Genetic heterogeneity among intertidal habitats in the flat periwinkle, Littorina obtusata

Comparisons among patterns exhibited by functionally distinct genetic markers have been widely used to infer the impacts of demography and selection in structuring genetic variation in natural populations. However, such multilocus comparisons remain an indirect evaluation of selection at particular candidate loci; ideally, the identification of a candidate gene by comparative genetic methodologies should be complemented by functional analyses and experimental manipulations of genotypes in the laboratory or field. We examined genotype frequency variation among replicated intertidal habitats at two spatial scales in the grazing snail Littorina obtusata. Both of the candidate allozyme markers varied predictably with environment, and these patterns were consistent at both spatial scales. Three of four reference loci were spatially homogeneous, but one microsatellite exhibited significant structure at both geographical and mesoscales. To initiate a direct examination of whether the observed genotype frequency variation at one of the candidate markers, mannose-6-phosphate isomerase (MPI), was impacted by differential survivorship of genotypes, we conducted a series of laboratory-based thermal stress assays using snails from two geographically disparate source populations. When snails were exposed to bouts of thermal/desiccation stress, patterns of mortality were nonrandom with respect to MPI genotype. Furthermore, patterns of mortality in the laboratory manipulation coincided with the observed distribution of genotypes in the field. The data suggest the operation of selection at the Mpi or a linked locus, but functional studies and further experimentation are required to establish the relationship between MPI genotype and fitness across heterogeneous intertidal environments.     

Environmental heterogeneity and balancing selection in the acorn barnacle Semibalanus balanoides

The northern acorn barnacle Semibalans banlanoides occupies several intertidal microhabitats which vary greatly in their degree of physical stress. This environmental heterogeneity creates distinct selection regimes which can maintain genetic variation in natural populations. Despite considerable attention placed on the link between spatial variation in fitness and balancing selection at specific loci, experimental manipulations and fitness estimates for molecular polymorphisms have rarely been conducted in the wild. The aim of this transplant experiment was to manipulate the level of physical stress experienced by a cohort of barnacles in the field and then investigate the spatial variation in fitness for genotypes at three loci: two candidate allozymes and the mitochondrial DNA control region. The viability of mannose-6-phosphate isomerase (Mpi) genotypes was dependent on the level of physical stress experienced in the various treatments; alternative homozygotes were favoured in alternative high stress-low stress environments. In contrast, the fitness of genotypes at other loci was equivalent among treatments and unaffected by the manipulation. Evaluated in the light of balancing selection models, these data indicate that the presence of multiple environmental niches is sufficient to promote a stable Mpi polymorphism in barnacle populations and that allelic variation at this locus reflects the process of adaptation to the heterogeneous intertidal landscape.     

Adaptive maintenance of genetic polymorphism in an intertidal barnacle: habitat- and life-stage-specific survivorship of Mpi genotypes

In the northern acorn barnacle, Semibalanus balanoides, genotype frequencies of three genetic markers were tracked over time in four types of intertidal habitats. These habitats were selected to represent natural variation in several environmental parameters, specifically the degree of physical stress experienced by barnacles. Frequencies for one allozyme locus (Gpi) and a presumably neutral mtDNA marker were homogeneous among habitats in each temporal sample. Similarly, no temporal stratification in genotype frequencies was evident across the five sampling intervals: from planktonic larvae sampled in March to juveniles collected at the end of June. In contrast to the Gpi and mtDNA loci, Mpi genotypes significantly changed in frequency in two habitats in the high intertidal zone. On exposed substrate, the Mpi-FF homozygote increased in frequency, whereas the alternative homozygote, Mpi-SS, significantly decreased in frequency. Barnacles that were protected from environmental stress at high intertidal heights by the Ascophyllum nodosum algal canopy demonstrated the opposite pattern. In both habitats, the change in frequency of the heterozygote was intermediate to that of the homozygous genotypes. Furthermore, these patterns of genotype-by-environment association reflected a pulse of genotype-specific mortality that occurred over a two-week interval subsequent to metamorphosis from the larval to the adult form. These data indicate that each Mpi homozygote is the highest fitness genotype in some portion of the intertidal environment. Using the Levene (1953) model to evaluate the spatial variation in genotypic fitness, the stable maintenance of the Mpi polymorphism is predicted under certain subsets of conditions. Environmental heterogeneity in the intertidal zone translates to spatial variation in selection pressures, which may result in the active maintenance of the Mpi polymorphism in this species.     

Ecological Genetics of Mpi and Gpi Polymorphisms in the Acorn Barnacle and the Spatial Scale of Neutral and Non-neutral Variation

Different allozyme genotypes at the mannose phosphate isomerase(Mpi) locus in the northern acorn barnacle (Semibalanus balanoides)show a strong association with distinct intertidal microhabitats.In estuaries along the Maine Coast, the FF homozygote has higherfitness in exposed, high-tide level microhabitats while theSS homozygote has higher fitness under algal cover or at low-tidemicrohabitats. These patterns are consistent with a Levene (1953)model of balancing selection. In these same samples, polymorphismsat the glucose phosphate isomerase locus (Gpi) and mitochondrialDNA (mtDNA) show no fitness differences among microhabitats,providing intra-genomic controls supporting selection at ornear Mpi. Here we report a similar analysis of genotype-by-microhabitatassociations at sites in Narragansett Bay, Rhode Island, closeto the southern range limit of S. balanoides. Genotype zonationat Mpi between high- and low-tide microhabitats is significantlydifferent between Maine and Narragansett Bay due to oppositezonation patterns for the SF and FF genotypes. Enzyme activitydata are consistent with this "reverse" zonation. At Gpi, thereis significant microhabitat zonation in Narragansett Bay, whilethis locus behaves as a neutral marker in Maine. Mt DNA showsno significant microhabitat zonation in either Rhode Islandor Maine. The Mpi data suggest that Levene-type selection foralternative genotypes in alternative habitats may operate atscales of both 10's of meters and 100's of kilometers. The Gpidata show how an apparently neutral locus can exhibit non-neutralvariation under different environmental conditions. We arguethat both Mpi and Gpi provide important genetic variation foradaptation to environmental heterogeneity that is recruitedunder distinct conditions of stress and carbohydrate substrateavailability.     

Adaptation at Specific Loci. II. Demographic and Biochemical Elements in the Maintenance of the Colias Pgi Polymorphism

Demographically oriented sampling in the wild and biochemical study of allozymes in the laboratory have been used to probe maintenance of the phosphoglucose isomerase polymorphism of Colias butterflies.—The several alleles at this locus show negative or no covariation among their frequencies in the wild. This rules out Wahlund effects as a cause of observations of heterozygote excess at this locus in broods that fly as single cohorts. Unusually heavy mortality among adults, due to drought stress or other causes, can preclude manifestation of differential survivorship among phosphoglucose isomerase genotypes. In broods composed of overlapping cohorts, heterozygote deficiency, apparently due to Wahlund effects in time as cohorts of different survivorship experience mix, can be found. Heterozygotes at this locus fly under a broader range of weather conditions than other genotypes.—Previously detected kinetic differentiation among the genotypes extends in greater magnitude to the glycolytic reaction direction, as well as to a broader range of test conditions than examined before. The heterozygote 3/4 is strikingly heterotic for several measures of kinetic functional effectiveness. Other heterozygotes are sometimes heterotic, more often intermediate (but not exactly so, nor additive in any sense) in properties between homozygotes.—Predictions are made from the biochemical analysis and from the insects' thermal ecology concerning distributions of the genotypes in the wild. Some agree with facts already established. Others are tested and confirmed from data already on hand. Still others are to be tested as reported in an accompanying paper.—All available evidence points to a combination of heterozygote advantage and fluctuating-environment selection as responsible for maintaining this polymorphism. There is considerable evidence for the operation of protein-structural constraint on the range of adaptations possible at this locus.     

Genetic variation in the acorn barnacle from allozymes to population genomics

Understanding the patterns of genetic variation within and among populations is a central problem in population and evolutionary genetics. We examine this question in the acorn barnacle, Semibalanus balanoides, in which the allozyme loci Mpi and Gpi have been implicated in balancing selection due to varying selective pressures at different spatial scales. We review the patterns of genetic variation at the Mpi locus, compare this to levels of population differentiation at mtDNA and microsatellites, and place these data in the context of genome-wide variation from high-throughput sequencing of population samples spanning the North Atlantic. Despite considerable geographic variation in the patterns of selection at the Mpi allozyme, this locus shows rather low levels of population differentiation at ecological and trans-oceanic scales (F(ST)?～?5%). Pooled population sequencing was performed on samples from Rhode Island (RI), Maine (ME), and Southwold, England (UK). Analysis of more than 650 million reads identified approximately 335,000 high-quality SNPs in 19 million base pairs of the S. balanoides genome. Much variation is shared across the Atlantic, but there are significant examples of strong population differentiation among samples from RI, ME, and UK. An F(ST) outlier screen of more than 22,000 contigs provided a genome-wide context for interpretation of earlier studies on allozymes, mtDNA, and microsatellites. F(ST) values for allozymes, mtDNA and microsatellites are close to the genome-wide average for random SNPs, with the exception of the trans-Atlantic F(ST) for mtDNA. The majority of F(ST) outliers were unique between individual pairs of populations, but some genes show shared patterns of excess differentiation. These data indicate that gene flow is high, that selection is strong on a subset of genes, and that a variety of genes are experiencing diversifying selection at large spatial scales. This survey of polymorphism in S. balanoides provides a number of genomic tools that promise to make this a powerful model for ecological genomics of the rocky intertidal.     

Four new alleles at the mannose-6-phosphate isomerase locus in rabbit

Four new alleles at the rabbit ( Oryctolagus cuniculus ) mannose-6-phosphate isomerase (E. C. 5. 3. 1. 8, MPI) locus are proposed to account for phenotypes observed after starch gel electrophoresis and enzymatic staining of red cell lysates and tissues. Population data from various wild and domestic rabbit populations are presented.     

Ablation of mouse phosphomannose isomerase (Mpi) causes mannose 6-phosphate accumulation, toxicity, and embryonic lethality

MPI encodes phosphomannose isomerase, which interconverts fructose 6-phosphate and mannose 6-phosphate (Man-6-P), used for glycoconjugate biosynthesis. MPI mutations in humans impair protein glycosylation causing congenital disorder of glycosylation Ib (CDG-Ib), but oral mannose supplements normalize glycosylation. To establish a mannose-responsive mouse model for CDG-Ib, we ablated Mpi and provided dams with mannose to rescue the anticipated defective glycosylation. Surprisingly, although glycosylation was normal, Mpi(-/-) embryos died around E11.5. Mannose supplementation even hastened their death, suggesting that man-nose was toxic. Mpi(-/-) embryos showed growth retardation and placental hyperplasia. More than 90% of Mpi(-/-) embryos failed to form yolk sac vasculature, and 35% failed chorioallantoic fusion. We generated primary embryonic fibroblasts to investigate the mechanisms leading to embryonic lethality and found that mannose caused a concentration- and time-dependent accumulation of Man 6-P in Mpi(-/-) fibroblasts. In parallel, ATP decreased by more than 70% after 24 h compared with Mpi(+/+) controls. In cell lysates, Man-6-P inhibited hexokinase (70%), phosphoglucose isomerase (65%), and glucose-6-phosphate dehydrogenase (85%), but not phosphofructokinase. Incubating intact Mpi(-/-) fibroblasts with 2-[(3)H]deoxyglucose confirmed mannose-dependent hexokinase inhibition. Our results in vitro suggest that mannose toxicity in Mpi(-/-) embryos is caused by Man-6-P accumulation, which inhibits glucose metabolism and depletes intracellular ATP. This was confirmed in E10.5 Mpi(-/-) embryos where Man-6-P increased more than 10 times, and ATP decreased by 50% compared with Mpi(+/+) littermates. Because Mpi ablation is embryonic lethal, a murine CDG-Ib model will require hypomorphic Mpi alleles.     

INTERTIDAL MICROHABITAT AND SELECTION AT MPI: INTERLOCUS CONTRASTS IN THE NORTHERN ACORN BARNACLE,SEMIBALANUS BALANOIDES

Barnacles were sampled from various microhabitats in the rocky intertidal at multiple sites in two years. At sites in which there were large differences among microhabitats in temperature profiles, Mpi genotype frequencies were consistently and significantly different. Genotype frequencies for another allozyme locus (Gpi) as well as a DNA marker shown to be neutral (the mtDNA control region) were statistically homogeneous among thermal microhabitats at all sites in both years. The data indicate that temperature and/or desiccation mediated selection is operating at Mpi or a linked locus and that Mpi genotypes experience differential mortality in the various habitat types. If the relative fitness of genotypes is dependent on habitat type, the Mpi polymorphism may be actively maintained by a Levene model of balancing selection (Levene 1953). Because barnacle larvae are produced in abundance each year and spend five to eight weeks dispersing in the water column, there is little opportunity for the accumulation of adaptive divergence over the environmental grain size relevant in intertidal habitats. The Mpi polymorphism may be an important component of a suite of traits involved in the adaptation of barnacles to heterogeneous environments. Due to the relatively high concentration of mannose in a variety of algal groups, the metabolism of mannose may substantially affect individual performance and fitness in this and other species that feed on algae and phytoplankton. Because the Mpi locus is one of the most strongly polymorphic in marine organisms, these findings may be relevant for a diversity of other such species.     

Interactions of the receptor for insulin-like growth factor II with mannose-6-phosphate and antibodies to the mannose-6-phosphate receptor

Recently, the sequence of the human receptor for insulin-like growth factor II (IGF-II) was found to be 80% identical [Morgan et al., (1987) Nature 329, 301-307] to the sequence of a partial clone of the bovine cation-independent mannose-6-phosphate receptor [Lobel et al., (1987) Proc. Natl. Acad. Sci. USA 84, 2233-2237]. In the present study, the purified receptor for insulin-like growth factor II (IGF-II) was found to react with two different polyclonal antibodies to the purified mannose-6-phosphate receptor. Moreover, mannose-6-phosphate was found to stimulate the binding of labeled IGF-II to the IGF-II receptor by two-fold. This effect had the same specificity and affinity as the reported binding of mannose-6-phosphate to its receptor; mannose-1-phosphate and mannose had no effect on the binding of labeled IGF-II to its receptor, and the half-maximally effective concentration of mannose-6-phosphate was 0.3 mM. Also, mannose-6-phosphate did not affect labeled IGF-II binding to the insulin receptor. These results support the hypothesis that a single protein of Mr-250,000 binds both IGF-II and mannose-6-phosphate. Furthermore, they indicate that mannose-6-phosphate can modulate the interaction of IGF-II to its receptor.     

Arf regulates interaction of GGA with mannose-6-phosphate receptor

The role of ADP-ribosylation factor (Arf) in Golgi associated, γ-adaptin homologous, Arf-interacting protein (GGA)-mediated membrane traffic was examined. GGA is a clathrin adaptor protein that binds Arf through its GAT domain and the mannose-6-phosphate receptor through its VHS domain. The GAT and VHS domains interacted such that Arf and mannose-6-phosphate receptor binding to GGA were mutually exclusive. In vivo , GGA bound membranes through either Arf or mannose-6-phosphate receptor. However, mannose-6-phosphate receptor excluded Arf from GGA-containing structures outside of the Golgi. These data are inconsistent with predictions based on the model for Arf's role in COPI veside coat function. We propose that Arf recruits GGA to a membrane and then, different from the current model, 'hands-off' GGA to mannose-6-phosphate receptor. GGA and mannose-6-phosphate receptor are then incorporated into a transport intermediate that excludes Arf .     

Phosphomannose isomerase: A versatile selectable marker forArabidopsis thaliana germ-line transformation

A new selection system using mannose has been evaluated for germ-line transformation ofArabidopsis thaliana. Although mannose itself has no adverse effects on plant cells, it leads to an accumulation of mannose-6-phosphate, which depletes intracellular stores of inorganic phosphate. This results in an inhibition of plant cell growth. The selection system uses theEscherichia coli pmi gene that encodes phosphomannose isomerase (PMI). Transgenic plants carrying thepmi gene can detoxify mannose-6-phosphate by conversion to fructose-6-phosphate, an intermediate of glycolysis, via the PMI activity. Germ-line transformation ofA. thaliana followed by sterile selection on 2–5 mM of mannose resulted in the isolation of mannose-6-phosphate-resistant progeny in about 2.5% of the treated seed, consistent with transformation rates using other selection schemes. Integrative transformation was confirmed by Southern hybridization. Analysis of PMI enzyme activity demonstrated a 5-fold range of activity levels, although these differences had little effect on the ability to select transformed plants or on the growth of transformed plants on mannose. Finally, mannose selection using thepmi gene could be accomplished in sterile plates and in soil, making this an extremely versatile tool forA. thaliana transformation.     

Molecular recognition and targeting of lysosomal proteins.

Recent studies have established that in mammalian cells insulin-like growth factor-II can couple the large mannose-6-phosphate receptor to a GTP-binding protein and that the insulin-like growth factor-II-induced activation of the GTP-binding protein is inhibited by mannose-6-phosphate and lysosomal enzymes. In mouse, the gene for the large mannose-6-phosphate receptor is maternally imprinted.     

Parameters interacting with mannose selection employed for the production of transgenic sugar beet

The mannose selection system employs the phosphomannose isomerase (PMI) gene as selectable gene and mannose, converted to mannose-6-phosphate by endogenous hexokinase, as selective agent. The transgenic PMI-expressing cells have acquired the ability to convert mannose-6-phosphate to fructose-6-phosphate, while the non-transgenic cells accumulate mannose-6-phosphate with a concomitant consumption of the intracellular pools of phosphate and ATP. Thus, certain steps of mannose selection depend on the cells’ own metabolism which may be affected by a number of factors, some of which are studied here using Agrobacterium tumefaciens-mediated gene transfer to sugar beet cotyledonary explants. Four frequently employed saccharides (sucrose, glucose, fructose, and maltose) were tested at various concentrations and were found to interact strongly with the phytotoxic effect of mannose, glucose being able to counteract nearly 100% of an almost complete mannose-induced growth inhibition. Sucrose, maltose, and fructose also alleviated significantly the mannose-induced growth inhibition, but were 4-, 5-, and 7-fold less potent than glucose, respectively (calculated as hexose equivalents). The transformation frequencies were also dependent on the nature and concentration of the added carbohydrates, but in this respect sucrose resulted in the highest transformation frequencies, about 1.0%, while glucose and fructose gave significantly lower frequencies. The selection efficiencies were highest in the presence of maltose where no non-transgenic escapes were found over a range of concentrations. The effect of the light intensity was also investigated and the transformation frequencies were positively correlated to light intensity, although the relative impact of light on growth in the presence of mannose appeared not to be dependent on the mannose concentration. Additional phosphate in the selection media had a strong positive effect on the transformation frequencies, suggesting phosphate limitation during selection. The mannose selection system was found to be relatively genotype-independent, provided a slight optimization of the mannose concentrations during selection. Analysis of F₁-offspring showed that all studied primary transformants resulted in PMI-expressing plantlets and that the segregational patterns were in accordance with expectations in at least 50% of the transformants, confirming the stable and active inheritance of the PMI-gene.     

Inhibition by lysosomal enzymes and mannose-6-phosphate of the phosphorylation of the lysosomal enzyme binding receptor protein from monkey brain

The lysosomal enzymes beta-glucuronidase and alpha-L-fucosidase and mannose-6-phosphate inhibited the phosphorylation of the lysosomal enzyme binding receptor protein prepared from monkey brain. Inhibition of both serine and tyrosine phosphorylation was observed. A non-lysosomal glycoprotein enzyme butyrylcholinesterase, mannose or glucose did not inhibit phosphorylation. Tyrosine phosphorylation of histone by the receptor protein was also inhibited by the lysosomal enzymes and mannose-6-phosphate.     

Interaction of mannose-6-phosphate with the hysteretic transition in glucose-6-phosphate hydrolysis in intact liver microsomes.

We showed previously that glucose-6-phosphatase activity was characterised in intact liver microsomes by a hysteretic transition between a rapid and a slower catalytic form of the enzyme. We have now further investigated the substrate specificity of these two kinetic forms. It was found that the pre-incubation of intact microsomes with mannose-6-phosphate or glucose-6-phosphate (50 microM for 30 s) suppressed the burst in glucose-6-phosphatase activity, that the hysteretic transition was reversible and that mannose-6-phosphate inhibited glucose-6-phosphate hydrolysis during the first seconds of incubation, but not anymore after the burst. Our results indicate (i) that mannose-6-phosphate is recognised by the enzyme and can promote the hysteretic transition and (ii) that the transient phase is part of the catalytic mechanism itself.     

Electrophoretic variation at flight-related enzyme loci and its possible association with flight capacity inEpiphyas postvittana

Variations at three flight-related enzyme loci, -glycerophosphate dehydrogenase (-Gpdh), glucose-6-phosphate dehydrogenase (G6pd), and phosphoglucomutase (Pgm), ofEpiphyas postvittana (Walker) moths were investigated using starch gel electrophoresis. Among the three enzyme loci, -Gpdh andG6pd were found to be monomorphic, butPgm was polymorphic, with a total of seven different genotypes and five alleles identified in this study. Comparisons of allozyme variability at thePgm locus showed significant differentiation among five natural populations sampled from geographically distinct localities in New Zealand and Australia and between laboratory populations differentiated by artificial selection on flight capacity. ThePgm polymorphism was shown to be associated with the variation of flight capacity, but the role of the enzyme locus in the evolution of flight behavior is to be demonstrated in this species.This work was supported by the research scholarship of the University of New England.     

The evolution of mate choice in a fluctuating environment

This paper analyzes the evolutionary dynamics of a locus controlling the degree of female mating preference in a temporally fluctuating environment. Preference for mating with males with respect to their genotypes at a locus that is subject to temporally varying natural selection pressure is considered first. With weak selection and free recombination between the choice locus and the selected locus, preference for mating with heterozygotes appears to be favored. With strong selection, preference for homozygous mates may be favored. In each case, choice alleles may increase from very low initial frequencies to near fixation, in contrast to previous models of mate choice in varying environments. Linkages between the two loci has complex effects on the strength and direction of selection for mate choice. Preference for mating with males with the currently fitter genotypes at the locus under natural selection is also modelled. Provided that the environmental period is not too short, a rare allele conferring such preference may be favored and spread to fixation. Strong natural selection, tight linkage and a short environmental period may produce polymorphism for the level of mate choice.     

Contrasting histories of G6PD molecular evolution and malarial resistance in humans and chimpanzees

Although mutations in the glucose-6-phosphate dehydrogenase (G6PD) gene result in several blood-related diseases in humans, they also confer resistance to malarial infection. This association between G6PD and malaria was supported by population genetic analyses of the G6PD locus, which indicated that these mutations may have recently risen in frequency in certain geographic regions as a result of positive selection. Here we characterize nucleotide sequence variation in a 5.2-kb region of the G6PD locus in a population sample of 56 chimpanzees, as well as among 7 other nonhuman primates, to compare with that in humans in determining whether other primates that are impacted by malaria also exhibit patterns of G6PD polymorphism or divergence consistent with positive selection. We find that chimpanzees have several amino acid variants but that the overall pattern at G6PD in chimpanzees, as well as in Old and New World primates in general, can be explained by recent purifying selection as well as strong functional constraint dating back to at least 30-40 MYA. These comparative analyses suggest that the recent signature of positive selection at G6PD in humans is unique.