Transcriptome sequencing reveals both neutral and adaptive genome dynamics in a marine invader.

Species invasions cause significant ecological and economic damage, and genetic information is important to understanding and managing invasive species. In the ocean, many invasive species have high dispersal and gene flow, lowering the discriminatory power of traditional genetic approaches. High-throughput sequencing holds tremendous promise for increasing resolution and illuminating the relative contributions of selection and drift in marine invasion, but has not yet been used to compare the diversity and dynamics of a high-dispersal invader in its native and invaded ranges. We test a transcriptome-based approach in the European green crab (Carcinus maenas), a widespread invasive species with high gene flow and a well-known invasion history, in two native and five invasive populations. A panel of 10 809 transcriptome-derived nuclear SNPs identified significant population structure among highly bottlenecked invasive populations that were previously undifferentiated with traditional markers. Comparing the full data set and a subset of 9246 putatively neutral SNPs strongly suggested that non-neutral processes are the primary driver of population structure within the species' native range, while neutral processes appear to dominate in the invaded range. Non-neutral native range structure coincides with significant differences in intraspecific thermal tolerance, suggesting temperature as a potential selective agent. These results underline the importance of adaptation in shaping intraspecific differences even in high geneflow marine invasive species. They also demonstrate that high-throughput approaches have broad utility in determining neutral structure in recent invasions of such species. Together, neutral and non-neutral data derived from high-throughput approaches may increase the understanding of invasion dynamics in high-dispersal species.

Big and slow: phylogenetic estimates of molecular evolution in baleen whales (suborder mysticeti).

Baleen whales are the largest animals that have ever lived. To develop an improved estimation of substitution rate for nuclear and mitochondrial DNA for this taxon, we implemented a relaxed-clock phylogenetic approach using three fossil calibration dates: the divergence between odontocetes and mysticetes approximately 34 million years ago (Ma), between the balaenids and balaenopterids approximately 28 Ma, and the time to most recent common ancestor within the Balaenopteridae approximately 12 Ma. We examined seven mitochondrial genomes, a large number of mitochondrial control region sequences (219 haplotypes for 465 bp) and nine nuclear introns representing five species of whales, within which multiple species-specific alleles were sequenced to account for within-species diversity (1-15 for each locus). The total data set represents >1.65 Mbp of mitogenome and nuclear genomic sequence. The estimated substitution rate for the humpback whale control region (3.9%/million years, My) was higher than previous estimates for baleen whales but slow relative to other mammal species with similar generation times (e.g., human-chimp mean rate > 20%/My). The mitogenomic third codon position rate was also slow relative to other mammals (mean estimate 1%/My compared with a mammalian average of 9.8%/My for the cytochrome b gene). The mean nuclear genomic substitution rate (0.05%/My) was substantially slower than average synonymous estimates for other mammals (0.21-0.37%/My across a range of studies). The nuclear and mitogenome rate estimates for baleen whales were thus roughly consistent with an 8- to 10-fold slowing due to a combination of large body size and long generation times. Surprisingly, despite the large data set of nuclear intron sequences, there was only weak and conflicting support for alternate hypotheses about the phylogeny of balaenopterid whales, suggesting that interspecies introgressions or a rapid radiation has obscured species relationships in the nuclear genome.

Parallel amino acid replacements in the rhodopsins of the rockfishes (Sebastes spp.) associated with shifts in habitat depth.

Among various groups of fishes, a shift in peak wavelength sensitivity has been correlated with changes in their photic environments. The genus Sebastes is a radiation of marine fish species that inhabit a wide range of depths from intertidal to over 600 m. We examined 32 species of Sebastes for evidence of adaptive amino acid substitution at the rhodopsin gene. Fourteen amino acid positions were variable among these species. Maximum likelihood analyses identify several of these to be targets of positive selection. None of these correspond to previously identified critical amino acid sites, yet they may in fact be functionally important. The occurrence of independent parallel changes at certain amino acid positions reinforces this idea. Reconstruction of habitat depths of ancestral nodes in the phylogeny suggests that shallow habitats have been colonized independently in different lineages. The evolution of rhodopsin appears to be associated with changes in depth, with accelerated evolution in lineages that have had large changes in depth.

Humans as the world's greatest evolutionary force.

In addition to altering global ecology, technology and human population growth also affect evolutionary trajectories, dramatically accelerating evolutionary change in other species, especially in commercially important, pest, and disease organisms. Such changes are apparent in antibiotic and human immunodeficiency virus (HIV) resistance to drugs, plant and insect resistance to pesticides, rapid changes in invasive species, life-history change in commercial fisheries, and pest adaptation to biological engineering products. This accelerated evolution costs at least $33 billion to $50 billion a year in the United States. Slowing and controlling arms races in disease and pest management have been successful in diverse ecological and economic systems, illustrating how applied evolutionary principles can help reduce the impact of humankind on evolution.

Tactics of acclimation: morphological changes of sponges in an unpredictable environment.

Reciprocal transplants of genetically identical fragments of intertidal sponges between environments of high and low wave action exhibit great variability in the timing of their responses to environmental change. Sponges quickly begin production of stiffer and stronger tissues in high wave energy environments but delay formation of new, weak tissues in calm habitats. This may be due to the risks of forming wave-intolerant tissue in a temporally variable, unpredictable environment. These results suggest that the evolution of acclimatory control is linked to environmental predictability and concomitantly to risks of acclimatory errors.

Speciation and the evolution of gamete recognition genes: pattern and process.

Proteins on gamete surfaces are major determinants of fertilization success, particularly in free-spawning animals. Molecular analyses of these simple genetic systems show rapid evolution, positive selection, accelerated coalescence and, sometimes, extensive polymorphism. Careful analysis of the behavior of sperm produced by males with different gamete alleles shows that these alleles can deliver significant functional differences. Three forms of allele-specific fertilization advantage have been shown: assortative mating based on gamete type, rare allele advantage and heterozygote superiority. Models suggest that sperm and egg proteins may be coevolutionary partners that can alternate between directional selection for high fertilization ability and cyclic adaptation of eggs and sperm driven by sexual conflict. These processes act within allopatric populations and may accelerate their divergence if gamete adaptations in separate demes reduce cross-fertilization. Reproductive character displacement by reinforcement may play a diversifying role when previously allopatric populations rejoin. In circumstance that might prove to be common, divergence in sympatry can be driven by sexual conflict or by association of mating types with ecological differences. The ecology of fertilization, especially the degree of sperm competition and egg death via polyspermy, are important determinants of the strength and direction of selection on gametes. Free-spawning animals allow careful analysis of gamete recognition -from the behavior of adults and interactions of gametes, to molecular patterns of allele divergence. Future research efforts on the evolutionary consequences of fertilization ecology, and the interaction between extensive variation in egg surface proteins and sperm fertilization ability, are particularly needed.

Extranuclear differentiation and gene flow in the finite island model.

Use of sequence information from extranuclear genomes to examine deme structure in natural populations has been hampered by lack of clear linkage between sequence relatedness and rates of mutation and migration among demes. Here, we approach this problem in two complementary ways. First, we develop a model of extranuclear genomes in a population divided into a finite number of demes. Sex-dependent migration, neutral mutation, unequal genetic contribution of separate sexes and random genetic drift in each deme are incorporated for generality. From this model, we derive the relationship between gene identity probabilities (between and within demes) and migration rate, mutation rate and effective deme size. Second, we show how within- and between-deme identity probabilities may be calculated from restriction maps of mitochondrial (mt) DNA. These results, when coupled with our results on gene flow and genetic differentiation, allow estimation of relative interdeme gene flow when deme sizes are constant and genetic variants are selectively neutral. We illustrate use of our results by reanalyzing published data on mtDNA in mouse populations from around the world and show that their geographic differentiation is consistent with an island model of deme structure.

Predicting nuclear gene coalescence from mitochondrial data: the three-times rule.

Coalescence theory predicts when genetic drift at nuclear loci will result in fixation of sequence differences to produce monophyletic gene trees. However, the theory is difficult to apply to particular taxa because it hinges on genetically effective population size, which is generally unknown. Neutral theory also predicts that evolution of monophyly will be four times slower in nuclear than in mitochondrial genes primarily because genetic drift is slower at nuclear loci. Variation in mitochondrial DNA (mtDNA) within and between species has been studied extensively, but can these mtDNA data be used to predict coalescence in nuclear loci? Comparison of neutral theories of coalescence of mitochondrial and nuclear loci suggests a simple rule of thumb. The "three-times rule" states that, on average, most nuclear loci will be monophyletic when the branch length leading to the mtDNA sequences of a species is three times longer than the average mtDNA sequence diversity observed within that species. A test using mitochondrial and nuclear intron data from seven species of whales and dolphins suggests general agreement with predictions of the three-times rule. We define the coalescence ratio as the mitochondrial branch length for a species divided by intraspecific mtDNA diversity. We show that species with high coalescence ratios show nuclear monophyly, whereas species with low ratios have polyphyletic nuclear gene trees. As expected, species with intermediate coalescence ratios show a variety of patterns. Especially at very high or low coalescence ratios, the three-times rule predicts nuclear gene patterns that can help detect the action of selection. The three-times rule may be useful as an empirical benchmark for evaluating evolutionary processes occurring at multiple loci.

Concordant evolutionary patterns among Indo-West Pacific butterflyfishes.

Genetic differences within a 495 base pair section of the mitochondrial cytochrome b gene reveal a striking concordance among species in two monophyletic groups of Indo-west Pacific butterflyfishes. In both species groups, an approximately 2.0% genetic break clearly partitions individuals between the Indian Ocean and Pacific Ocean. However, levels of intra-Pacific mtDNA variation are low, on average less than 1.0%, and fail to cluster by species boundaries defined by colour pattern. Individuals from different species, separated by thousands of kilometers, often possess identical cytochrome b sequences, whereas conspecifics from the same reefs can show up to 1.5% difference. The discrepancy between the mtDNA gene tree and species boundaries may reflect retained ancestral variation or may be the result of hybridization. The strong temporal and phylogenetic concordance between these two independent species groups suggests that genetic differentiation was influenced by common environmental factors. Low levels of within- and between-species genetic differences imply a recent divergence time and suggest a link between speciation within each group and Pleistocene climatic fluctuations. These results paint a turbulent picture of the recent evolutionary history of the Indo-West Pacific.

Rapid recovery of genetic diversity of stomatopod populations on Krakatau: temporal and spatial scales of marine larval dispersal.

Although the recovery of terrestrial communities shattered by the massive eruption of Krakatau in 1883 has been well chronicled, the fate of marine populations has been largely ignored. We examined patterns of genetic diversity in populations of two coral reef-dwelling mantis shrimp, Haptosquilla pulchella and Haptosquilla glyptocercus (Stomatopoda: Protosquillidae), on the islands of Anak Krakatau and Rakata. Genetic surveys of mitochondrial cytochrome oxidase c (subunit 1) in these populations revealed remarkably high levels of haplotypic and nucleotide diversity that were comparable with undisturbed populations throughout the Indo-Pacific. Recolonization and rapid recovery of genetic diversity in the Krakatau populations indicates that larval dispersal from multiple and diverse source populations contributes substantially to the demographics of local populations over intermediate temporal (tens to hundreds of years) and spatial scales (tens to hundreds of kilometres). Natural experiments such as Krakatau provide an excellent mechanism to investigate marine larval dispersal and connectivity. Results from stomatopods indicate that marine reserves should be spaced no more than 50-100 km apart to facilitate ecological connectivity via larval dispersal.

Predicted decline of protected whales based on molecular genetic monitoring of Japanese and Korean markets.

We present a two-tiered analysis of molecular genetic variation in order to determine the origins of whale' products purchased from retail markets in Japan and the Republic of (South) Korea during 1993-1999. This approach combined phylogenetic analysis of mitochondrial DNA sequences for identification of protected species with a statistical comparison of intraspecific haplotype frequencies for distinguishing regional subpopulations or 'stocks' hunted for scientific research by the Japanese and killed incidentally in coastal fisheries by the Koreans. The phylogenetic identification of 655 products included eight species or subspecies of baleen whales, sperm whales, a pygmy sperm whale, two species of beaked whales, porpoises, killer whales and numerous species of dolphins as well as domestic sheep and horses. Six of the baleen whale species (the fin, sei, common-form and small-form Bryde's, blue or blue/fin hybrid, and humpback) and the sperm whale are protected by international agreements dating back to at least 1989 for all species and 1966 for some species. We compared the haplotype frequencies from the Japanese market sample to those reported from scientific hunting in the western North Pacific stock for products derived from the exploited North Pacific minke whale. The market sample differed significantly from the scientific catch (p < 0.001), showing a greater than expected frequency of haplotypes characteristic of the protected Sea of Japan stock. We used a 'mixed-stock' analysis and maximum-likelihood methods to estimate that 31% (95% confidence interval 19-43%) of the market for this species originated from the Sea of Japan stock. The source of these products was assumed to be undocumented 'incidental takes' from fisheries' by-catch, although we cannot exclude the possibility of illegal hunting or smuggling. The demographic impact of this undocumented exploitation was evaluated using the model of population dynamics adopted by the Scientific Committee of the International Whaling Commission. For the range of exploitation consistent with the market sample, this protected stock was predicted to decline towards extinction over the next few decades. These results confirmed the power of molecular methods in monitoring retail markets and pointed to the inadequacy of the current moratorium for ensuring the recovery of protected species. More importantly, the integration of genetic evidence with a model of population dynamics identified an urgent need for actions to limit undocumented exploitation of a 'protected' stock of whales.

Human health significance of organochlorine and mercury contaminants in Japanese whale meat.

The concentrations of total mercury, polychlorinated biphenyls (PCBs), and organochlorine pesticides (SigmaDDT, dieldrin, hexachlorobenzene [HCB], and SigmaHCH) were determined in 61 whale meat products (bacon, blubber, red meat, liver, intestine, and tongue) purchased from retail outlets across Japan. Mean (range) concentrations of contaminants in all samples were: total mercury 4.17 (0.01-204); SigmaPCB 1.14 (0-8.94); SigmaDDT 0.98 (0-7.46); dieldrin 0.07 (0-0.35); HCB 0.06 (0-0.22); and SigmaHCH 0.07 (0-0.19) micro g/g (wet weight). The data were used to calculate estimated daily intakes (EDIs) of contaminants at two hypothetical levels of whale meat consumption. These EDIs were compared with FAO/WHO "tolerable daily intake" (TDI) values for each chemical. EDIs calculated for higher levels of whale meat consumption were in some cases exceptionally high and for many products exceeded FAO/ WHO-TDIs for total mercury, PCBs, and dieldrin, with exceedance factor values (EDI/TDI) for total mercury, PCBs, and dieldrin reaching maxima of 175, 5.36, and 2.1, respectively. For sensitive consumers and those with high-level consumption (e.g., whaling communities), exposure to mercury and to a lesser extent PCBs from certain whale blubber and bacon and striped dolphin liver products could lead to chronic health effects. The Japanese community should therefore exercise a precautionary approach to the consumption of such foods in excess, particularly by high-risk members of the population.

Signs of adaptation to local pH conditions across an environmental mosaic in the California Current Ecosystem.

Little is known about the potential for rapid evolution in natural populations in response to the high rate of contemporary climatic change. Organisms that have evolved in environments that experience high variability across space and time are of particular interest as they may harbor genetic variation that can facilitate evolutionary response to changing conditions. Here we review what is known about genetic capacity for adaptation in the purple sea urchin, Strongylocentrotus purpuratus, a species that has evolved in the upwelling ecosystem of the Northeast Pacific Ocean. We also present new results testing for adaptation to local pH conditions in six populations from Oregon to southern California. We integrate data on 19,493 genetic polymorphisms with data on local pH conditions. We find correlations between allele frequency and rank average time spent at pH <7.8 in 318 single-nucleotide polymorphisms in 275 genes. Two of the genes most correlated with local pH are a protein associated with the cytoskeleton and a proton pump, with functional roles in maintenance of cell volume and with internal regulation of pH, respectively. Across all loci tested, high correlations with local pH were concentrated in genes related to transport of ions, biomineralization, lipid metabolism, and cell-cell adhesion, functional pathways important for maintaining homeostasis at low pH. We identify a set of seven genes as top candidates for rapid evolutionary response to acidification of the ocean. In these genes, the putative low-pH-adapted allele, based on allele frequencies in natural populations, rapidly increases in frequency in purple sea urchin larvae raised at low pH. We also found that populations from localities with high pH show a greater change in allele frequency toward putative low-pH-adapted alleles under experimental acidification, compared with low-pH populations, suggesting that both natural and artificial selection favor the same alleles for response to low pH. These results illustrate that purple sea urchins may be adapted to local pH and suggest that this species may possess the genetic capacity for rapid evolution in response to acidification. This adaptive capacity likely comes from standing genetic variation maintained in nature by balancing selection across the spatial and temporal environmental mosaic that characterizes the California Current Ecosystem.

Marine speciation on a small planet.

The scale of population structure in many marine species is on the order of thousands to tens of thousands of kilometers. How does speciation take place in oceans that are only about this same size? Recent results suggest an important role for transient isolation, gamete ecology and molecular evolution at gamete recognition loci. These factors have long been appreciated by plant biologists, and are likely to be a fruitful area of research for marine biologists as well.

Rates of molecular evolution and the fraction of nucleotide positions free to vary.

Selective constraints on DNA sequence change were incorporated into a model of DNA divergence by restricting substitutions to a subset of nucleotide positions. A simple model showed that both mutation rate and the fraction of nucleotide positions free to vary are strong determinants of DNA divergence over time. When divergence between two species approaches the fraction of positions free to vary, standard methods that correct for multiple mutations yield severe underestimates of the number of substitutions per site. A modified method appropriate for use with DNA sequence, restriction site, or thermal renaturation data is derived taking this fraction into account. The model also showed that the ratio of divergence in two gene classes (e.g., nuclear and mitochondrial) may vary widely over time even if the ratio of mutation rates remains constant. DNA sequence divergence data are used increasingly to detect differences in rates of molecular evolution. Often, variation in divergence rate is assumed to represent variation in mutation rate. The present model suggests that differing divergence rates among comparisons (either among gene classes or taxa) should be interpreted cautiously. Differences in the fraction of nucleotide positions free to vary can serve as an important alternative hypothesis to explain differences in DNA divergence rates.

All males are not created equal: fertility differences depend on gamete recognition polymorphisms in sea urchins.

Behaviors, morphologies, and genetic loci directly involved in reproduction have been increasingly shown to be polymorphic within populations. Explaining how such variants are maintained by selection is crucial to understanding the genetic basis of fertility differences, but direct tests of how alleles at reproductive loci affect fertility are rare. In the sea urchin genus Echinometra, the protein bindin mediates sperm attachment to eggs, evolves quickly, and is polymorphic within species. Eggs exposed to experimental sperm mixtures show strong discrimination on the basis of the males' bindin genotype. Different females produce eggs that nonrandomly select sperm from different males, showing that variable egg-sperm interactions determine fertility. Eggs select sperm with a bindin genotype similar to their own, suggesting strong linkage between female choice and male trait loci. These experiments demonstrate that alleles at a single locus can have a strong effect on fertilization and that reproductive loci may retain functional polymorphisms through epistatic interactions between male and female traits. They also suggest that positive selection at gamete recognition loci like bindin involves strong selection within species on mate choice interactions.