Gene Frequency Comparisons Between Taxa: Support for the Natural Selection of Protein Polymorphisms

The hypothesis has been advanced that the pervasive protein variation found in natural populations of many organisms is adaptively neutral, and thus not subject to natural selection. This neutrality hypothesis predicts that at polymorphic gene loci different configurations of allelic frequencies will occur in different species. Results of an extensive study of protein variation in several species of Drosophila show that any two species have very similar allelic frequencies at a substantial proportion of all gene loci, while at many other loci the species have very different sets of alleles. Genetic distances have been calculated between pairs of subspecies, morphologically similar species, and morphologically different species. The distribution of genetic distances is strikingly different from the predictions of the neutrality theory. Protein variation appears to be maintained by natural selection.     

Genetic Differentiation Within and Between Species of the Drosophila willistoni Group

We describe allelic variation at 28 loci in six Caribbean populations of four sympatric species of Drosophila. Within any one species the allelic frequencies are very similar from population to population, although there is evidence of local as well as regional genetic differentiation. The genetic distance is greater between populations from different islands than between populations of the same island. When the allelic frequencies are compared between different species, a remarkable pattern appears. In any pair of species nearly half of the loci have essentially identical allelic frequencies, while nearly the other half of the loci have different alleles and in different frequencies. The loci with nearly identical allelic frequencies are different when different pairs of species are compared. The patterns of allelic variation within and between species are inconsistent with the hypothesis that the variation is adaptively neutral. Migration or mutation cannot explain the patterns of genetic variation, either. Balancing natural selection is the main process maintaining protein polymorphisms in natural populations.     

Complexities in the genetic structure of Anopheles gambiae populations in west Africa as revealed by microsatellite DNA analysis

Chromosomal forms of Anopheles gambiae, given the informal designations Bamako, Mopti, and Savannah, have been recognized by the presence or absence of four paracentric inversions on chromosome 2. Studies of karyotype frequencies at sites where the forms occur in sympatry have led to the suggestion that these forms represent species. We conducted a study of the genetic structure of populations of An. gambiae from two villages in Mali, west Africa. Populations at each site were composed of the Bamako and Mopti forms and the sibling species, Anopheles arabiensis. Karyotypes were determined for each individual mosquito and genotypes at 21 microsatellite loci determined. A number of the microsatellites have been physically mapped to polytene chromosomes, making it possible to select loci based on their position relative to the inversions used to define forms. We found that the chromosomal forms differ at all loci on chromosome 2, but there were few differences for loci on other chromosomes. Geographic variation was small. Gene flow appears to vary among different regions within the genome, being lowest on chromosome 2, probably due to hitchhiking with the inversions. We conclude that the majority of observed genetic divergence between chromosomal forms can be explained by forces that need not involve reproductive isolation, although reproductive isolation is not ruled out. We found low levels of gene flow between the sibling species Anopheles gambiae and Anopheles arabiensis, similar to estimates based on observed frequencies of hybrid karyotypes in natural populations.     

Enzyme Variability in the Drosophila willistoni Group, I. Genetic Differentiation Among Sibling Species

We have studied by gel electrophoresis the variability of 14 structural genes in four sibling species, Drosophila willistoni, D. paulistorum, D. equinoxialis, and D. tropicalis. Samples of about 30 populations from different parts of the distribution areas of each species were examined. Genetic variants are found at every locus; 67% of the loci are polymorphic, having two or more alleles, the rarer of which has a frequency of 5% or higher.The gene frequencies are fairly uniform over the distribution area of each species, but considerably different in different species. It is estimated that individuals which belong to the different species differ on the average in somewhat more than one half of their gene loci. The morphological similarity of the four sibling species contrasts with the extensive diversity in their genetic materials.     

A test of the background selection hypothesis based on nucleotide data from Drosophila ananassae

We estimated DNA sequence variation within and between four populations of Drosophila ananassae at Om(1D) and vermilion (v) by using single-strand conformation polymorphism analysis and direct DNA sequencing. Om(1D) is located on the X chromosome in a region with a normal recombination rate; v is in a region of low recombination. In each population, levels of nucleotide diversity at v are reduced 10- to 25-fold relative to those at Om(1D). Divergence between D. ananassae and its sibling species D. pallidosa, however, is comparable for both loci. This lack of correlation between levels of polymorphism and divergence led to the rejection of a constant-rate, neutral model. To distinguish among alternative models, we propose a test of the background selection hypothesis based on the observed pattern of differentiation between populations. Although the degree of differentiation (measured by F_ST) among all pairs of subpopulations is similar at Om(1D), we found substantial differences at v. The two northern populations from Burma and Nepal are very homogeneous, whereas comparisons between northern and southern populations (e.g., between Nepal and middle India) produced large F_ST values. A coalescent-based simulation of the background selection model (in a geographically structured species with a finite number of demes) showed that the observed homogeneity among the northern populations is inconsistent with the background selection hypothesis. Instead, it may have been caused by a recent hitchhiking event that was limited to the northern species range.     

Insecticide Resistance Allele Frequencies in Anopheles gambiae before and after Anti-Vector Interventions in Continental Equatorial Guinea

Anti-malaria interventions that rely on insecticides can be compromised by insecticide-resistance alleles among malaria vectors. We examined frequency changes of resistance alleles at two loci, knockdown resistance (kdr) and acetylcholinesterase-1 (ace-1), which confer resistance to pyrethroids and DDT, and carbamates, respectively. A total of 7,059 Anopheles gambiae sensu stricto mosquitoes were analyzed from multiple sites across continental Equatorial Guinea. A subset of sites included samples collected pre-intervention (2007) and post-intervention (2009–2011). Both L1014S and L1014F resistance alleles were observed in almost all pre-intervention collections. In particular, L1014F was already at substantial frequencies in M form populations (17.6–74.6%), and at high frequencies (> 50%) in all but two S form populations. Comparison before and throughout anti-vector interventions showed drastic increases in L1014F, presumably caused by intensified selection pressure imposed by pyrethroids used in vector control efforts. In light of these findings, inclusion of other insecticide classes in any anti-vector intervention can be considered prudent.     

Preliminary data on the genetic differentiation of Onchocerca volvulus in Africa (Nematoda: Filarioidea)

Data are reported on the genetic structure of three Onchocerca volvulus populations, respectively from Mali (savanna), Ivory Coast (forest), and Zaire (forest gallery in savanna). Electrophoretic analysis, carried out on 25 gene-enzyme systems, has shown a remarkable genetic heterogeneity existing within O.volvulus. Zaire and West Africa populations appear chiefly differentiated at Mdh-1 and 6Pgdh loci, their average Nei's genetic distance being 0.11. In West Africa Nei's D found between the savanna and forest samples is 0.04. The savanna population from Zaire is more similar to the savanna one from Mali (D = 0.09) than to the forest one from Ivory Coast (D = 0.13). This appears mainly due to the loci Ldh and Hbdh (possibly linked), some alleles of which seem to be selected for in forest populations (Ldh110, Hbdh108), while others in the savanna ones (Ldh100, Hbdh100). The hypothesis that the discrepant epidemiological patterns of human onchocerciasis are related to intrinsic differences in the parasite seems supported by the obtained data. The differences in allele frequencies found at the reported loci appear strong enough to allow biochemical identification of O. volvulus populations from different geographic regions and different habitats.     

Population structure of multilocus associations

A method of analysis is presented whereby the structure of multilocus associations among and within several populations can be partitioned into its components. The components are measured by their contributions to the variance in the number of heterozygous loci in two randomly chosen gametes. The singlelocus components are the average and the variation among populations in gene diversity and the variance among populations in allele frequency. The two-locus components include the mean and variance of disequilibria, the covariance of allele frequencies over populations, and various interactions. When applied to allozyme data from populations of wild (Hordeum spontaneum) and cultivated barley (H. vulgare), the analysis highlighted the repetitive pattern of the multilocus associations in the composite crosses whereas it emphasized the regionally localized and geographically variable pattern present in the natural populations of the wild species. The analysis is flexible and applicable to multilocus gametic data from any set of populations, without regard to the number of alleles per locus or the reproductive method of the organism.     

Occurrence of sibling species of Lutzomyia longipalpis (Diptera: Psychodidae) in Venezuela: first evidence from reproductively isolated sympatric populations

The delimitation of cryptic species within the main vector of the American visceral leishmaniasis, Lutzomyia longipalpis, remains a topic of controversy. An analysis of genetic variability based on 8 enzymatic loci revealed fixed differences in 2 diagnostic loci, adenylate kinase (Ak) and hexokinase (Hk), between sympatric and allopatric populations at 4 localities in Venezuela. The absence of heterozygotes for these 2 loci within 1 locality indicates, for the first time, the presence of 2 sympatric reproductively isolated populations or cryptic species within L. longipalpis. Significant differences were also detected between these cryptic species in the allele frequencies of glucose-6-phosphate isomerase (Gpi) and malate dehydrogenase, decarboxylating (Me). One species showed mean heterozygosities that ranged between 6.6% and 6.7%, with 1.6-1.9 alleles detected per locus, while the other had mean heterozygosities that ranged from 4.3% to 6.3%, with 1.3-1.6 alleles per locus. Comparisons of isozyme profiles with published data suggests that 1 species is similar to the L. longipalpis described in Colombian and Brazilian populations, whereas the other has not been previously reported.     

Founder Effects and Linkage Disequilibria in Experimental Populations of Drosophila

Six laboratory populations of Drosophila paulistorum were examined for changes in gene frequencies at an enzyme locus, tetrazolium oxidase (To). In some of the populations, the alleles were introduced on over 100 independently derived chromosomes. These populations showed considerable stability in gene frequencies although they were at widely different starting frequencies. Other populations were begun with only a few (about 6) independently derived chromosomes. These populations showed significant and somewhat erratic changes in To gene frequencies. The difference in behavior of the two sets of populations was almost certainly caused by linkage effects due to sample size. The implication of these studies in understanding the role of the founder effect in natural populations is briefly discussed.     

Strong balancing selection at HLA loci: Evidence from segregation in South Amerindian families

The genotypic proportions for major histocompatibility complex loci, HLA-A and HLA-B, of progeny in families in 23 South Amerindian tribes in which segregation for homozygotes and heterozygotes could occur are examined. Overall, there is a large deficiency of homozygotes compared with Mendelian expectations (for HLA-A, 114 observed and 155.50 expected and for HLA-B 110 observed and 144.75 expected), consistent with strong balancing selection favoring heterozygotes. There is no evidence that these deficiencies were associated with particular alleles or with the age of the individuals sampled. When these families were divided into four mating types, there was strong selection against homozygotes, averaging 0.462 for three of the mating types over the two loci. For the other mating type in which the female parent is homozygous and shares one allele with the heterozygous male parent, there was no evidence of selection against homozygotes. A theoretical model incorporating these findings surprisingly does not result in a stable polymorphism for two alleles but does result in an excess of heterozygotes and a minimum fitness at intermediate allele frequencies. However, for more than two alleles, balancing selection does occur and the model approaches the qualities of the symmetrical heterozygote advantage model as the number of alleles increases.     

Differential escape of HCV from CD8+ T cell selection pressure between China and Germany depends on the presenting HLA class I molecule

Adaptation of hepatitis C virus (HCV) to CD8⁺ T cell selection pressure is well described; however, it is unclear if HCV differentially adapts in different populations. Here, we studied HLA class I‐associated viral sequence polymorphisms in HCV 1b isolates in a Chinese population and compared viral substitution patterns between Chinese and German populations. We identified three HLA class I‐restricted epitopes in HCV NS3 with statistical support for selection pressure and found evidence for differential escape pathways between isolates from China and Germany depending on the HLA class I molecule. The substitution patterns particularly differed in the epitope VTLTHPITK_1635‐1643, which was presented by HLA‐A*03 as well as HLA‐A*11, two alleles with highly different frequencies in the two populations. In Germany, a substitution in position seven of the epitope was the most frequent substitution in the presence of HLA‐A*03, functionally associated with immune escape and nearly absent in Chinese isolates. In contrast, the most frequent substitution in China was located at position two of the epitope and became the predominant consensus residue. Moreover, substitutions in position one of the epitope were significantly enriched in HLA‐A*11‐positive individuals in China and associated with different patterns of CD8⁺ T cell reactivity. Our study confirms the differential escape pathways selected by HCV that depended on different HLA class I alleles in Chinese and German populations, indicating that HCV differentially adapts to distinct HLA class I alleles in these populations. This result has important implications for vaccine design against highly variable and globally distributed pathogens, which may require matching antigen sequences to geographic regions for T cell‐based vaccine strategies.     

Polymorphisms in Continental and Island Populations of Drosophila willistoni

A comparative study of genic allozyme and chromosomal polymorphisms in four continental (South American) and six oceanic island (West Indies) populations of Drosophila willistoni has been made. The pattern of genic polymorphism is closely similar in all populations. Although regional and local differences in gene frequencies are found, generally the same alleles occur at high, intermediate, and low frequencies in all populations. An average individual is heterozygous at 18.4 and 16.2% of its loci in the continental and island populations, respectively. By contrast, chromosomal polymorphism is sharply reduced on the islands compared to most continental populations, and some chromosomal inversions are more frequent on some islands than on others. The observations are not compatible with the hypothesis that most of the gene variants are adaptively neutral. Balancing natural selection is responsible for most of the genic polymorphism in natural populations of D. willistoni.     

Adaptive Linkage Disequilibrium Between Two Esterase Loci of a Salamander

In some populations of the salamander Plethodon cinereus, two polymorphic esterase loci are in linkage disequilibrium. Short-term stability of the linkage disequilibrium is demonstrated by an age class analysis. Long, perhaps very long, term stability is suggested by its distribution. This stability and concordant geographic variation in allelic frequencies imply selective origin and maintenance. Data on the frequencies of two color morphs suggest that formation of the linkage disequilibrium is dependent on the genetic background.     

Using rare mutations to estimate population divergence times: A maximum likelihood approach

In this paper we propose a method to estimate by maximum likelihood the divergence time between two populations, specifically designed for the analysis of nonrecurrent rare mutations. Given the rapidly growing amount of data, rare disease mutations affecting humans seem the most suitable candidates for this method. The estimator RD, and its conditional version RDc, were derived, assuming that the population dynamics of rare alleles can be described by using a birth–death process approximation and that each mutation arose before the split of a common ancestral population into the two diverging populations. The RD estimator seems more suitable for large sample sizes and few alleles, whose age can be approximated, whereas the RDc estimator appears preferable when this is not the case. When applied to three cystic fibrosis mutations, the estimator RD could not exclude a very recent time of divergence among three Mediterranean populations. On the other hand, the divergence time between these populations and the Danish population was estimated to be, on the average, 4,500 or 15,000 years, assuming or not a selective advantage for cystic fibrosis carriers, respectively. Confidence intervals are large, however, and can probably be reduced only by analyzing more alleles or loci.     

New microsatellite markers for multi-scale genetic studies on Phlebotomus ariasi Tonnoir,vector of Leishmania infantum in the Mediterranean area

The population structure of Phlebotomus ariasi, a proven vector of Leishmania infantum in the Mediterranean area, is still poorly understood. Previously, only two microsatellite loci had been developed to study the population genetics of this species. Herein we use these loci and determined fourteen novel microsatellite loci, useful for the characterization of P. ariasi populations. These loci were tested on three populations of P. ariasi, two from France and one from Portugal. In addition, the usefulness of these markers was also evaluated on seven other sandfly species.     

From the Cover: Longer or shorter spines: Reciprocal trait evolution in stickleback via triallelic regulatory changes in Stanniocalcin2a

Vertebrates have repeatedly modified skeletal structures to adapt to their environments. The threespine stickleback is an excellent system for studying skeletal modifications, as different wild populations have either increased or decreased the lengths of their prominent dorsal and pelvic spines in different freshwater environments. Here we identify a regulatory locus that has a major morphological effect on the length of stickleback dorsal and pelvic spines, which we term Maser (major spine enhancer). Maser maps in a closely linked supergene complex that controls multiple armor, feeding, and behavioral traits on chromosome IV. Natural alleles in Maser are differentiated between marine and freshwater sticklebacks; however, alleles found among freshwater populations are also differentiated, with distinct alleles found in short- and long-spined freshwater populations. The distinct freshwater alleles either increase or decrease expression of the bone growth inhibitor gene Stanniocalcin2a in developing spines, providing a simple genetic mechanism for either increasing or decreasing spine lengths in natural populations. Genomic surveys suggest many recurrently differentiated loci in sticklebacks are similarly specialized into three or more distinct alleles, providing multiple ancient standing variants in particular genes that may contribute to a range of phenotypes in different environments.

Similar ecological conditions often result in parallel evolution of the same phenotypic traits in independent populations (1–3). However, ecological conditions typically vary in detail between locations, leading to the evolution of interesting phenotypic differences among evolving populations (4, 5). The contrast between convergence and divergence during adaptive radiations has contributed to decades of work seeking to better understand the principles underlying evolution (6, 7).The threespine stickleback (Gasterosteus aculeatus) provides an opportunity to study the mechanisms that contribute to both parallel and divergent evolution. Migratory marine stickleback have been colonizing and adapting to new freshwater environments for millions of years, with the most recent wide-scale radiation occurring in the countless new freshwater environments generated by glacial recession since the last Ice Age, ∼12 Kya (8). Newly derived freshwater populations typically evolve similar phenotypic changes, including reduced bony armor plates and less robust spines. However, characteristic differences also evolve repeatedly among populations in diverse freshwater environments. Decades of work have analyzed the diverging ecological pressures between lakes and streams (9), large and small lakes (10), benthic and limnetic trophic niches within a lake (11), habitats with different water chemistry and light environments (12), and presence or absence of different types of predators (13). Consequently, freshwater stickleback exhibit exceptional phenotypic diversity, including changes in body size, body shape, color, feeding structures, armor plates, and bony dorsal and pelvic spines (8). Despite recent progress on the genetics of some stickleback traits, the molecular mechanisms underlying many phenotypic specializations remain poorly understood.A key unanswered question is whether diverse evolutionary outcomes occur by modifying different genes in different environments or by modifying the same genes in different ways. Ancient alleles have been identified at particular loci that allow rapid evolution of common marine-freshwater differences by repeated selection of standing variants that already preexist at low frequencies in marine ancestors (14–18). Repeated fixation of preexisting variants favors the reuse of not only the same gene, but also the same freshwater haplotypes in derived populations that share traits. However, it is still not clear whether the distinct phenotypes seen among many freshwater populations are controlled by additional alleles of the same loci that control common marine–freshwater differences, by changes in additional loci, or both.Phenotypic variability among different stickleback populations is particularly pronounced in the dorsal and pelvic spines for which the species is named. Ossified spines are a key evolutionary innovation that spurred a massive radiation of acanthomorph fish, the remarkably diverse fish group that contains about one-third of all living vertebrate species (19, 20). Threespine stickleback typically have three eponymous dorsal spines, but their length can differ greatly among populations and some populations have more than three while others have fewer than three (8, 21). Paired pelvic fins or hindlimbs are found in both fish and tetrapods. In stickleback, the pelvic fin consists of one fin ray and a large, serrated, locking pelvic spine that articulates with an underlying pelvis and can be raised and lowered as a defense against predators (22). The length of the pelvic spine varies dramatically among stickleback populations, and is sometimes lost entirely (8, 21). Although the Pitx1 locus has been identified as a major locus controlling major reduction and even complete loss of the pelvic apparatus in stickleback (23, 24), the genes controlling quantitative variation in pelvic spine length in pelvic-complete individuals are still largely unknown. Based on the significance and diversity of dorsal and pelvic spine phenotypes in Gasterosteus and other fish, we decided to further investigate the genetic mechanisms underlying spine evolution and development.     

A rapid method of haplotyping HFE mutations and linkage disequilibrium in a Caucasoid population

Background—HFE mutations are associated with hereditary haemochromatosis. However, a simple method capable of demonstrating the cis/trans arrangement of alleles is lacking, and linkage disequilibrium between HFE alleles and classic HLA loci is unknown. These are important issues as the pathogenic role of the mutations is not known.
Aims—To develop a simple method of genotyping HFE mutations suitable for clinical use in addition to large disease studies.
Patients—A total of 330 Caucasoid cadaveric organ donor controls were examined. Ten individuals previously HLA-H genotyped by polymerase chain reaction using restriction fragment length polymorphism (PCR-RFLP) were also examined to validate the method.
Methods—A simple polymerase chain reaction using sequence specific primers (PCR-SSP) capable of haplotyping the mutations was developed. HFE allele and haplotype frequencies and linkage disequilibrium with eight HLA class I and II loci were examined in the control population.
Results—27% and 19.7% of patients were positive for the 63D and 282Y alleles, respectively. No chromosome carried both 63D and 282Y. Linkage disequilibrium between 282Y and HLA-A*03 was confirmed, but was not straightforward: some A*03-associated alleles (DRB1*15, DQB1*06), but not all (B*07, Cw*0702), were associated with 282Y.
Conclusions—Linkage disequilibrium data suggest that an HLA-B*07 containing haplotype contains an element affording protection from haemochromatosis and may suggest the timing of the founder 282Y mutation.

     

Divergent and conserved features in the spatial expression of the Drosophila pseudoobscura esterase-5B gene and the esterase-6 gene of Drosophila melanogaster

The regulatory regions of homologous genes encoding esterase 6 (Est-6) of Drosophila melanogaster and esterase 5B (Est-5B) of Drosophila pseudoobscura show very little similarity. We have undertaken a comparative study of the pattern of expression directed by the Est-5B and Est-6 5′-flanking DNA to attempt to reveal conserved elements regulating tissue-specific expression in adults. Esterase regulatory sequences were linked to a lacZ reporter gene and transformed into D. melanogaster embryos. Est-5B, 5′ upstream elements, give rise to a β-galactosidase expression pattern that coincides with the wild-type expression of Est-5B in D. pseudoobscura. The expression patterns of the Est-5B/lacZ construct are different from those of a fusion gene containing the upstream region of Est-6. Common sites of expression for both kinds of constructs are the third segment of antenna, the maxillary palps, and salivary glands. In vitro deletion mutagenesis has shown that the two genes have a different organization of regulatory elements controlling expression in both the third segment of antenna and maxillary palps. The results suggest that the conservation of the expression pattern in genes that evolved from a common ancestor may not be accompanied by preservation of the corresponding cis-regulatory elements.     

Breeding structure of the sand fly Lutzomyia longipalpis (Lutz & Neiva) in Brazil.

Eleven populations of Lutzomyia longipalpis (Lutz & Neiva), the sand fly vector of Leishmania chagasi, from different areas of Brazil were analyzed for genetic variation at 16 enzyme loci. In this region, the prevalence of visceral leishmaniasis (VL) caused by L. chagasi is spotty and reproductive isolation among populations of Lu. longipalpis has been reported. It is thought that morphologically similar cryptic species with varying vectorial capacity may be responsible for the discontinuous distribution of VL. The aim was to study the genetic structure of populations within this region and to identify demes that may represent sibling species. Genotypic frequencies within populations were in close compliance to Hardy-Weinberg expectations, suggesting there are no sympatric species among these 11 populations. Levels of genetic distance between pairs of populations were very low (< 0.03), consistent with local populations within a single sand fly species. When genotypic frequency data for all populations were pooled, 9 of the 13 polymorphic loci deviated from Hardy-Weinberg expectations, indicating some degree of genetic substructuring. Estimates of effective migration rates (N(e)m) among all populations were low, 2.73, suggesting that gene flow is restricted among populations, which is probably the reason for the observed genetic substructuring.