Can we continue to neglect genomic variation in introgression rates when inferring the history of speciation? A case study in a Mytilus hybrid zone.

The use of molecular data to reconstruct the history of divergence and gene flow between populations of closely related taxa represents a challenging problem. It has been proposed that the long-standing debate about the geography of speciation can be resolved by comparing the likelihoods of a model of isolation with migration and a model of secondary contact. However, data are commonly only fit to a model of isolation with migration and rarely tested against the secondary contact alternative. Furthermore, most demographic inference methods have neglected variation in introgression rates and assume that the gene flow parameter (Nm) is similar among loci. Here, we show that neglecting this source of variation can give misleading results. We analysed DNA sequences sampled from populations of the marine mussels, Mytilus edulis and M. galloprovincialis, across a well-studied mosaic hybrid zone in Europe and evaluated various scenarios of speciation, with or without variation in introgression rates, using an Approximate Bayesian Computation (ABC) approach. Models with heterogeneous gene flow across loci always outperformed models assuming equal migration rates irrespective of the history of gene flow being considered. By incorporating this heterogeneity, the best-supported scenario was a long period of allopatric isolation during the first three-quarters of the time since divergence followed by secondary contact and introgression during the last quarter. By contrast, constraining migration to be homogeneous failed to discriminate among any of the different models of gene flow tested. Our simulations thus provide statistical support for the secondary contact scenario in the European Mytilus hybrid zone that the standard coalescent approach failed to confirm. Our results demonstrate that genomic variation in introgression rates can have profound impacts on the biological conclusions drawn from inference methods and needs to be incorporated in future studies.

Positive Darwinian selection in gamete recognition proteins of Strongylocentrotus sea urchins.

Gamete recognition proteins commonly experience positive Darwinian selection and evolve more rapidly than nonreproductive proteins, but the selective forces responsible for their adaptive diversification remain unclear. We examined the patterns of positive selection in the cognate interacting pair of proteins formed by sperm bindin and its egg receptor (EBR1) and in two regions of the sea urchin sperm receptor for egg jelly suREJ3 gene (exons 22 and 26) among four species of Strongylocentrotus sea urchins (S. purpuratus, S. droebachiensis, S. pallidus and S. franciscanus). The signatures of selection differed at each reproductive protein. A strong signal of positive selection was detected at bindin in all lineages even though the species compared had highly variable gamete traits and experience different intensities and forms of sexual selection and sexual conflict in nature. Weaker selection was observed at EBR1 but the small region studied precluded a clear understanding of the extent of sexual conflict between bindin and the EBR1 protein. At the suREJ3 locus, diversifying selection was observed in exon 22 but not exon 26, suggesting that these regions experience different selective pressures and evolutionary constraints. Positive selection was also detected within S. pallidus at suREJ-22 because of the presence of 12 amino acid replacement mutations segregating at frequencies >0.10. Our results suggest that sexual conflict may be the predominant evolutionary mechanism driving the rapid diversification of reproductive proteins between, and polymorphism within, strongylocentrotid sea urchins.

Isolation by distance in the Atlantic cod, Gadus morhua, at large and small geographic scales.

Genetic isolation by distance (IBD) has rarely been described in marine species with high potential for dispersal at both the larval and adult life-history stages. Here, we report significant relationships between inferred levels of gene flow and geographic distance in the Atlantic cod, Gadus morhua, at 10 nuclear restriction-fragment-length-polymorphism (RFLP) loci at small regional scales in the western north Atlantic region (< 1,600 km) that mirror those previously detected over its entire geographic range (up to 7,300 km). Highly significant allele frequency differences were observed among eight northwestern Atlantic populations, although the mean FST for all 10 loci was only 0.014. Despite this weak population structuring, the distance separating populations explained between 54% and 62% of the variation in gene flow depending on whether nine or 10 loci were used to estimate Nm. Across the species' entire geographic range, highly significant differences were observed among six regional populations at nine of the 10 loci (mean FST = 0.068) and seven loci exhibited significant negative relationships between gene flow and distance. At this large geographic scale, natural selection acting in the vicinity of one RFLP locus (GM798) had a significant effect on the correlation between gene flow and distance, and eliminating it from the analysis caused the coefficient of determination to increase from 17% to 62%. The role of vicariance was assessed by sequentially removing populations from the analysis and was found to play a minor role in contributing to the relationship between gene flow and distance at either geographic scale. The correlation between gene flow and distance detected in G. morhua at small and large spatial scales suggests that dispersal distances and effective population sizes are much smaller than predicted for the species and that the recent age of populations, rather than extensive gene flow, may be responsible for its weak population structure. Our results suggest that interpreting limited genetic differences among populations as reflecting high levels of ongoing gene flow should be made with caution.

Nucleotide polymorphism and natural selection at the pantophysin (Pan I) locus in the Atlantic cod, Gadus morhua (L.).

Molecular studies of nucleotide sequence variation have rarely attempted to test hypotheses related to geographically varying patterns of natural selection. The present study tested the role of spatially varying selection in producing significant linkage disequilibrium and large differences in the frequencies of two common alleles at the pantophysin (Pan I) locus among five populations of the Atlantic cod, Gadus morhua. Nucleotide sequences of 124 Pan I alleles showed strong evidence for an unusual mix of balancing and directional selection but no evidence of stable geographically varying selection. The alleles were highly divergent at both the nucleotide level (differing on average by 19 mutations) and at amino acid level (each having experienced three amino acid substitutions since diverging from a common ancestral allele). All six amino acid substitutions occurred in a 56-residue intravesicular loop (IV1 domain) of the vesicle protein and each involved a radical change. An analysis of molecular variation revealed significant heterogeneity in the frequencies of recently derived mutations segregating within both allelic classes, suggesting that two selective sweeps may be presently occurring among populations. The dynamic nature of the Pan I polymorphism in G. morhua and clear departure from equilibrium conditions invalidate a simple model of spatially varying selection.

Parallel evolution of the melanic form of the California legless lizard, Anniella pulchra, inferred from mitochondrial DNA sequence variation.

The phylogenetic relationships among populations of the fossorial California legless lizard, Anniella pulchra, were examined by sequencing a 990-bp region of the mitochondrial cytochrome b gene. The mitochondrial DNA gene tree was then compared with the geographic distributions of two currently recognized subspecies: A. p. nigra, a melanic form restricted to two disjunct coastal populations, and A. p. pulchra, a more widely distributed, silvery form. We tested the null hypothesis that all A. p. nigra form a clade that is monophyletic with respect to A. p. pulchra. Our results strongly reject the monophyletic origin of the melanic forms and suggest that the two populations of the nominal subspecies A. p. nigra may have arisen independently from different ancestral populations in a parallel evolutionary response to selection in cool, coastal habitats.

The protective effects of hypoxia-induced hypometabolism in the Nautilus.

Specimens of Nautilus pompilius were trapped at depths of 225-300 m off the sunken barrier reef southeast of Port Moresby, Papua New Guinea. Animals transported to the Motupore Island laboratory were acclimated to normal habitat temperatures of 18 degrees C and then cannulated for arterial and venous blood sampling. When animals were forced to undergo a period of progressive hypoxia eventually to encounter ambient partial pressure of oxygen (PO2) levels of approximately 10 mmHg (and corresponding arterial PO2's of approximately 5 mmHg), they responded by lowering their aerobic metabolic rates to 5-10% of those seen in resting normoxic animals. Coincident with this profound metabolic suppression was an overall decrease in activity, with brief periods of jet propulsion punctuating long periods of rest. Below ambient PO2 levels of 30-40 mmHg, ventilatory movements became highly periodic and at the lowest PO2 levels encountered, ventilation occasionally ceased altogether. Cardiac output estimated by the Fick equation decreased during progressive hypoxia by as much as 75 80%, and in the deepest hypometabolic states heart rates slowed to one to two cycles of very low amplitude per minute. By the end of 500 min exposure to ambient PO2 levels of 10 mmHg or less, the anaerobic end products octopine and succinate had increased significantly in adductor muscle and heart, respectively. Increased concentrations of octopine in adductor muscle apparently contributed to a small intracellular acidosis and to the development of a combined respiratory and metabolic acidosis in the extracellular compartment. On the other hand, increases in succinate in heart muscle occurred in the absence of any change in cardiac pHi. Taken together, we estimate that these anaerobic end products would make up less than 2% of the energy deficit arising from the decrease in aerobic metabolism. Thus, metabolic suppression is combined with a massive downregulation of systemic O2 delivery to match metabolic supply to demand.

Genetic population structure and gene flow in the Atlantic cod Gadus morhua: a comparison of allozyme and nuclear RFLP loci.

High levels of gene flow have been implicated in producing uniform patterns of allozyme variation among populations of many marine fish species. We have examined whether gene flow is responsible for the limited population structure in the Atlantic cod, Gadus morhua L., by comparing the previously published patterns of variation at 10 allozyme loci to 17 nuclear restriction fragment length polymorphism (RFLP) loci scored by 11 anonymous cDNA clones. Unlike the allozyme loci, highly significant differences were observed among all populations at the DNA markers in a pattern consistent with an isolation-by-distance model of population structure. The magnitude of allele frequency variation at the nuclear RFLP loci significantly exceeded that observed at the protein loci (chi 2 = 24.6, d.f. = 5, P < 0.001). Estimates of gene flow from the private alleles method were similar for the allozymes and nuclear RFLPs. From the infinite island model, however, estimates of gene flow from the DNA markers were fivefold lower than indicated by the proteins. The discrepancy between gene flow estimates, combined with the observation of a large excess of rare RFLP alleles, suggests that the Atlantic cod has undergone a recent expansion in population size and that populations are significantly displaced from equilibrium. Because gene flow is a process that affects all loci equally, the heterogeneity observed among populations at the DNA level eliminates gene flow as the explanation for the homogeneous allozyme patterns. Our results suggest that a recent origin of cod populations has acted to constrain the extent of population differentiation observed at weakly polymorphic loci and implicate a role for selection in affecting the distribution of protein variation among natural populations in this species.

Allozyme and RFLP heterozygosities as correlates of growth rate in the scallop Placopecten magellanicus: a test of the associative overdominance hypothesis.

Several studies have reported positive correlations between the degree of enzyme heterozygosity and fitness-related traits. Notable among these are the correlations between heterozygosity and growth rate in marine bivalves. Whether the correlation is the result of intrinsic functional differences between enzyme variants at the electrophoretic loci scored or arises from non-random genotypic associations between these loci and others segregating for deleterious recessive genes (the associative overdominance hypothesis) is a matter of continuing debate. A prediction of the associative overdominance hypothesis, not shared by explanations that treat the enzyme loci as causative agents of the correlation, is that the correlation is not specific to the type of genetic marker used. We have tested this prediction by scoring heterozygosity at single locus nuclear restriction fragment length polymorphisms (RFLPs) in a cohort of juvenile scallops (Placopecten magellanicus) in which growth rate was known to be positively correlated with an individual's degree of allozyme heterozygosity. A total of 222 individuals were scored for their genotypes at seven allozyme loci, two nonspecific protein loci of unknown function and eight nuclear RFLPs detected by anonymous cDNA probes. In contrast to the enzyme loci, no correlation was observed between growth rate and the degree of heterozygosity at the DNA markers. Furthermore, there was no relationship between the magnitude of heterozygote deficiency at a locus and its effect on the correlation. The differences observed between the effects of allozyme and RFLP heterozygosity on growth rate provide evidence against the associative overdominance hypothesis, but a strong case against this explanation must await corroboration from similar studies in different species.

Direct evidence for extensive paternal mitochondrial DNA inheritance in the marine mussel Mytilus.

Inheritance of mitochondrial DNA in animals was thought to be strictly maternal. Recently, evidence for incidental paternal mtDNA leakage was obtained in hybrid crosses of Drosophila and mice. In mice, the frequency of paternal mtDNA contributions was estimated at 10(-4), compared with maternal contributions. The common occurrence in the marine mussel Mytilus of heteroplasmic individuals with two or more types of highly diverged mtDNA molecules was interpreted as strong evidence for biparental mtDNA inheritance by some, but not by others. We report here results from pair-matings involving two species of mussels, Mytilus edulis and Mytilus trossulus. Extensive contribution of paternal mtDNA, amounting to several orders of magnitude higher than that inferred for Drosophila or mice, was observed in both intra- and interspecific crosses.

APPARENT SELECTIVE NEUTRALITY OF MITOCHONDRIAL DNA SIZE VARIATION: A TEST IN THE DEEP-SEA SCALLOP PLACOPECTEN MAGELLANICUS.

Animal mitochondrial DNA (mtDNA) is believed to have evolved under intense selection for economy of the size of the molecule. Among scallop species mtDNA size may vary by a factor of two and among conspecific individuals by as much as 25%. We have examined the possibility that large mtDNA size differences may be associated with fitness in the deep sea scallop Placopecten magellanicus by comparing shell lengths of individuals with different copy numbers of a large mtDNA repeated sequence. Among juvenile cohorts of same age, shell length is known to be a good index of overall fitness in marine bivalves and it is shown here to be affected by differences in nuclear genotype, expressed as the degree of enzyme heterozygosity. We have observed no correlation between shell length and mtDNA length and interpreted this to mean that variation in the size of animal mtDNA is effectively neutral to the forces of natural selection acting on the individual. This type of mtDNA variation must, therefore, be explained in terms of biases in the molecular mechanisms causing expansion or contraction of the molecule, differential replication rates of mtDNA molecules of different size, and the stochastic assortment of mtDNA size classes among individuals.

Expression of overdominance for specific activity at the phosphoglucomutase-2 locus in the Pacific oyster, Crassostrea gigas.

Environmental and genetic components of specific activity variation at the phosphoglucomutase-2 locus in the Pacific oyster, Crassostrea gigas, were examined to assess the direct role played by this polymorphism in a heterozygosity/growth relationship. Both environmental variables studied, season and intertidal position, exerted highly significant effects on phosphoglucomutase specific activity but no interactions occurred between these factors and Pgm-2 genotype. Highly significant differences were also detected between Pgm-2 genotypes. The three most common heterozygotes (Pgm-2(92/100), Pgm-2(96/100) and Pgm-2(100/104) consistently expressed greater specific activities than the Pgm-2(92/92), Pgm-2(96/96), Pgm-2(100/100) and Pgm-2(104/104) homozygotes. Overall, the specific activities of heterozygotes for the Pgm-2(100) allele exceeded heterozygotes by 24% and 20% in the mantle and adductor muscle tissues, respectively. Heterozygotes formed between the three less frequent Pgm-2(92), Pgm-2(96) and Pgm-2(104) alleles differed sharply from those possessing the Pgm-2(100) allele in being indistinguishable from homozygotes. The possibility of these patterns arising from the undetected presence of an inactive Pgm-2 allele was examined and found to be inconsistent with all of its predicted effects on the specific activity data. Genuine overdominance was shown to be capable of explaining the specific activities of ten structural locus genotypes, allelic frequency distributions in natural populations, and the maintenance of the enzyme polymorphism in a balanced state. The results provide evidence favoring the overdominance explanation for one locus involved in a heterozygosity/growth relationship and suggest that the reported effects of this locus on adult body weight may have been caused by the greater flux capacities of heterozygotes for the Pgm-2(100) allele.

Biochemical characterization of genotypes at the phosphoglucomutase-2 locus in the Pacific oyster, Crassostrea gigas.

The four most common allozymes at the Pgm-2 locus in Crassostrea gigas were purified and characterized over physiological ranges of temperature and pH. Significant differences were observed between genotypes in their apparent Michaelis constants for glucose-1-phosphate and glucose-1,6-diphosphate, V max/Km ratios, pH-dependent activities, and temperature stabilities. These functional differences were caused almost exclusively by the divergent properties of the Pgm-292 allozyme; limited differentiation existed among the Pgm-296, Pgm-2100, and Pgm-2104 variants. Heterozygotes displayed strict intermediacy for all kinetic and structural properties examined. The results are discussed in light of their ability to account for the overdominant body weights of Pgm-2 heterozygotes reported by Fujio (1982). It is concluded that overdominance is unlikely to arise at this locus as a consequence of these biochemical differences because of their limited magnitude and incompatibility with allelic frequencies in natural populations.