Latitudinal clines for nucleotide polymorphisms in the Esterase 6 gene of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Previous studies have found non-neutral patterns of nucleotide polymorphism in the promoter and coding regions of Est6 in D. melanogaster. Coding region polymorphism peaks around two closely linked replacement differences associated with the EST6-F/EST6-S allozyme polymorphism. The promoter contains two common, highly diverged haplotype groups, P1 and P7, that differentially affect Est6 expression. Allozyme studies have also revealed latitudinal clines in EST6-F and EST6-S frequencies that recur across continents. Here we analyse nucleotide polymorphisms across the promoter and the region of peak coding sequence polymorphism in 10 Australian populations along a 25° latitudinal gradient in order to examine the basis for the allozyme clines. As with the earlier studies, we find an excess of intermediate to high frequency variants in both the P1/P7 region and around the two EST6-F/EST6-S replacements in some populations. The two EST6-F/EST6-S replacement polymorphisms show latitudinal clines whereas the P1 and P7 groups of promoter haplotypes do not. However the strongest clines are for three co-segregating silent site polymorphisms in a 4 bp stretch at the 3′ end of the sequenced region. Monte Carlo simulations show that the clines for those three sites can explain all others in the data but none of the others can explain those three. Thus the allozyme clines may not reflect selection on either the P1/P7 polymorphism or the two replacements previously associated with the EST6-F/EST-S difference.     

Allelic and haplotypic diversity at the major histocompatibility class II within domesticated Australian Atlantic salmon (Salmo salar L.)

Variation within the major histocompatibility (MH) class II alpha gene ( Sasa-DAA ) was compared between domesticated Australian Atlantic salmon and their ancestral Canadian population. The level of Sasa-DAA and MH class II beta gene ( Sasa-DAB ) sequence variation was also examined within the Australian population and compared with that published for European Atlantic salmon populations. In contrast to variation previously reported for non-coding microsatellite loci, a high level of MH class II allelic variation has been maintained within the domesticated Australian populations. Furthermore, a high level of Sasa-DAA and Sasa-DAB allele sequence diversity was also observed and exceeded that reported for other cultured Atlantic salmon populations. The number of Sasa-DAB allele sequences (14) surpassed the number of Sasa-DAA allele sequences (9) to produce 14 unique class II haplotypes. We conclude that the Australian Atlantic salmon populations show high MH class II allelic and haplotypic variation compared with both its ancestral Canadian population and other cultured Atlantic salmon populations.     

Studies of esterase 6 in Drosophila melanogaster. XVIII. Biochemical differences between the slow and fast allozymes

Most natural populations of Drosophila melanogaster are polymorphic for two major electrophoretic variants at the esterase-6 locus. The frequency of the EST 6F allozyme is greatest in populations in warmer latitudes, whereas the EST 6S allozyme is predominant in colder latitudes. Latitudinal clines in electromorph frequencies are found on three continents. Purified preparations of the allozymes have been characterized for their pH optimum, substrate specificity, organophosphate inhibition, alcohol activation, thermal stability, and kinetic parameters. These and previous analyses of the EST 6 allozymes reveal that the two variants have differences in their physical and kinetic properties that may provide a basis for the selective maintenance of the polymorphisms and an explanation of the clinal variation observed in natural populations.      

Nucleotide Polymorphism in the 5'' Promoter Region of Esterase 6 in Drosophila Melanogaster and Its Relationship to Enzyme Activity Variation

A 974-bp region immediately 5' of the esterase 6 gene was sequenced in 17 field derived third chromosome isoallelic lines. Twenty-three polymorphisms were identified, only two in the first 400 bp 5' but 16 in a 325-bp region from -494 to -819 bp. This distribution differs from previously published patterns in Drosophila simulans and D. mauritiana, where the first 800 bp are highly conserved. Fourteen common polymorphisms in the 325-bp region above are all in strong linkage disequilibrium with each other. Moreover, most of the haplotypes defined by the total of 23 polymorphisms fall into two groups that differ as a block at all 14 of these latter sites. Sequence differences between the two groups include some restriction sites that were scored in an earlier study of RFLPs and EST6 enzyme phenotypes among 42 isoallelic lines from the same population. By collating the two studies, we show that one haplotype group yields ~15% lower EST6 enzyme activity in adult males than the other. The promoter haplotypes show only weak disequilibrium with the esterase 6 fast/slow allozyme polymorphism, so it seems unlikely that previously reported latitudinal clines in the allozyme frequencies are due to their hitchhiking along with selection on the promoter difference.     

Microsatellite variation in natural Drosophila melanogaster populations from New South Wales (Australia) and Tasmania

Microsatellite variation was studied at 48 microsatellite loci in 10 Drosophila melanogaster populations to investigate the population structure on the Australian east coast. Low, but statistically significant population differentiation was observed among most populations. The populations on the Australian mainland did not show evidence for isolation by distance. We conclude that the population structure of D. melanogaster on the Australian mainland is probably the result of a shared history (recent colonization). The observed differences between local D. melanogaster populations probably reflect variation in effective population sizes rather than patterns of gene flow. Two populations from Tasmania were more differentiated from the Australian mainland than a population from Israel, raising the question whether they are derived from the Australian mainland or colonized from a different source population.     

Nucleotide variation at the hypervariable esterase 6 isozyme locus of Drosophila simulans

Esterase 6 (Est-6/EST6) is polymorphic in both Drosophila melanogaster and D. simulans for two common allozyme forms, as well as for several other less common variants. Parallel latitudinal clines in the frequencies of the common EST6-F and EST6-S allozymes in these species have previously been interpreted in terms of a shared amino acid polymorphism that distinguishes the two variants and is subject to selection. Here we compare the sequences of four D. simulans Est-6 isolates and show that overall estimates of nucleotide heterozygosity in both coding and 5' flanking regions are more than threefold higher than those obtained previously for this gene in D. melanogaster. Nevertheless, the ratio of replacement to exon silent-site polymorphism in D. simulans is less than the ratio of replacement to silent divergence between D. simulans and D. melanogaster, which could be the result of increased efficiency of selection against replacement polymorphisms in D. simulans or to divergent selection between the two species. We also find that the amino acid polymorphisms separating EST6- F and EST6-S in D. simulans are not the same as those that separate these allozymes in D. melanogaster, implying that the shared clines do not reflect shared molecular targets for selection. All comparisons within and between the two species reveal a remarkable paucity of variation in a stretch of nearly 400 bp immediately 5' of the gene, indicative of strong selective constraint to retain essential aspects of Est-6 promoter function.      

Thermal adaptive significance of ADH and EST-6 allozymes in Indian geographical populations of Drosophila melanogaster

Indian geographical populations of Drosophila melanogaster exhibit significant correlation (r 0.95) of allelic frequencies at Est -6 and Adh loci with latitude as well as altitude. Est -6^S and AdhF allozymes are well adapted to colder environments while Est -6^F and AdhS are warm adapted. The data on allozymic clines match climatic conditions on the Indian subcontinent. On the basis of multiple regression analysis, one major conclusion is that coefficient of variation of temperature ( T _CV) along latitude/altitude accounts for alterations in allelic frequency at the Adh locus while T _max and T _max explain changes at the Est -6 locus. Thus, climatic conditions lead to thermal selection of allozymes in Indian populations of D. melanogaster .     

Phylogeography and population structure of the Dall's porpoise,Phocoenoides dalli,in Japanese waters revealed by mitochondrial DNA

We investigated genetic diversity and phylogenetic relationships among three morphologically distinct populations of Dall's porpoise (Phocoenoides dalli) in Japanese waters by analyzing mitochondrial DNA variation. These populations, the Sea of Japan-Okhotsk dalli-type population, the truei-type population and the standard dalli-type population in the northwestern North Pacific, are clearly discriminated from each other by differences in the size of their white flank patch. A total of 479 bp of the mitochondrial control region and flanking tRNA genes was sequenced for 103 individuals. Haplotypic diversity was high (h = 0.968), but these haplotypes differed by only a few nucleotides (pi = 0.0106). Although many haplotypes were shared between populations, analysis of molecular variance (AMOVA) indicated genetic subdivision among the three populations (overall F (ST) = 0.023, P < 0.001; phi(ST) = 0.026, P = 0.029). Pairwise comparisons indicated a low but significant difference between the Sea of Japan-Okhotsk and the other two populations, whereas there was no significant difference between the latter. These results suggest that there is a close evolutionary relationship among these populations despite their consistent differences in coloration. This may reflect genetic polymorphism in the common ancestral population, which subsequently underwent a rapid divergence. The low genetic variability and haplotypic differentiation of the Sea of Japan-Okhotsk population suggest that it originated from a small population that colonized the Sea of Japan or that experienced population reduction when this Sea was isolated from the North Pacific in the last glacial period.     

Non-African populations of Drosophila melanogaster have a unique origin

Drosophila melanogaster is widely used as a model in DNA variation studies. Patterns of polymorphism have, however, been affected by the history of this species, which is thought to have recently spread out of Africa to the rest of the world. We analyzed DNA sequence variation in 11 populations, including four continental African and seven non-African samples (including Madagascar), at four independent X-linked loci. Variation patterns at all four loci followed neutral expectations in all African populations, but departed from it in all non-African ones due to a marked haplotype dimorphism at three out of four loci. We also found that all non-African populations show the same major haplotypes, though in various frequencies. A parsimonious explanation for these observations is that all non-African populations are derived from a single ancestral population having undergone a substantial reduction of polymorphism, probably through a bottleneck. Less likely alternatives involve either selection at all four loci simultaneously (including balancing selection at three of them), or admixture between two divergent populations. Small but significant structure was observed among African populations, and there were indications of differentiation across Eurasia for non-African ones. Since population history may result in non-equilibrium variation patterns, our study confirms that the search for footprints of selection in the D. melanogaster genome must include a sufficient understanding of its history.     

Molecular Variation of Adh and P6 Genes in an African Population of Drosophila Melanogaster and Its Relation to Chromosomal Inversions

Four-cutter molecular polymorphism of Adh and P6, and chromosome inversion polymorphism of chromosome II were investigated in 95 isogenic lines of an Ivory Coast population of Drosophila melanogaster, a species assumed to have recently spread throughout the world from a West African origin. The P6 gene showed little linkage disequilibrium with the In(2L)t inversion, although it is located within this inversion. This suggests that the inversion and the P6 locus have extensively exchanged genetic information through either double crossover or gene conversion. Allozymic variation in ADH was in linkage disequilibrium with In(2L)t and In(2R)NS inversions. Evidence suggests either that inversion linkage with the Fast allele is selectively maintained, or that this allele only recently appeared. Molecular polymorphism at the Adh locus in the Ivory Coast is not higher than in North American populations. New haplotypes specific to the African population were found, some of them connect the ``Wa(s)-like' haplotypes found at high frequencies in the United States to the other slow haplotypes. Their relation with In(2L)t supports the hypothesis that Wa(s) recently recombined away from an In(2L)t chromosome which may be the cause of its divergence from the other haplotypes.     

History and structure of sub-Saharan populations of Drosophila melanogaster

Drosophila melanogaster is an important model organism in evolutionary genetics, yet little is known about the population structure and the demographic history of this species within sub-Saharan Africa, which is thought to contain its ancestral range. We surveyed nucleotide variation at four 1-kb fragments in 240 individual lines representing 21 sub-Saharan and 4 Palearctic population samples of D. melanogaster. In agreement with recent studies, we find a small but significant level of genetic differentiation within sub-Saharan Africa. A clear geographic pattern is observed, with eastern and western African populations composing two genetically distinct groups. This pattern may have resulted from a relatively recent establishment of D. melanogaster in western Africa. Eastern populations show greater evidence for long-term stability, consistent with the hypothesis that eastern Africa contains the ancestral range of the species. Three sub-Saharan populations show evidence for cosmopolitan introgression. Apart from those cases, the closest relationships between Palearctic and sub-Saharan populations involve a sample from the rift zone (Uganda), suggesting that the progenitors of Palearctic D. melanogaster might have come from this region. Finally, we find a large excess of singleton polymorphisms in the full data set, which is best explained by a combination of population growth and purifying selection.     

Haplotype analysis in Australian hemochromatosis patients: evidence for a predominant ancestral haplotype exclusively associated with hemochromatosis.

Hemochromatosis (HC), an inherited disorder of iron metabolism, shows a very strong founder effect in Australia, with the majority of patients being of Celtic (Scots/Irish) origin. Australian HC patients thus provide an ideal group in which to examine HC-gene-region haplotypes, to analyze the extent of linkage disequilibrium and genetic heterogeneity in HC. We have analyzed chromosomes from 26 multiply affected HC pedigrees, and we were able to assign HC status unambiguously to 107 chromosomes--64 as affected and 43 as unaffected. The haplotypes examined comprise the following highly polymorphic markers: the serological marker HLA-A and the microsatellites D6S248, D6S265, HLA-F, and D6S105. All show highly significant allelic association with HC and no evidence of separation from the disease locus by recombination. Analysis identified a predominant ancestral haplotype comprising alleles 5-1-3-2-8 (marker order: D6S248-D6S265-HLA-A-HLA-F-D6S105), present in 21 (33%) of 64 affected chromosomes, and exclusively associated with HC (haplotype relative risk 903). No other common haplotype was significantly associated with HC. Haplotype analysis in Australian HC patients thus provides strong evidence for (a) the introduction of HC into this population on an ancestral haplotype, (b) a common mutation associated with HC in Australian patients, and (c) a candidate HC-gene region extending between and including D6S248 and D6S105.     

Localization of sequences regulating ancestral and acquired sites of esterase6 activity in Drosophila melanogaster

We have broadly defined the DNA regions regulating esterase6 activity in several life stages and tissue types of D. melanogaster using P- element-mediated transformation of constructs that contain the esterase6 coding region and deletions or substitutions in 5' or 3' flanking DNA. Hemolymph is a conserved ancestral site of EST6 activity in Drosophila and the primary sequences regulating its activity lie between -171 and -25 bp relative to the translation initiation site: deletion of these sequences decrease activity approximately 20-fold. Hemolymph activity is also modulated by four other DNA regions, three of which lie 5' and one of which lies 3' of the coding region. Of these, two have positive and two have negative effects, each of approximately twofold. Esterase6 activity is present also in two male reproductive tract tissues; the ejaculatory bulb, which is another ancestral activity site, and the ejaculatory duct, which is a recently acquired site within the melanogaster species subgroup. Activities in these tissues are at least in part independently regulated: activity in the ejaculatory bulb is conferred by sequences between -273 and -172 bp (threefold decrease when deleted), while activity in the ejaculatory duct is conferred by more distal sequences between -844 and -614 bp (fourfold decrease when deleted). The reproductive tract activity is further modulated by two additional DNA regions, one in 5' DNA (-613 to -284 bp; threefold decrease when deleted) and the other in 3' DNA (+1860 to +2731 bp; threefold decrease when deleted) that probably overlaps the adjacent esteraseP gene. Collating these data with previous studies suggests that expression of EST6 in the ancestral sites is mainly regulated by conserved proximal sequences while more variable distal sequences regulate expression in the acquired ejaculatory duct site.      

Allelic variation and haplotype structure of the dopamine receptor gene DRD2 in nine Indian populations

The human dopaminergic system is a significant focal point of study in the fields of neuropsychiatry and pharmacology, plus it is also a promising nuclear DNA marker in studies of human genome diversity. In this study, we assayed six polymorphic markers in the dopamine D2 receptor gene (DRD2) in 482 unrelated individuals from nine ethnic populations of India. Our results demonstrate that the six markers are highly polymorphic in all populations and the constructed haplotypes show a high level of heterozygosity. Out of the eight possible three-site haplotypes, all populations commonly shared only three haplotypes. The haplotypes exhibited fairly high frequencies across multiple populations; Kurumba population showed all eight three-site haplotypes. The ancestral haplotype (B2-D2-Al) was observed at high frequency only in the Siddi population. Haplotypes based on all six markers revealed 16 haplotypes, out of which only 6 are most common with a frequency of greater than 5% in at least one of the nine populations. But only three haplotypes were shared by all nine populations with the cumulative frequency ranging from 80.8% (Kurumba) to 96.6% (Onge). Great variation in levels of linkage disequilibrium (LD) was detected, ranging from complete LD in the Badaga to virtually no LD in the Siddi. This range of LD likely reflects different population histories, such as African ancestry in the Siddi and recent founding events in the population isolates, Badaga and Kota.     

Genetic variation and population differentiation in the lichen-forming ascomycete Xanthoria parietina on the island Storfosna, central Norway

Genetic diversity and fine-scale population structure in the lichen-forming ascomycete Xanthoria parietina was investigated using sequence variation in part of the intergenic spacer (IGS) and the complete internal transcribed spacer (ITS) regions of the nuclear ribosomal DNA. Sampling included 213 and 225 individuals, respectively, from seven populations in two different habitats, bark and rock, on the island Storfosna off the central west coast of Norway. Both markers revealed significant variation and a total of 10 IGS and 16 ITS haplotypes were found. There were no signs of significant positive spatial autocorrelation at any spatial size class down to 10% of transect length, nor did we find significant deviations from neutrality or signs of historical population expansion. Analysis of molecular variance (amova) indicated that most of the genetic variance observed was within populations, but when populations were grouped according to habitat, more than a quarter of the variance was explained among groups. Pairwise comparisons of populations (F(ST), exact tests of population differentiation) revealed significant differentiation between populations in different habitats (on bark or rock), but not between populations in the same habitat. Haplotype networks show that internal and presumably old haplotypes are shared between habitats, whereas terminal haplotypes tend to be unique to a habitat, mostly bark. We interpret the observed pattern to mean that there is no evidence of restricted gene flow between populations in the same habitat at the present spatial scale (interpopulation distances one or a few kilometres). On the other hand, differentiation between habitats is considerable, which we attribute to restricted gene flow between habitats (habitat isolation). Evidence suggests that the observed differentiation did not evolve locally. Estimates of divergence time between populations in the respective habitats indicate that an ancestral population started to diverge at least 34,000 years ago but probably much further back in time.     

Parallel selection on TRPV6 in human populations

We identified and examined a candidate gene for local directional selection in Europeans, TRPV6, and conclude that selection has acted on standing genetic variation at this locus, creating parallel soft sweep events in humans. A novel modification of the extended haplotype homozygosity (EHH) test was utilized, which compares EHH for a single allele across populations, to investigate the signature of selection at TRPV6 and neighboring linked loci in published data sets for Europeans, Asians and African-Americans, as well as in newly-obtained sequence data for additional populations. We find that all non-African populations carry a signature of selection on the same haplotype at the TRPV6 locus. The selective footprints, however, are significantly differentiated between non-African populations and estimated to be younger than an ancestral population of non-Africans. The possibility of a single selection event occurring in an ancestral population of non-Africans was tested by simulations and rejected. The putatively-selected TRPV6 haplotype contains three candidate sites for functional differences, namely derived non-synonymous substitutions C157R, M378V and M681T. Potential functional differences between the ancestral and derived TRPV6 proteins were investigated by cloning the ancestral and derived forms, transfecting cell lines, and carrying out electrophysiology experiments via patch clamp analysis. No statistically-significant differences in biophysical channel function were found, although one property of the protein, namely Ca(2+) dependent inactivation, may show functionally relevant differences between the ancestral and derived forms. Although the reason for selection on this locus remains elusive, this is the first demonstration of a widespread parallel selection event acting on standing genetic variation in humans, and highlights the utility of between population EHH statistics.     

Mitochondrial DNA variation in Indo-Pacific populations of the giant tiger prawn,Penaeus monodon

Surveys of mitochondrial DNA (mtDNA) variation in the giant tiger prawn, Penaeus monodon, using restriction fragment length polymorphisms have provided the first clear evidence that the Indo-West Pacific region is a site of accumulation of genetic diversity rather than a site of origin of genetic diversity. No haplotyes were found in common between a group of five southeast African populations and a group of five Australian (including Western Australia) and three southeast Asian populations. The dominant haplotype was different in the Australian and southeast Asian population groups. Genetic diversity (pi) was greatest in Indonesia (pi averaged 0.05), less in the Philippines and Australia (pi averaged 0.01), and markedly less in the southeast African and the West Australian populations (pi averaged 0.003). The high diversity of the southeast Asian populations resulted from the occurrence in those populations of a set of haplotypes found only in southeast Asia but derived from the southeast African haplotypes. These genetic variants therefore evolved in the Indian Ocean and later migrated into the Indo-West Pacific region. Low genetic variation in the geographically marginal populations in southeast Africa and Western Australia is considered to be the result of bottlenecks, but mismatch distributions suggest that large population sizes have been maintained in Indonesian populations for long periods.     

A Reanalysis of Protein Polymorphism in Drosophila Melanogaster, D. Simulans, D. Sechellia and D. Mauritiana: Effects of Population Size and Selection

Comparison of synonymous and nonsynonymous variation/substitution within and between species at individual genes has become a widely used general approach to detect the effect of selection versus drift. The sibling species group comprised of two cosmopolitan (Drosophila melanogaster and Drosophila simulans) and two island (Drosophila mauritiana and Drosophila sechellia) species has become a model system for such studies. In the present study we reanalyzed the pattern of protein variation in these species, and the results were compared against the patterns of nucleotide variation obtained from the literature, mostly available for melanogaster and simulans. We have mainly focused on the contrasting patterns of variation between the cosmopolitan pair. The results can be summarized as follows: (1) As expected the island species D. mauritiana and D. sechellia showed much less variation than the cosmopolitan species D. melanogaster and D. simulans. (2) The chromosome 2 showed significantly less variation than chromosome 3 and X in all four species which may indicate effects of past selective sweeps. (3) In contrast to its overall low variation, D. mauritiana showed highest variation for X-linked loci which may indicate introgression from its sibling, D. simulans. (4) An average population of D. simulans was as heterozygous as that of D. melanogaster (14.4% v.s. 13.9%) but the difference was large and significant when considering only polymorphic loci (37.2% v.s. 26.1%). (5) The species-wise pooled populations of these two species showed similar results (all loci = 18.3% v.s. 20.0%, polymorphic loci = 47.2% v.s. 37.6%). (6) An average population of D. simulans had more low-frequency alleles than D. melanogaster, and the D. simulans alleles were found widely distributed in all populations whereas the D. melanogaster alleles were limited to local populations. As a results of this, pooled populations of D. melanogaster showed more polymorphic loci than those of D. simulans (48.0% v.s. 32.0%) but the difference was reduced when the comparison was made on the basis of an average population (29.1% v.s. 21.4%). (7) While the allele frequency distributions within populations were nonsignificant in both D. melanogaster and D. simulans, melanogaster had fewer than simulans, but more than expected from the neutral theory, low frequency alleles. (8) Diallelic loci with the second allele with a frequency less than 20% had similar frequencies in all four species but those with the second allele with a frequency higher than 20% were limited to only melanogaster the latter group of loci have clinal (latitudinal) patterns of variation indicative of balancing selection. (9) The comparison of D. simulans/D. melanogaster protein variation gave a ratio of 1.04 for all loci and 1.42 for polymorphic loci, against a ratio of approximately 2-fold difference for silent nucleotide sites. This suggests that the species ratios of protein and silent nucleotide polymorphism are too close to call for selective difference between silent and allozyme variation in D. simulans. In conclusion, the contrasting levels of allozyme polymorphism, distribution of rare alleles, number of diallelic loci and the patterns of geographic differentiation between the two species suggest the role of natural selection in D. melanogaster, and of possibly ancient population structure and recent worldwide migration in D. simulans. Population size differences alone are insufficient as an explanation for the patterns of variation between these two species.