Experimental microevolution: transplantation of pink salmon into the European North

Human-mediated translocations of species beyond their native ranges can enhance evolutionary processes in populations introduced to novel environments. We studied such processes in several generations of pink salmon Oncorhynchus gorbuscha introduced to the European North of Russia using a set of morphological and life-history traits as well as molecular genetic markers with different selective values: protein-coding loci, mtDNA, microsatellites, and MHC. The introduction of reproductively isolated pink salmon broodlines of odd and even years yielded different results. The odd-year broodline established self-reproducing local populations in many rivers of new range, but sustainable changes in external morphology, reproduction, and life-history, as well as the impoverishment of the gene pool occurred. Their successful colonisation of the new range resulted in specialisation manifested in the rapid directional shifts in some highly heritable phenotypic traits accompanied by increased homozygosity at molecular markers as a consequence of genetic drift and selective processes. The returns of transplanted pink salmon of even-year broodline decreased sharply already in the second generation, but there was no marked reduction of genetic diversity. Our data, as well as the analysis of the history of all pink salmon transplantations beyond the species range, demonstrate comparatively greater success of introduced odd-year broodline and permit to assume different adaptive plasticity of the even- and odd-year broodlines in pink salmon, what is most likely determined by differences in their evolutionary histories. Population genetic data suggest that the even-year broodline probably diverged from the odd-year broodline relatively recently and, due to the founder effect, may have lost a part of its genetic variation with which adaptive plasticity potential is associated.     

Comparative phylogeography of the two pink salmon broodlines: an analysis based on a mitochondrial DNA genealogy

Over most of their natural northern Pacific Ocean range, pink salmon (Oncorhynchus gorbuscha) spawn in a habitat that was repeatedly and profoundly affected by Pleistocene glacial advances. A strictly two-year life cycle of pink salmon has resulted in two reproductively isolated broodlines, which spawn in alternating years and evolved as temporal replicates of the same species. To study the influence of historical events on phylogeographical and population genetic structure of the two broodlines, we first reconstructed a fine-scale mtDNA haplotype genealogy from a sample of 80 individuals and then determined the geographical distribution of the major genealogical assemblages for 718 individuals sampled from nine Alaskan and eastern Asian even- and nine odd-year pink salmon populations. Analysis of restriction site states in seven polymerase chain reaction (PCR)-amplified mtDNA regions (comprising 97% of the mitochondrial genome) using 13 endonucleases resolved 38 haplotypes, which clustered into five genealogical lineages that differed from 0.065 to 0.225% in net sequence divergence. The lineage sorting between broodlines was incomplete, which suggests a recent common ancestry. Within each lineage, haplotypes exhibited star-like genealogies indicating recent population growth. The depth of the haplotype genealogy is shallow ( approximately 0.5% of nucleotide sequence divergence) and probably reflects repeated decreases in population size due to Pleistocene glacial advances. Nested clade analysis (NCA) of geographical distances showed that the geographical distribution observed for mitochondrial DNA (mtDNA) haplotypes resulted from alternating influences of historical range expansions and episodes of restricted dispersal. Analyses of molecular variance showed weak geographical structuring of mtDNA variation, except for the strong subdivision between Asian and Alaskan populations within the even-year broodline. The genetic similarities observed among and within geographical regions probably originated from postglacial recolonizations from common sources rather than extensive gene flow. The phylogeographical and population genetic structures differ substantally between broodlines. This can be explained by stochastic lineage sorting in glacial refugia and perhaps different recolonization routes in even- and odd-year broodlines.     

Genetic linkage mapping of allozyme loci in even- and odd-year pink salmon (Oncorhynchus gorbuscha)

We constructed genetic linkage maps of allozyme loci in even- and odd-year pink salmon (Oncorhynchus gorbuscha), using the total of 320 families (each female was crossed with two different males, and 80 females and 160 males were used for each of even year and odd year). The maps include eight linkage groups involving 22 loci. We observed substantial variation in recombination frequencies among different families within broodline and between sexes within broodlines. In the linkage analysis between sAAT-3* and sMDH-B1,2*, two even-year families and one odd-year family exhibited evidence of association, but two even-year and one odd-year families did not. Recombination rate tends to be reduced in males in pink salmon. The ratio of recombination rate (female/male), which ranged from 1.7 to infinity, averaged 2.8 in the even-year crosses and 3.2 in the odd-year crosses. The linkage groups (LG) I and II involving sAAT and mAH loci, which probably duplicated in the recent tetraploidization event, and the orders of loci in the LGs I (sAAT-3* --> mAH-4*) and II (mAH-3* --> sAAT-4*) were reversed, suggesting the possible paracentric inversion during salmonid evolution after the duplication.     

Estimates of population differentiation in pink salmon Oncorhynchus gorbuscha with using of the microsatellite markers may be underestimated due to high population sizes

Data on the variation at eight microsatellite loci in the Far East salmon Oncorhynchus gorbuscha samples caught in 1984–1985 and 2001–2006 are analyzed. F-statistics indices at all levels of the hierarchical spatial structures are very small. At the same time, the differentiation between populations (according to F _ST estimates) in the odd-year broodline of pink salmon does not exceed the temporal variation within populations. In the even-year broodline, the F-statistics indices at the interregional and intraregional levels are significantly greater than those in the odd-year broodline. F _ST estimates (averaged over the same set of loci) vary widely within the range: the highest values are observed in populations of the coast of North America (except Alaska) and in the new range in the European North of Russia, whereas in the populations of the Asian part of the range and Alaska they are one order of magnitude smaller. The causes of the heterogeneity of the estimates of genetic differentiation within the range and between the broodlines of odd and even years are discussed. Since the mean population size estimates were correlated with the F _ST values, it was assumed that the effect of random genetic drift, the main factor of population divergence in selectively neutral loci, weakens with an increase in the population size. Because of the greater population sizes in pink salmon compared to other salmon species, as well due to the uneven distribution of populations of different size, the usage of microsatellite markers may lead to an underestimation of the true divergence of populations and their regional groups and, consequently, to an overestimation of genetic migration.     

Assessment of concordance among genealogical reconstructions from various mtDNA segments in three species of Pacific salmon (genus Oncorhynchus)

Seven segments of mitochondrial DNA (mtDNA), comprising 97% of the mitochondrial genome, were amplified by polymerase chain reaction (PCR) and examined for restriction site variation using 13 restriction endonucleases in three species of Pacific salmon: pink (Oncorhynchus gorbuscha), chum (O. keta) and sockeye (O. nerka) salmon. The distribution of variability across the seven mtDNA segments differed substantially among species. Little similarity in the distribution of variable restriction sites was found even between the mitochondrial genomes of the even- and odd-year broodlines of pink salmon. Significantly different levels of nucleotide diversity were detected among three groups of genes: six NADH-dehydrogenase genes had the highest; two rRNA genes had the lowest; and a group that included genes for ATPase and cytochrome oxidase subunits, the cytochrome b gene, and the control region had intermediate levels of nucleotide diversity. Genealogies of mtDNA haplotypes were reconstructed for each species, based on the variation in all mtDNA segments. The contributions of variation within different segments to resolution of the genealogical trees were compared within each species. With the exception of sockeye salmon, restriction site data from different genome segments tended to produce rather different trees (and hence rather different genealogies). In the majority of cases, genealogical information in different segments of mitochondrial genome was additive rather than congruent. This finding has a relevance to phylogeographic studies of other organisms and emphasizes the importance of not relying on a limited segment of the mtDNA genome to derive a phylogeographic structure.     

Population structure of odd-broodline Asian pink salmon and its contrast to the even-broodline structure

Most of the variation (99%) of Asian odd-broodline pink salmon Oncorhynchus gorbuscha , based on data at 32 variable (46 total) allozyme loci from 35 populations, occurred within populations. The remaining interpopulation variation was attributable to: (1) differences between northern (the northern Sea of Okhotsk, eastern Kamchatka Peninsula and western Kamchatka Peninsula) and southern (Hokkaido Island, Kuril Islands and Sakhalin Island) populations; (2) differences between the southern areas; (3) low variation among populations within some areas. The pattern contrasted strongly with that observed for Asian even-broodline populations, which had a strong structure, possibly related to geographic and oceanographic influences. Isolation-by-distance analyses of each of the two broodlines showed a stronger relationship (x 4·8) among even- than odd-broodline populations. Allele frequency differences between even- and odd-broodlines reflected the reproductive isolation of the broodlines. However, there were no fixed frequency differences which, considered with the differing population structures, suggests that migration-drift equilibrium has not yet obtained in one or both broodlines. The structural differences also suggest it is likely that the even- and odd-broodlines are of different ages and that one is derived from the other. Allozyme data do not provide a genealogical basis for identifying the ancestral lineage.     

Genetic divergence in pink salmon introduced into the European North of Russia: Microsatellite and allozyme variation analysis

The 1985 introduction into the European North of Russia resulted in the formation of a large stock of pink salmon of the odd-year broodline. To assess the divergence of the new population and the role of various microevolutionary factors, variation of four microsatellite loci and fifteen genes encoding proteins (allozymes) in samples of fish, running for spawning in rivers of the new area, and in samples from the donor population of the Ola River (Magadan region). In the generations 8 and 9 of the introduced pink salmon of the odd-year line, the genetic diversity (the number of alleles and the mean heterozygosity) both at allozyme and at microsatellite loci was significantly lower, than that in the donor population. The explanations of the decline in diversity are discussed. The first evidence for spatial genetic divergence in transplanted fish within the new area has been obtained; the divergence level may be comparable with that characteristic of native populations.     

Genetic Changes in Pink Salmon Oncorhynchus gorbuscha Walbaum during Acclimatization in the White Sea Basin

Genetic parameters of pink salmon introduced into the White Sea basin in 1985 and 1998 were compared to the corresponding parameters of the donor population from the Ola River (Magadan oblast). The detected genetic differences indicate that colonization of a new area is accompanied by impoverishment of the gene pool of the native population. This effect was particularly marked in the odd-year broodline of pink salmon introduced in 1985. The probable causes of these genetic changes are discussed.     

Timing of Development During Epiboly in Embryos of Second-Generation Crosses and Backcrosses between Odd- and Even-Broodyear Pink Salmon, Oncorhynchus gorbuscha

Synopsis Odd- and even-year-spawning pink salmon (Oncorhynchus gorbuscha) are genetically isolated; their broodlines differ even in the same natal stream. Hybrids between broodlines exhibit outbreeding depression in survival. Variation in the time to completion of epiboly in embryos appears to be adaptive in both broodlines. We compared stage of development at a time near the completion of epiboly in families of second-generation offspring from crosses between odd- and even-year broodlines with development stages of within-broodyear controls and of backcrossed families. We observed embryos derived from matings of mature fish that were the results of fertilizations made 2 years earlier of eggs from females from the even brood year with semen from males from the even broodyear and with cryopreserved semen from males of the odd broodyear. The resulting fry had been released to the Pacific Ocean and recovered at maturity. Second generation embryos were produced by factorial matings of these mature fish involving (1) female and male controls, (2) female and male hybrids, and (3) both backcrosses. Analysis of variation of development time detected no effect of outbreeding, i.e., differences between controls and second generation hybrids (p > 0.05), but did detect variation between individual female parents (p < 0.03). Neither epistatic nor additive outbreeding depression could be detected in the rate of early embryonic development of pink salmon. However, effects on development rate attributable to female parents indicate that either a maternal effect or early additive genetic effects occurred before the expression of the paternal genome in embryos.     

Gene–Centromere Distances of Allozyme loci in Even- and Odd-year Pink Salmon, (Oncorhynchus gorbuscha)

We produced gynogenetic progeny families to estimate gene-centromere (G-C) distances of allozyme loci in even-year and odd-year pink salmon (Oncorhynchus gorbuscha). G-C distances of 37 loci distributed on a chromosome ranged from 1 cM at LDH-A1* to 49 cM at ADA-2*, DIA-2*, and sMDH-B1,2*. The distribution of the G-C distances along the chromosome arm was not even and appears telomeric. Eight loci in even-year and seven in odd-year showed high G-C distances (>45 cM), indicating that one crossover per chromosome arm is usual in pink salmon. Variation was observed in the results from different families; 14 loci out of 21 tested, showed heterogeneity. At mAH-3*, G-C distances from five odd-year families ranged from 6 to 37 cM; the widest range observed in this study. At isoloci such as sMDH-A 1,2* and sMDH-B1,2* the distances from different families were grouped into statistically discrete distributions, suggesting that it may be a reflection polymorphism at both isoloci. It appears G-C distances in salmonid species are well conserved with some minor differences.     

Outbreeding Depression in Hybrids Between Spatially Separated Pink Salmon, Oncorhynchus gorbuscha, Populations: Marine Survival, Homing ability, and Variability in Family Size

Hybridization between distinct populations and introgression of nonnative genes can erode fitness of native populations through outbreeding depression, either by producing a phenotype intermediate to that of both contributing genomes (and maladapted in either population's environment) or by disrupting distinct coadapted complexes of epistatic genes. In salmon, fitness-related traits such as homing ability or family-size distribution may be eroded. We investigated geographically separated pink salmon populations in repeated trials in independent broodyears (odd and even). Hybrids were made between female Auke Creek (Southeast Alaska) pink salmon and Pillar Creek (Kodiak Island, ～1 000 km away) males; hybrids and their offspring were compared to offspring of control crosses of the same females with Auke Creek males. Parentage assignment from microsatellite analysis was used to improve estimates of survival and straying and to examine variation of family size. Hybridization reduced return rates of adults (a proxy for survival at sea) in the F₁ generation in the odd-year broodline (p < 0.0001) but not in the even-year broodline (p = 0.678). Hybridization reduced survival in both the odd- and even-broodyear F₂ (p < 0.005 and p < 0.0001). Hybridization did not appear to impair homing ability; weekly surveys revealed similar straying rates (～2%) by both hybrid and control fish into nearby (～1 km) Waydelich Creek in both generations in both trials. Hybridization did not increase the index of variability (σ²/μ) in family size. Decreased survival in the hybrid F₂ generation supports an epistatic model of outbreeding depression; nonepistatic effects may have contributed to reduced survival in the odd-broodyear F₁ hybrid fish. Outbreeding depression in hybrids of geographically separated populations demonstrates that introgression of nonnative fish can erode fitness, and should be recognized as a potential detriment of both aquaculture and management practices.     

Geographical and temporal mitochondrial DNA variability in populations of pink salmon

Pink salmon Oncorhynchus gorbuscha from odd and even year generations in rivers of Sakhalin Island, Kuril Island, Kamchatka Peninsula, and Alaska were investigated with five informative restriction endonucleases for mtDNA variation. The odd and even generations from the same rivers of South Sakhalin differed greatly. The time of divergence between the two broodlines was estimated at 0.9-1.1 Myr. The variability of mtDNA in odd year generations was higher than in even year generations and may have been due to' founder' and/or' bottleneck' effects. The differences among river populations within the Sakhalin region in 1991-1993 were not significant and this confirms the highly migratory nature of pink compared with other Pacific salmon. The mtDNA samples revealed statistically significant differences between regions. The northern populations (Kamchatka, Alaska) were less diverse in number and frequency of haplotypes than the southern populations (Sakhalin). This suggests that pink salmon originated in the Sakhalin-Kuril region and that a founder effect during the spread of this species may have restricted the mtDNA variability in other regions.     

Genetic divergence in pink salmon introduced into the European north of Russia: microsatellite and allozyme variation analysis

The 1985 introduction into the European North of Russia resulted in the formation of a large stock of pink salmon of the odd-year breeding line. To assess the divergence of the new population and the role of various microevolutionary factors, variation of four microsatellite loci and fifteen genes encoding proteins (allozymes) in samples of fish, running for spawning in rivers of the new area, and in samples from the donor population of the Ola River (Magadan oblast). In the generations 8 and 9 of the introduced pink salmon of the odd-year line, the genetic diversity (the number of alleles and the mean heterozygosity) both at allozyme and at microsatellite loci was significantly lower, than that in the donor population. The explanations of the decline in diversity are discussed. The first evidence for spatial genetic divergence in transplanted fish within the new area has been obtained; the divergence level may be comparable with that characteristic of native populations.     

Genetic differentiation of pink salmon oncorhynchus gorbuscha Walbaum in the Asian part of the range

Genetic variation at 19 allozyme (including 11 polymorphic) and 10 microsatellite loci was examined in the population samples of odd- and even-broodline pink salmon from the southern part of Sakhalin Island, Southern Kuril Islands, and the northern coast of the Sea of Okhotsk. The estimates of relative interpopulation component of genetic variation over the allozyme loci, per broodline, were on average 0.43% (GST), while over the microsatellite loci it was 0.26% (the theta(ST) coefficient, F-statistics based on the allele frequency variance), and 0.90% (the rho(ST) coefficient, R-statistics based on the allele size variance). The values of interlinear component constituted 2.34, 0.31, and 1.05% of the total variation, respectively. Using the allozyme loci, statistically significant intralinear heterogeneity was demonstrated among the regions, as well as among the populations of Southern Sakhalin Island. Multivariate scaling based on the allozyme data demonstrated regional clustering of the sample groups, representing certain populations during the spawning run or in different years. Most of the microsatellite loci examined were found to be highly polymorphic (mean heterozygosity > 0.880). The estimates of interlinear, interregional, and interpopulation variation over these loci in terms of theta(ST) values were substantially lower than in terms of rho(ST) values. Regional genetic differentiation, mostly expressed at the allozyme loci among the populations from the northern and southern parts of the Sea of Okhotsk (i.e., between the Sakhalin and Kuril populations), was less expressed at the microsatellite loci. The differentiation between these regions observed can be considered as the evidence in favor of a large-scale isolation by distance characterizing Asian pink salmon. It is suggested that in pink salmon, low genetic differentiation at neutral microsatellite loci can be explained by extremely high heterozygosity,of the loci themselves, as well as by the migration gene exchange among the populations (the estimate of the genetic migration coefficient inferred from the "private" allele data constituted 2.6 to 3.4%), specifically, by the ancient migration exchange, which occurred during postglacial colonization and colonization of the range.     

Genetic Population Structure of Chum Salmon in the Pacific Rim Inferred from Mitochondrial DNA Sequence Variation

We examined the genetic population structure of chum salmon, Oncorhynchus keta, in the Pacific Rim using mitochondrial (mt) DNA analysis. Nucleotide sequence analysis of about 500 bp in the variable portion of the 5′ end of the mtDNA control region revealed 20 variable nucleotide sites, which defined 30 haplotypes of three genealogical clades (A, B, and C), in more than 2,100 individuals of 48 populations from Japan (16), Korea (1), Russia (10), and North America (21 from Alaska, British Columbia, and Washington). The observed haplotypes were mostly associated with geographic regions, in that clade A and C haplotypes characterized Asian populations and clade B haplotypes distinguished North American populations. The haplotype diversity was highest in the Japanese populations, suggesting a greater genetic variation in the populations of Japan than those of Russia and North America. The analysis of molecular variance and contingency χ² tests demonstrated strong structuring among the three geographic groups of populations and weak to moderate structuring within Japanese and North American populations. These results suggest that the observed geographic pattern might be influenced primarily by historic expansions or colonizations and secondarily by low or restricted gene flow between local groups within regions. In addition to the analysis of population structure, mtDNA data may be useful for constructing a baseline for stock identification of mixed populations of high seas chum salmon.     

Analysis of mtDNA indicates weak temporal genetic heterogeneity in pink salmon spawning runs in two rivers on Sakhalin Island

Both odd- and even-year pink salmon populations were sampled during the spawning runs in Firsovka and Bakhura rivers on Sakhalin Island. Four collections of 30 fish spaced at 2-week intervals were taken from each river in 2 consecutive years. Four restriction endonucleases were used to examine 2·15% of the mitochondrial genome. Eighteen variable sites and three types of insertion defined 37 haplotypes among 449 fish. Heterogeneity tests showed highly significant differences among temporal collections taken in the odd year from Bakhura River, and no significant differences among temporal collections from the other three spawning runs. However, probabilities of homogeneity among temporal collections were low in all tests, and an integral estimate of the probability of homogeneity for the total set of tests was less than 0·001, indicating highly significant overall temporal heterogeneity. Analysis of molecular variance (AMOVA), revealed that a small portion of the variance was distributed among temporal collections, and small φ_ST values that differed significantly from zero only in the odd-year population in Bakhura River. Temporal differences in spawning are probably adapative, and allow greater productivity in areas of high spawning densities.     

Genetic differentiation of pink salmon Oncorhynchus gorbuscha Walbaum in the Asian part of the range

Genetic variation at 19 enzyme (including 11 polymorphic) and 10 microsatellite loci was examined in the population samples of odd-and even-broodline pink salmon from the southern part of Sakhalin Island, Southern Kuril Islands, and the northern coast of the Sea of Okhotsk. The estimates of relative interpopulation component of genetic variation for the allozyme loci, per broodline, were on average 0.43% (G _ST), while over the microsatellite loci it was 0.26% (the ?_ST coefficient, F-statistics based on the allele frequency variance), and 0.90% (the ρ_ST coefficient, R-statistics based on the allele size variance). The values of interlinear component constituted 2.34, 0.31, and 1.05% of the total variation, respectively. Using the allozyme loci, statistically significant intralinear heterogeneity was demonstrated among the regions, as well as among the populations of southern Sakhalin. Multidimensional scaling based on the allozyme data demonstrated regional clustering of the sample groups, representing certain populations during the spawning run or in different years. Most of the microsatellite loci examined were found to be highly polymorphic (mean heterozygosity > 0.880). The estimates of interlinear, interregional, and interpopulation variation over these loci in terms of ?_ST values were substantially lower than in terms of ρ_ST values. Regional genetic differentiation, mostly expressed at the allozyme loci between the populations from the northern Sea of Okhotsk and the Sakhalin and Kuril group of populations, was less expressed at the microsatellite loci. The differentiation between these regions observed can be considered as the evidence in favor of a large-scale isolation by distance characterizing Asian pink salmon. It is suggested that in pink salmon, low genetic differentiation at neutral microsatellite loci can be explained by extremely high heterozygosity of the loci themselves, as well as by the migration gene exchange among the populations (the estimate of the gene migration coefficient inferred from the “private” allele data constituted 2.6 to 3.4%), specifically, by the ancient migration exchange, which occurred during postglacial colonization of the range     

Out crosses between seasonally different segments of a Pacific salmon population reveal local adaptation

In salmon populations, local adaptation to seasonally varying incubation temperature is characterized by temperature-adjusted development times [measured in degree days – accumulated temperature units (ATUs)] that differ between control and F₁ hybrid crosses that were made between temporally separated population segments, a contrast not expected in a panmictic population. We examined adaptation of embryo development time to seasonally cooling temperature in a population of pink salmon by estimating genetic components of variation in control and hybrid F₁ crosses made between members of early- and late-spawning subpopulations, and replicated our observations in independent odd- and even-year brood lines. In each brood line, both sire and dam components of variation of development time were significant and accounted for a substantially larger portion of variation than their interactions, which suggested that natural selection has acted primarily on additive genetic variation. The implications of these results are that (1) spatially or temporally proximate salmon populations may be structured by distinct adaptations; (2) artificial relaxation of local geneflow barriers may lead to depression of fitness; and (3) populations of salmon genetically structured by local adaptation may carry variation that enhances their persistence during rapid climate change.     

Genetic changes in pink salmon Oncorhynchus gorbuscha Walbaum during acclimatization in the White sea basin

Genetic parameters of pink salmon introduced into the White Sea basin in 1985 and 1998 were compared to the corresponding parameters of the donor population from the Ola River (Magadan oblast). The detected genetic differences indicate that colonization of a new area is accompanied by impoverishment of the gene pool of the native population. This effect was particularly marked in the odd-year line of pink salmon introduced in 1985. The probable causes of these genetic changes are discussed.     

Acclimatization of salmon Oncorhynchus gorbuscha (Walbaum) in the European north: data from restriction analysis of mtDNA

Pink salmon spawners introduced into the White Sea basin (the Umba River) were compared to the spawners from the basin of the Sea of Okhotsk (the Ola River) using restriction analysis of two fragments of mitochondrial DNA (mtDNA). One of the fragments included genes ND5/ND6, the other, the cytochrome b gene and the control region. It was found that mtDNA variation and diversity at the earlier examined nuclear allozyme genes significantly decreased in the odd broodline of pink salmon 8 years after the introduction. The haplotype diversity in the even broodline was considerably lower than in the odd broodline exhibiting virtually no change two generations after the introduction. Based on the results obtained, a possible role of these changes in adaptation of White Sea pink salmon from the odd broodline to the new environment is discussed.