Phylogeography and conservation of the bull shark (Carcharhinus leucas) inferred from mitochondrial and microsatellite DNA

The bull shark (Carcharhinus leucas) is a widely distributed, large coastal shark species known to travel long distances. These characteristics, coupled with the species?? long life span and late age of maturity, would lead one to predict significant global genetic exchange among bull shark populations. By contrast, data show localized depletion in some areas of large coastal shark fisheries, indicating some geographic isolation may exist. We examined genetic variation in the control region of mitochondrial DNA and at five nuclear microsatellite loci in bull sharks sampled from the western Atlantic to investigate the degree of population subdivision. The average per sample haplotype and nucleotide diversity in the mtDNA (0.51 ± 0.26 and 0.12% ± 0.12, respectively) and expected heterozygosity (0.84) in the microsatellite loci contrast sharply in having lower and higher values (respectively) relative to many other shark species. Significant structure exists between the Brazilian and all northern populations at the mtDNA control region (pairwise ??_ST > 0.8, P < 0.001), but not at the nuclear microsatellite loci. Adjacent northern populations show weak to no genetic differentiation for both markers. These results are congruent with restricted maternal gene flow between populations caused by female site fidelity to nursery areas. We estimate the current effective population size to be around 160,000 and 221,000 individuals for the southern and northern Atlantic populations, respectively. The philopatric habits and the relatively low levels of mtDNA genetic diversity observed in bull sharks must be considered in the conservation of this species. Our results indicate that effective bull shark management strategies will require local, regional, and international attention and cooperation.     

Strong genetic isolation despite wide distribution in a commercially exploited coastal shark

The common smoothhound, Mustelus mustelus, is an epibenthic species targeted by fisheries around the world driven by the increasing demand for shark products. Given the wide-spread occurrence of this species and corresponding lack of molecular data in many areas of said distribution, baseline molecular assessments of this commercially important shark may contribute to finer-scale analyses in areas in which this species is targeted. Therefore, population genetic analyses were conducted along the East Atlantic, from the Mediterranean Sea to the south-east coast of Africa, using microsatellite markers and the mitochondrial control region (mtCR). Overall, M. mustelus displayed low to moderate genetic diversity, with the Mediterranean populations appearing to exhibit the lowest mitochondrial diversity, and the west African populations displaying the lowest nuclear diversity. Microsatellite analysis indicated strong genetic differentiation between the three regions, with finer-scale population structure in each region, without correlation between genetic and geographical distance. For the mtCR sequences, a total of 18 haplotypes were identified, with a high degree of divergence discernable between the regions, largely in accordance with the microsatellite data. The study documents a remarkable level of population isolation across a vast area, suggesting little or no present-day connectivity among extant populations. The findings may serve as an essential baseline for global population management and commercial traceability of this threatened shark.

     

Population structure in the endangered cyprinid fish Pararasbora moltrechti in Taiwan, based on mitochondrial and microsatellite DNAs

The genetic structure of four populations of Pararasbora moltrechti, an endemic species of the Cyprinidae in Taiwan, was investigated based on the genetic variation of mtDNA Cyt b gene and five microsatellite loci. High haplotype diversity (h = 0.92) but low nucleotide diversity (0.004) in mtDNA was detected in this endangered species. In total, 33 haplotypes and four clusters were identified in its mtDNA. Nevertheless, low correspondence was found between geographical division and mtDNA clusters. In contrast, Bayesian cluster analysis of the microsatellite data identified four genetic groups and revealed highly structured populations. Significantly negative Tajima's D statistics and mismatch distribution analyses suggest that P. moltrechti populations may have experienced a demographic expansion. In light of the results of a nested clade analysis of mtDNA haplotypes, we conclude that recent population fluctuations and restricted gene flow played major roles in shaping the spatial genetic structure of P. moltrechti populations.     

Invasion success and genetic diversity of introduced populations of guppies Poecilia reticulata in Australia

High genetic diversity is thought to characterize successful invasive species, as the potential to adapt to new environments is enhanced and inbreeding is reduced. In the last century, guppies, Poecilia reticulata, repeatedly invaded streams in Australia and elsewhere. Quantitative genetic studies of one Australian guppy population have demonstrated high additive genetic variation for autosomal and Y-linked morphological traits. The combination of colonization success, high heritability of morphological traits, and the possibility of multiple introductions to Australia raised the prediction that neutral genetic diversity is high in introduced populations of guppies. In this study we examine genetic diversity at nine microsatellite and one mitochondrial locus for seven Australian populations. We used mtDNA haplotypes from the natural range of guppies and from domesticated varieties to identify source populations. There were a minimum of two introductions, but there was no haplotype diversity within Australian populations, suggesting a founder effect. This was supported by microsatellite markers, as allelic diversity and heterozygosity were severely reduced compared to one wild source population, and evidence of recent bottlenecks was found. Between Australian populations little differentiation of microsatellite allele frequencies was detected, suggesting that population admixture has occurred historically, perhaps due to male-biased gene flow followed by bottlenecks. Thus success of invasion of Australia and high additive genetic variance in Australian guppies are not associated with high levels of diversity at molecular loci. This finding is consistent with the release of additive genetic variation by dominance and epistasis following inbreeding, and with disruptive and negative frequency-dependent selection on fitness traits.     

Global phylogeography with mixed-marker analysis reveals male-mediated dispersal in the endangered scalloped hammerhead shark (Sphyrna lewini)

Background

The scalloped hammerhead shark, Sphyrna lewini, is a large endangered predator with a circumglobal distribution, observed in the open ocean but linked ontogenetically to coastal embayments for parturition and juvenile development. A previous survey of maternal (mtDNA) markers demonstrated strong genetic partitioning overall (global Φ_ST = 0.749) and significant population separations across oceans and between discontinuous continental coastlines.

Methodology/Principal Findings

We surveyed the same global range with increased sample coverage (N = 403) and 13 microsatellite loci to assess the male contribution to dispersal and population structure. Biparentally inherited microsatellites reveal low or absent genetic structure across ocean basins and global genetic differentiation (F _ST = 0.035) over an order of magnitude lower than the corresponding measures for maternal mtDNA lineages (Φ_ST = 0.749). Nuclear allelic richness and heterozygosity are high throughout the Indo-Pacific, while genetic structure is low. In contrast, allelic diversity is low while population structure is higher for populations at the ends of the range in the West Atlantic and East Pacific.

Conclusions/Significance

These data are consistent with the proposed Indo-Pacific center of origin for S. lewini, and indicate that females are philopatric or adhere to coastal habitats while males facilitate gene flow across oceanic expanses. This study includes the largest sampling effort and the most molecular loci ever used to survey the complete range of a large oceanic predator, and findings emphasize the importance of incorporating mixed-marker analysis into stock assessments of threatened and endangered shark species.     

Hierarchical metapopulation structure in a highly mobile marine predator: the southern Australian coastal bottlenose dolphin (<Emphasis Type="Italic">Tursiops</Emphasis> cf. <Emphasis Type="Italic">australis</Emphasis>)

Little is known about the population ecology of the recently described bottlenose dolphin species Tursiops australis. The classification of this species is still under debate, but this putative species is thought to be comprised of small and genetically distinct populations (including sub-populations under increasing anthropogenic threats) and is likely endemic to coastal southern Australia. Mitochondrial DNA (mtDNA) control region sequences and microsatellite loci were used to assess genetic variation and hierarchical population structure of coastal T. cf. australis across a range of spatial scales and environmental discontinuities between southern Western Australia (WA) and central South Australia (SA). Overall, genetic diversity was similar to that typically found for bottlenose dolphins, although very low mtDNA diversity was found in Gulf St. Vincent (GSV) dolphins. We found historical genetic subdivision and likely differences in colonisation between GSV and Spencer Gulf, outer- and inner-gulf locations, and SA/WA and previously identified Victorian/Tasmanian populations. A hierarchical metapopulation structure was revealed along southern Australia, with at least six genetic populations occurring between Esperance, WA and southern Tasmania. In addition, fine-scale genetic subdivision was observed within each SA/WA population. In general, contemporary migration was limited throughout southern Australia, but an important gene flow pathway was identified eastward along the Great Australian Bight. Management strategies that promote gene flow among populations should be implemented to assist with the maintenance of the inferred metapopulation structure. Further research into the population ecology of this species is needed to facilitate well-informed management decisions.     

Population differentiation within and among Asian elephant (Elephas maximus) populations in southern India

Southern India, one of the last strongholds of the endangered Asian elephant (Elephas maximus), harbours about one-fifth of the global population. We present here the first population genetic study of free-ranging Asian elephants, examining within- and among-population differentiation by analysing mitochondrial DNA (mtDNA) and nuclear microsatellite DNA differentiation across the Nilgiris-Eastern Ghats, Anamalai, and Periyar elephant reserves of southern India. Low mtDNA diversity and 'normal' microsatellite diversity were observed. Surprisingly, the Nilgiri population, which is the world's single largest Asian elephant population, had only one mtDNA haplotype and lower microsatellite diversity than the two other smaller populations examined. There was almost no mtDNA or microsatellite differentiation among localities within the Nilgiris, an area of about 15,000 km2. This suggests extensive gene flow in the past, which is compatible with the home ranges of several hundred square kilometres of elephants in southern India. Conversely, the Nilgiri population is genetically distinct at both mitochondrial and microsatellite markers from the two more southerly populations, Anamalai and Periyar, which in turn are not genetically differentiated from each other. The more southerly populations are separated from the Nilgiris by only a 40-km-wide stretch across a gap in the Western Ghats mountain range. These results variably indicate the importance of population bottlenecks, social organization, and biogeographic barriers in shaping the distribution of genetic variation among Asian elephant populations in southern India.     

Historic hybridization and introgression between two iconic Australian anemonefish and contemporary patterns of population connectivity

Endemic species on islands are considered at risk of extinction for several reasons, including limited dispersal abilities, small population sizes, and low genetic diversity. We used mitochondrial DNA (D-Loop) and 17 microsatellite loci to explore the evolutionary relationship between an endemic anemonefish, Amphiprion mccullochi (restricted to isolated locations in subtropical eastern Australia) and its more widespread sister species, A. akindynos. A mitochondrial DNA (mtDNA) phylogram showed reciprocal monophyly was lacking for the two species, with two supported groups, each containing representatives of both species, but no shared haplotypes and up to 12 species, but not location-specific management units (MUs). Population genetic analyses suggested evolutionary connectivity among samples of each species (mtDNA), while ecological connectivity was only evident among populations of the endemic, A. mccullochi. This suggests higher dispersal between endemic anemonefish populations at both evolutionary and ecological timeframes, despite separation by hundreds of kilometers. The complex mtDNA structure results from historical hybridization and introgression in the evolutionary past of these species, validated by msat analyses (NEWHYBRIDS, STRUCTURE, and DAPC). Both species had high genetic diversities (mtDNA h > 0.90, π = 4.0%; msat genetic diversity, gd > 0.670). While high gd and connectivity reduce extinction risk, identifying and protecting populations implicated in generating reticulate structure among these species should be a conservation priority.     

World phylogeography and male‐mediated gene flow in the sandbar shark,Carcharhinus plumbeus

The sandbar shark, Carcharhinus plumbeus, is a large, cosmopolitan, coastal species. Females are thought to show philopatry to nursery grounds while males potentially migrate long distances, creating an opportunity for male‐mediated gene flow that may lead to discordance in patterns revealed by mitochondrial DNA (mtDNA) and nuclear markers. While this dynamic has been investigated in elasmobranchs over small spatial scales, it has not been examined at a global level. We examined patterns of historical phylogeography and contemporary gene flow by genotyping 329 individuals from nine locations throughout the species’ range at eight nuclear microsatellite markers and sequencing the complete mtDNA control region. Pairwise comparisons often resulted in fixation indices and divergence estimates of greater magnitude using mtDNA sequence data than microsatellite data. In addition, multiple methods of estimation suggested fewer populations based on microsatellite loci than on mtDNA sequence data. Coalescent analyses suggest divergence and restricted migration among Hawaii, Taiwan, eastern and western Australia using mtDNA sequence data and no divergence and high migration rates, between Taiwan and both Australian sites using microsatellite data. Evidence of secondary contact was detected between several localities and appears to be discreet in time rather than continuous. Collectively, these data suggest complex spatial/temporal relationships between shark populations that may feature pulses of female dispersal and more continuous male‐mediated gene flow.     

The genetic consequences of a demographic bottleneck in an introduced biological control insect

Population bottlenecks may result in the loss of genetic diversity, with potentially negative consequences for species of interest in conservation biology, including rare species, invasive species and biological control agents. We examined mtDNA sequence data and four variable microsatellite loci (SSRs) in the melaleuca psyllid Boreioglycaspis melaleucae, which was introduced from Australia to Florida as a biological control agent of the invasive plant Melaleuca quinquenervia. We sampled psyllids in the native and introduced ranges as well as individuals stored from the original founding population. There was a clear loss of mtDNA haplotype diversity, as well as a loss of rare microsatellite alleles, in the introduced range. However, there was little genetic differentiation between the home and introduced ranges, and no evidence for a genetic bottleneck based on an analysis of heterozygosity with the microsatellite markers. Overall, the data showed that the demographic bottleneck had a limited effect on the genetics of populations in the new range.     

Population differentiation in the Pacific white-sided dolphin Lagenorhynchus obliquidens inferred from mitochondrial DNA and microsatellite analyses

We investigated genetic diversity and differentiation of the Pacific white-sided dolphin (Lagenorhynchus obliquidens) in Japanese coastal waters and offshore North Pacific by analyzing mitochondrial DNA and nuclear microsatellite variation. A total of 519 bp of the mitochondrial control region was sequenced and five microsatellite locus were genotyped for 59 individuals. A high level of haplotypic diversity (h=96.1%), moderate level of nucleotide diversity (pi=1.65%) and average expected heterozygosity (HE=0.66-0.76) were within an extent of those reported for other odontocetes. Consistent genetic difference between the samples from Japanese coastal Pacific-Sea of Japan and offshore North Pacific was indicated by analyses of molecular variance (AMOVAs) based on mtDNA and microsatellite variations, comparison of genetic variabilities, and geographical distributions of mtDNA haplotypes and microsatellite alleles. This result suggests that Pacific white-sided dolphins in each of the above two areas belong to different populations between which gene flow has been severely restricted. The low genetic diversity and mtDNA genealogy of the population in Japanese coastal waters suggest that it originated from a small population that colonized the Sea of Japan or that experienced population reduction when this Sea was isolated from the North Pacific during a glacial period in the Late Pleistocene.     

Population structure of bottlenose dolphins (Tursiops aduncus) impacted by bycatch along the east coast of South Africa

Shark nets placed along the coast of KwaZulu-Natal to protect bathers from shark attacks result in an incidental by-catch of bottlenose dolphins (Tursiops aduncus) at twice the level suggested by the IWC as the maximum sustainable capture rate for a cetacean population. Observational data reported the presence of at least two putative populations, one a seasonal migratory population moving into the KwaZulu-Natal area following the movement of sardines, and the other a coastal, apparently resident population. Some observations indicated possible further subdivision of the coastal population. We analysed nine microsatellite loci and 599 bps of the mitochondrial control region and found small but significant differentiation within the putative coastal population (F_ST = 0.022) separated north and south of Ifafa. There was weaker evidence for differentiation between the southern coastal and migratory populations (F_ST = 0.012). Genetic diversity was low for both types of markers in all populations, and the pattern of mtDNA variation was consistent with a founder event. Considering the high capture rate, the low level of genetic diversity, and the evidence of possible population differentiation along the coast, we suggest that particular care should be taken in managing the take from shark nets especially in the north coastal area of KZN where the population showed the higher degree of differentiation.     

Microsatellite Analysis of Genetic Variation and Population Subdivision for the Black Muntjac, Muntiacus crinifrons

The black muntjac (Muntiacus crinifrons) is a rare deer found only in a restricted region in east China. Recent studies of mitochondrial DNA diversity have shown a markedly low level of nucleotide diversity for the species, and the Suichang population was genetically differentiated from the two other populations, in Huangshan and Tianmushan mountains. In this study, we extended the analysis of genetic diversity and population subdivision for the black muntjac using data from 11 highly polymorphic nuclear DNA microsatellite loci. Contrary to the results based on mtDNA data, the microsatellite loci revealed that the black muntjac retained a rather high nuclear genetic diversity (overall average H (E) = 0.78). Nevertheless, both types of markers supported the idea that the extant black muntjac population is genetically disrupted (overall phi (ST) = 0.16 for mtDNA and overall F (ST) = 0.053 for microsatellite, both P < 0.001). The correlation between genetic differentiation and geographic distance was not significant (Mantel test; P > 0.05), implying that the patterns of genetic differentiation observed in this study might result from recent habitat fragmentation or loss. Based on the results from the mtDNA and nuclear DNA data sets, two management units were defined for the species, Huangshan/Tianmushan and Suichang. We also recommend that a new captive population be established with individuals from the Suichang region as a founder source.     

Microsatellite markers and mtDNA data indicate two distinct groups in dwarf galaxias, Galaxiella pusilla (Mack) (Pisces: Galaxiidae), a threatened freshwater fish from south-eastern Australia

Galaxiella pusilla is a small, non-migratory freshwater fish, endemic to south-eastern Australia and considered nationally threatened. To assist in the conservation of the species, microsatellite markers were developed and used to characterize genetic variation in 20 geographically distinct populations across its range. Substantial genetic differentiation was found between an eastern (Victoria east of the Otway Ranges and Tasmania) and western (South Australia and Victoria west of, and including, the Otway Ranges) region. This major separation was also observed in data from a mitochondrial gene and supports a previously proposed split. Populations from the eastern region had overall lower genetic diversity for both the microsatellite and mtDNA markers. There was substantial genetic differentiation between populations within the two regions, suggesting that gene flow is limited by the isolation of freshwater streams. Genetic structure, consistent with an isolation-by-distance model, was also evident in both regions. Patterns of genetic variation in this threatened species are compared to those obtained for other taxa across the same region. The need to consider separate conservation strategies for the two sets of populations is emphasized.     

Microsatellite and mitochondrial DNA variation defines island genetic reservoirs for reintroductions of an endangered Australian marsupial, <Emphasis Type="Italic">Perameles bougainville</Emphasis>

Natural populations of the endangered western barred bandicoot (Perameles bougainville) now exist on only two islands in Shark Bay, Western Australia. Our aim was to investigate genetic diversity in natural, reintroduced, and captive populations of the bandicoots and to assess the extent of divergence between the populations. The contemporary isolation of the natural populations has resulted in heterogeneity of allele frequency between the islands, which has acted to maintain a higher combined diversity than would be expected from either population on its own. These findings highlight how remnant island populations can act as genetic reservoirs to maximize diversity for reintroductions into a species former range. Although diversity is high between island populations, diversity within populations, based on six microsatellite loci, are amongst the lowest ever recorded for populations of marsupials. The mtDNA sequence data indicate that the two remaining natural populations show only minor divergence from each other, with the five haplotypes separated by just single base pairs. The reintroduced population and captive colonies show evidence for the loss of diversity related to genetic drift operating on small isolated populations.     

Homing behaviour facilitates subtle genetic differentiation among river populations of Alosa sapidissima: microsatellites and mtDNA

Significant but subtle differentiation was detected for both microsatellite DNA and mitochondrial DNA among four populations of American shad Alosa sapidissima . The data indicate that straying among rivers is sufficient to permit only marginal population differentiation in this species, but suggest that individual river populations should be managed as distinct stocks. Comparison of the Hudson and Columbia populations, the latter derived from the former over 100 years ago, revealed only a slight reduction in microsatellite DNA variation for the founded population but halving of mitochondrial DNA, consistent with the haploid maternal inheritance of the latter marker. The depleted and endangered James River (Virginia) population and two other Atlantic coast populations exhibited similar levels of microsatellite DNA variation, but mtDNA diversity in the James River was marginally lower than in other Atlantic populations, again consistent with the low effective population size of mtDNA.     

Demography or selection on linked cultural traits or genes? Investigating the driver of low mtDNA diversity in the sperm whale using complementary mitochondrial and nuclear genome analyses

Mitochondrial DNA has been heavily utilized in phylogeography studies for several decades. However, underlying patterns of demography and phylogeography may be misrepresented due to coalescence stochasticity, selection, variation in mutation rates and cultural hitchhiking (linkage of genetic variation to culturally‐transmitted traits affecting fitness). Cultural hitchhiking has been suggested as an explanation for low genetic diversity in species with strong social structures, counteracting even high mobility, abundance and limited barriers to dispersal. One such species is the sperm whale, which shows very limited phylogeographic structure and low mtDNA diversity despite a worldwide distribution and large population. Here, we use analyses of 175 globally distributed mitogenomes and three nuclear genomes to evaluate hypotheses of a population bottleneck/expansion vs. a selective sweep due to cultural hitchhiking or selection on mtDNA as the mechanism contributing to low worldwide mitochondrial diversity in sperm whales. In contrast to mtDNA control region (CR) data, mitogenome haplotypes are largely ocean‐specific, with only one of 80 shared between the Atlantic and Pacific. Demographic analyses of nuclear genomes suggest low mtDNA diversity is consistent with a global reduction in population size that ended approximately 125,000 years ago, correlated with the Eemian interglacial. Phylogeographic analysis suggests that extant sperm whales descend from maternal lineages endemic to the Pacific during the period of reduced abundance and have subsequently colonized the Atlantic several times. Results highlight the apparent impact of past climate change, and suggest selection and hitchhiking are not the sole processes responsible for low mtDNA diversity in this highly social species.     

Reconciling molecular signatures across markers: mitochondrial DNA confirms founder effect in invasive North American house finches (<Emphasis Type="Italic">Carpodacus mexicanus</Emphasis>)

Well-characterized species introductions provide opportunities to compare the genetic signatures of known founder effects across classes of molecular markers. The release of small numbers of house finches (Carpodacus mexicanus) into the eastern United States in the 1940s led to substantial interest in the effects of this introduction on genetic diversity in this now abundant species, an issue that has been highlighted by a recent Mycoplasma disease epidemic that most intensively affects the introduced and potentially genetically depauperate house finch populations. Previous studies comparing genetic diversity levels in native and introduced house finch populations produced seemingly disparate results: comparisons based on amplified fragment length polymorphism, RFLP mtDNA, and allozyme markers found essentially equivalent levels of diversity in eastern and western populations, whereas microsatellite markers showed clear reductions in diversity in the introduced populations. Here we employ sequence variation at the ND2 mtDNA locus to further compare levels of diversity between the four native and five introduced house finch populations that were previously examined in the microsatellite study. We found substantially lower ND2 haplotype richness and diversity across all introduced populations of house finches. The majority of sequence variation (78%) was detected within subpopulations, with the remainder (22%) explained by the historical status of each population (native or introduced). Our results are consistent with previous microsatellite evidence for a founder effect during the introduction of eastern house finches, and suggest that the mtDNA founder effect was particularly severe, likely owing to a male-biased sex ratio at the time of introduction coupled with the lower effective population size of clonally inherited markers. We discuss how the inconsistencies between past studies of house finch diversity can inform the usefulness of distinct marker sets for detecting molecular signatures of founder events.     

Genetic Diversity and Population Structure of the Pelagic Thresher Shark (Alopias pelagicus) in the Pacific Ocean: Evidence for Two Evolutionarily Significant Units

There has been an increasing concern about shark overexploitation in the last decade, especially for open ocean shark species, where there is a paucity of data about their life histories and population dynamics. Little is known regarding the population structure of the pelagic thresher shark, Alopias pelagicus. Though an earlier study using mtDNA control region data, showed evidence for differences between eastern and western Pacific populations, the study was hampered by low sample size and sparse geographic coverage, particularly a lack of samples from the central Pacific. Here, we present the population structure of Alopias pelagicus analyzing 351 samples from six different locations across the Pacific Ocean. Using data from mitochondrial DNA COI sequences and seven microsatellite loci we found evidence of strong population differentiation between western and eastern Pacific populations and evidence for reciprocally monophyly for organelle haplotypes and significant divergence of allele frequencies at nuclear loci, suggesting the existence of two Evolutionarily Significant Units (ESU) in the Pacific Ocean. Interestingly, the population in Hawaii appears to be composed of both ESUs in what seems to be clear sympatry with reproductive isolation. These results may indicate the existence of a new cryptic species in the Pacific Ocean. The presence of these distinct ESUs highlights the need for revised management plans for this highly exploited shark throughout its range.     

Mitochondrial DNA evidence of an early Holocene population expansion of threespine sticklebacks from Scotland

In this study, we analyzed the cytochrome b gene in threespine stickleback (Gasterosteus aculeatus) populations from Scotland. We found evidence of a postglacial population expansion in Scotland and large differences in genetic diversity estimates among populations. Higher levels of genetic diversity are negatively correlated with distance from the ocean. In addition, distance from the ocean and predation risk both explain variation in plate count in Scottish populations. Overall, the mtDNA data support the racemic model of evolution in threespine stickleback.