequilibrium with the flanking loci. The action of two genetic process is required to account for this phenomenon: a recombinational hotspot operating between TAP1 and TAP2, to eliminate disequilibrium between these loci, and at the same time selection operating on particular combinations of alleles across the DR-DP region, to create disequilibrium in the favored haplotypes. The forces producing the patterns of disequilibrium observed here have implications for the mapping of train loci and disease genes: markers of TAP1, for example, would give a false impression as to the influence of DPB1 on a trait known to be associated with DQB1.     

Genetic drift vs. natural selection in a long-term small isolated population: major histocompatibility complex class II variation in the Gulf of California endemic porpoise (Phocoena sinus)

Although many studies confirm long-term small isolated populations (e.g. island endemics) commonly sustain low neutral genetic variation as a result of genetic drift, it is less clear how selection on adaptive or detrimental genes interplay with random forces. We investigated sequence variation at two major histocompatibility complex (Mhc) class II loci on a porpoise endemic to the upper Gulf of California, México (Phocoena sinus, or vaquita). Its unique declining population is estimated around 500 individuals. Single-strand conformation polymorphism analysis revealed one putative functional allele fixed at the locus DQB (n = 25). At the DRB locus, we found two presumed functional alleles (n = 29), differing by a single nonsynonymous nucleotide substitution that could increase the stability at the dimer interface of alphabeta-heterodimers on heterozygous individuals. Identical trans-specific DQB1 and DRB1 alleles were identified between P. sinus and its closest relative, the Burmeister's porpoise (Phocoena spinipinnis). Comparison with studies on four island endemic mammals suggests fixation of one allele, due to genetic drift, commonly occurs at the DQA or DQB loci (effectively neutral). Similarly, deleterious alleles of small effect are also effectively neutral and can become fixed; a high frequency of anatomical malformations on vaquita gave empirical support to this prediction. In contrast, retention of low but functional polymorphism at the DRB locus was consistent with higher selection intensity. These observations indicated natural selection could maintain (and likely also purge) some crucial alleles even in the face of strong and prolonged genetic drift and inbreeding, suggesting long-term small populations should display low inbreeding depression. Low levels of Mhc variation warn about a high susceptibility to novel pathogens and diseases in vaquita.     

New Methods for Detecting Positive Selection at Single Amino Acid Sites

Inferring positive selection at single amino acid sites is of particular importance for studying evolutionary mechanisms of a protein. For this purpose, Suzuki and Gojobori (1999) developed a method (SG method) for comparing the rates of synonymous and nonsynonymous substitutions at each codon site in a protein-coding nucleotide sequence, using ancestral codons at interior nodes of the phylogenetic tree as inferred by the maximum parsimony method. In the SG method, however, selective neutrality of nucleotide substitutions cannot be tested at codon sites, where only termination codons are inferred at any interior node or the number of equally parsimonious inferences of ancestral codons at all interior nodes exceeds 10,000. Here I present a modified SG method which is free from these problems. Specifically, I use the distance-based Bayesian method for inferring the single most likely ancestral codon from 61 sense codons at each interior node. In the computer simulation and real data analysis, the modified SG method showed a higher overall efficiency of detecting positive selection than the original SG method, particularly at highly polymorphic codon sites. These results indicate that the modified SG method is useful for inferring positive selection at codon sites where neutrality cannot be tested by the original SG method. I also discuss that the p-distance is preferable to the number of synonymous substitutions for inferring the phylogenetic tree in the SG method, and present a maximum likelihood method for detecting positive selection at single amino acid sites, which produced reasonable results in the real data analysis.     

Substantial functional diversity accompanies limited major histocompatibility complex class II variability in golden jackal (Canis aureus): A comparison between two wild Canis species in Croatia

The golden jackal (Canis aureus) is one of the less studied carnivores and research on its major histocompatibility complex (MHC) variability is just at its early stages. MHC genes encode cell-surface receptors that serve to bind and present antigens to T cells, which is essential to initiating specific immunological responses in vertebrates. In this paper we present for the first time patterns of genetic diversity and natural selection on MHC class II DLA-DRB1, DQA1 and DQB1 loci in the golden jackal using samples from two geographically distinct regions in Croatia and further compare them to the values found in its congener grey wolf (Canis lupus). Diversity of golden jackals at all three loci was markedly lower than that of grey wolves (allelic richness values were 4, 2 and 3 in jackal versus 11.9, 6.6 and 10.2 in wolves for DRB1, DQA1 and DQB1, respectively) and can be attributed to a genetic drift rather than to the lack of historical positive selection. The finding of high evolutionary distances (16.3% for DRB1 and 8.5% for DQB1) and a substantial number of codons predicted to be under the influence of positive selection (11 for DRB1 and 9 for DQB1) suggests that the investigated golden jackal population still contains considerable functional diversity necessary for the presentation of varied foreign peptides. In contrast to neutral genetic variation, our results suggest that the Dalmatian population has a higher MHC diversity than the Slavonian population, casting doubt on its supposed isolation and calling for a more extensive investigation of the MHC variability of southern Balkan jackal populations.     

Polymorphism and signatures of selection in the multimammate rat DQB gene

In order to test if DQB is a good candidate marker to investigate the relationship between major histocompatibility complex genes and pathogens in natural populations of Mastomys natalensis, we assessed the polymorphism and evolutionary history of this gene. Twenty-four individuals were genotyped for exon 2 of DQB using capillary electrophoresis single-strand conformation polymorphism, cloning, and sequencing. We found 21 different alleles. Four individuals show three alleles implying a duplication event in the history of this gene. Each distinct sequence translates to give a distinct amino acid sequence and there are strong signals of positive selection on peptide binding sites. Signals of recombination were found in the sequences suggesting that recombination has played a role in generating allelic diversity. Although trans-taxon polymorphism is present at the interspecific level in DQB exon 2 sequences of Mus species, we did not find any evidence of allele sharing among Muridae genera. This indicates a temporal limit of DQB allele sharing in Muridae of less than 8 Mya. In conclusion, although DQB seems to be a good marker to investigate pathogen-driven selection, the polymorphism of gene copy number may restrict its utility in natural populations.     

HLA class II DR-DQ amino acids and insulin-dependent diabetes mellitus: application of the haplotype method.

Insulin-dependent diabetes mellitus (IDDM) HLA class II DRB1-DQA1-DQB1 data from four populations (Norwegian, Sardinian, Mexican American, and Taiwanese) have been analyzed to detect the amino acids involved in the disease process. The combination of sites DRB1#67 and 86; DQA1#47; and DQB1#9, 26, 57, and 70 predicts the IDDM component in these four populations, when the results and criteria of the haplotype method for amino acids, developed in the companion paper in this issue of the Journal, are used. The following sites, either individually, or in various combinations, previously have been suggested as IDDM components: DRB1#57, 70, 71, and 86; DQA1#52; and DQB1#13, 45, and 57 (DQB1#13 and 45 correlates 100% with DQB1#9 and 26). We propose that DQA1#47 is a better predictor of IDDM than is the previously suggested DQA1#52, and we add DRB1#67 and DQB1#70 to the HLA DR-DQ IDDM amino acids. We do not claim to have identified all HLA DR-DQ amino acids-or highly correlated sites-involved in IDDM. The frequencies and predisposing/protective effects of the haplotypes defined by these seven sites have been compared, and the effects on IDDM are consistent across the populations. The strongest susceptible effects came from haplotypes DRB1 *0301/DQA1 *0501/ DQB1*0201 and DRB1*0401-5-7-8/DQA1*0301/ DQB1*0302. The number of strong protective haplotypes observed was larger than the number of susceptible ones; some of the predisposing haplotypes were present in only one or two populations. Although the sites under consideration do not necessarily have a functional involvement in IDDM, they should be highly associated with such sites and should prove to be useful in risk assessment.     

Duplication polymorphism at MHC class II DRB1 locus in the wild boar (<Emphasis Type="Italic">Sus scrofa</Emphasis>)

The origin of allelic polymorphism in genes of the major histocompatibility complex represents a central topic in evolutionary genetics as it is probably the most polymorphic region in the nuclear genome of vertebrates. Accordingly, the analyses of genetic variability at these loci provide evidence complementary to the population genetics studies based on neutral loci. In this study, four wild boar populations, two from Italy (Florence region and Castelporziano Presidential Reserve, outside Rome) and one each from Hungary and Poland, were characterized at a highly polymorphic fragment including part of intron 1 and exon 2 of swine leukocyte antigen (SLA) class II DRB1 gene by direct sequencing and by cloning. Excluding the false alleles, a total of 18 different sequences were observed in 57 individuals. The high ratio of nonsynonymous (dN) vs synonymous (dS) substitution rates in the peptide-binding region supports the hypothesis that balancing selection is operating at this locus. A duplication event at the DRB1 gene was documented only in one Italian population with both copies being putatively active. This is the first evidence of a polymorphism for the number of copies of an SLA gene.     

Heterozygosity excess at the cattle DRB locus revealed by large scale genotyping of two closely linked microsatellites

A method for MHC DRB typing in cattle based on two closely linked and highly polymorphic microsatellites is described. The two microsatellites DRBP1ms and DRB3ms are located in intron 2 of the corresponding DRB gene. The very strong linkage disequilibrium between the two loci made it possible to establish DRB microsatellite haplotypes. The typing results with this method on reference samples followed closely that obtained with RFLP and direct sequence analysis of DRB3 exon 2. The method is well suited for large scale genotyping and was successfully applied for typing more than 600 unrelated animals representing 23 breeds. The data were used to test whether the observed DRB allele frequency distributions were consistent with that expected for selectively neutral alleles in populations at mutation-drift equilibrium. A significant heterozygosity excess was detected and there was an obvious trend across breeds towards a more even allele frequency distribution than expected. The deviation may be due to balancing selection acting on the DRB locus or by recent population bottlenecks.     

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.