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The endangered fountain darter Etheostoma fonticola is found only in the Comal and San Marcos rivers in the Guadalupe River basin in central Texas, USA. Comal River fountain darters were believed to be extirpated following a severe drought in the 1950s and were reintroduced in the early 1970s using 457 darters from the San Marcos River. In this study we used 23 microsatellite loci to describe and evaluate the genetic diversity, population structure and effective population size (N e) of fountain darters. We also evaluated the genetic effect of the Comal River reintroduction and the influence of low-head dams (dams) on dispersal in both rivers. Bayesian analysis of individual genotypes and Analysis of Molecular Variation supported two distinct populations concordant with the two rivers. Estimates of N e were much smaller (

We developed and evaluated three qPCR assays for detecting environmental DNA from northern pike (Esox lucius). The assays target the cytochrome oxidase 1 (EluCOI), control region (EluCR), and cytochrome b (EluCytB) genes of the mitochondrial DNA. Target specificity, assessed using the fluorescence signal (at 45 cycles) to noise (at 1 cycle) ratio (S/N), showed strong amplification in northern pike (mean S/N = 2.62, 3.52, 2.69 for EluCOI, EluCR, EluCytB). The mean S/N estimates from fifteen non-esocid freshwater fishes were about 1.0, as expected for no amplification. EluCR showed evidence of amplification (mean S/N = 3.16) in muskellunge (Esox masquinongy). The sensitivity tests indicated EluCOI has a higher detection probability than EluCR and EluCytB at low (20 copies/reaction) copy number. The results favor using EluCOI although EluCytB and EluCR are viable assays for the detection of northern pike eDNA.

We examined the assumption that landscape heterogeneity similarly influences the spatial distribution of genetic diversity in closely related and geographically overlapping species. Accordingly, we evaluated the influence of watershed affiliation and nine habitat variables from four categories (spatial isolation, habitat size, climate, and ecology) on population divergence in three species of Pacific salmon (Oncorhynchus tshawytscha, O. kisutch, and O. keta) from three contiguous watersheds in subarctic North America. By incorporating spatial data we found that the three watersheds did not form the first level of hierarchical population structure as predicted. Instead, each species exhibited a broadly similar spatial pattern: a single coastal group with populations from all watersheds and one or more inland groups primarily in the largest watershed. These results imply that the spatial scale of conservation should extend across watersheds rather than at the watershed level which is the scale for fishery management. Three independent methods of multivariate analysis identified two variables as having influence on population divergence across all watersheds: precipitation in all species and subbasin area (SBA) in Chinook. Although we found general broad-scale congruence in the spatial patterns of population divergence and evidence that precipitation may influence population divergence in each species, we also found differences in the level of population divergence (coho > Chinook and chum) and evidence that SBA may influence population divergence only in Chinook. These differences among species support a species-specific approach to evaluating and planning for the influence of broad-scale impacts such as climate change.

Coregonine fishes have a circumpolar distribution in the Arctic and sub-Arctic Northern Hemisphere. This subfamily of Salmonidae consists of three genera: Prosopium, Stenodus and Coregonus, including over 30 species. Many species overlap spatially and are difficult to distinguish based on morphological characteristics, especially as larvae or juveniles. Here we present a method for rapid and cost-effective species identification for representatives of the three genera based on sequence variation at the mitochondrial cytochrome c oxidase subunit I gene (COI). We examined eight species common to North America with distributional overlap in Alaska. Mean pairwise sequence divergence for all eight species was 7.04% and ranged from 0.46% to 14.23%. This sequence variation was used to develop a genetic assay based on restriction fragment length polymorphism. In a blind test, this assay provided correct species assignment for 48 of 49 individuals representing all eight species. The single incorrect assignment may reflect hybridization between two closely related species. This DNA barcode-based assay promises to aid fishery managers and researchers by providing a cost-effective alternative to large-scale sequence analysis for identification of North American coregonine fishes.

In a cooperative project among three agencies, variation at 30 microsatellite loci was surveyed for 19 populations of Chinook salmon Oncorhynchus tshawytscha from the Yukon River. The observed number of alleles per locus ranged from 2 to 63. Loci with a greater number of alleles displayed lower genetic differentiation index (FST) values, but loci with lower FST values also tended to provide more-accurate estimates of stock composition. The observed number of alleles was related to the power of the locus for providing accurate estimates of stock composition of simulated single-population samples. Mean estimated stock compositions for these mixtures ranged from 38.9% for simulations of single loci with fewer than 10 alleles to 85.5% for simulated loci with more than 60 alleles. Reliable population-specific estimation of stock composition was obtained with a minimum of five loci. Comparison of microsatellite stock identification power with an existing nine-locus single-nucleotide polymorphism (SNP) ...

We examine sympatric anadromous (steelhead) and nonanadromous (resident) rainbow trout (Oncorhynchus mykiss) from neighboring locations to test three hypotheses: (1) the sympatric life history types are not genetically different; (2) fine-scale dispersal is the same for both sexes, and (3) fine-scale dispersal is the same for steelhead and resident individuals. Data from 13 microsatellite loci reveal no genetic difference between sympatric steelhead and resident O. mykiss but moderate population structure (FST=0.019–0.028) between adjacent samples, regardless of life history type. Our results provide further evidence of partial anadromy and suggest that geographic proximity and genetic history, more than migratory type, should be considered when identifying populations for use in restoration of local genetic diversity. We find evidence of resident-form dispersal bias on a fine spatial scale, however, we find no evidence that fine-scale dispersal varies by gender. Conservation strategies should aim to maintain resident and anadromous forms when they occur in sympatry, as they may be important in facilitating gene flow on small and large spatial scales, respectively.

Little is known about the genetic diversityof coho salmon in Alaska, although this arearepresents half of the species' North Americanrange. In this study, nine microsatellite lociwere used to genotype 32 putative coho salmonpopulations from seven regions of Alaska. Theprimary objectives were to estimate andevaluate the degree and spatial distribution ofneutral genetic diversity within and amongpopulations of Alaskan coho salmon. Geneticanalysis yielded four results that provideinsight into forces influencing geneticdiversity in Alaskan coho salmon and haveimportant conservation implications: 1)significant population differentiation wasfound within each region; 2) the degree ofdifferentiation (FST = 0.099) amongpopulations was as large or larger than thatreported for other Pacific salmon species inAlaska; 3) phenetic clustering of populationsshowed weak geographic concordance; 4) stronggenetic isolation by distance was only apparentat the finest geographic scale (within adrainage). These results suggest that cohosalmon populations are small relative topopulations of other Pacific salmon, and thegenetic diversity within and among coho salmonpopulations is influenced primarily by geneticdrift, and not gene flow. Resource managementand conservation actions affecting coho salmonin Alaska must recognize that the populationsare generally small, isolated, and probablyexhibit local adaptation to different spawningand freshwater rearing habitats. These factorsjustify managing and conserving Alaskan cohosalmon at a fine geographic scale.

This study used simulations and a known two-generation pedigree of chinook salmon (Oncorhynchus tshawytscha) to evaluate the effect of full sibs of parents on pedigree reconstruction. Parentage analysis was conducted on 100 parent pair-offspring relationships from pedigrees with unrelated (simulation) and related (chinook salmon) candidate parents. Parentage assignment success for the chinook salmon was lower than in the simulated populations. For example, the six most variable loci (mean H(E) = 0.87) provided a mean of 97% unambiguous assignments in the simulated population and 67% unambiguous assignments for the chinook salmon. Estimates of the pairwise relatedness coefficient ((xy)) for most nonexcluded false parents and true parents of chinook salmon offspring exceeded 0.50. These results support the conclusion that closely related candidate parents decrease the power of genetic markers for pedigree reconstruction based on exclusion. Ambiguous parentage may be resolved using single parent- and parent pair-offspring likelihood analysis, however, these methods should be used with caution and they are not replacements for using more loci when many candidate parents are full sibs.

We describe the inheritance of 460 PCR-based loci in the polyploid-derived pink salmon (Oncorhynchus gorbuscha) genome using gynogenetic haploid embryos. We detected a length polymorphism in a growth hormone gene (GH-2) intron that is caused by an 81 bp insertion homologous to the 3' end of the salmonid short interspersed repetitive element (SINE) SmaI. Such insertion polymorphisms within species bring into question the use of SINEs as phylogenetic markers. We confirmed that a microsatellite locus encodes a PCR-null allele that is responsible for an apparent deficit of heterozygotes in a population sample from Prince William Sound. Another set of microsatellite primers amplified alleles of the same molecular weight from both loci of a duplicated pair. In our analysis of several PCR-based multilocus techniques, we failed to detect evidence of comigrating fragments produced by duplicated loci. Segregation analysis of PCR-based markers using gynogenetic haploid embryos ensures that the interpretation of molecular variation is not complicated by heterozygosity, diploidy, or gene duplication. We urge investigators to test the inheritance of polymorphisms in salmonids prior to using them to measure genetic variation.

Herein we describe the development of eight microsatellite markers for the northern sea otter, Enhydra lutris kenyoni. A total of 45 primer pairs were developed and screened from enriched AAAT, CATC, TACA, and TAGA libraries derived from genomic DNA of E. lutris kenyoni. Of these, eight amplified successfully. The average observed heterozygosity, expected heterozygosity, and number of alleles per locus was 0.506, 0.537, and 3.4, respectively. These eight loci were tested across three additional genra; Vulpes lagopus, Martes americana, and Mustela nivalis. Based on the success of our results these loci will be useful for future studies across all sub-species of E. lutris.

We examined genetic variation at five microsatellite loci in 12 odd-year populations and one even-year population of pink salmon Oncorhynchus gorbuscha from six geographic regions of North America. The degree of polymorphism varied widely among loci. The total number of alleles in the odd-year samples varied from 4 (Oneμ3) to 53 (Ssa85). A probability test revealed significant heterogeneity in allele frequencies among all odd-year samples and among pooled odd-year samples from six regions. We compared estimates of a standard index of population structure (θ) based on variance in allele frequency with a new index suggested for microsatellites (ρST) based on variance in allele size. Our results suggest is a better estimator of intralineage (odd-year × odd-year) population structure, whereas is best suited for estimating interlineage (odd-year × even-year) population structure. The difference in performance of and for estimating intralineage and interlineage population structure suggests high migrat...

Determining the success of invasive species eradication efforts is challenging because populations at very low abundance are difficult to detect. Environmental DNA (eDNA) sampling has recently emerged as a powerful tool for detecting rare aquatic animals; however, detectable fragments of DNA can persist over time despite absence of the targeted taxa and can therefore complicate eDNA sampling after an eradication event. This complication is a large concern for fish eradication efforts in lakes since killed fish can sink to the bottom and slowly decay. DNA released from these carcasses may remain detectable for long periods. Here, we evaluated the efficacy of eDNA sampling to detect invasive Northern pike (Esox lucius) following piscicide eradication efforts in southcentral Alaskan lakes. We used field observations and experiments to test the sensitivity of our Northern pike eDNA assay and to evaluate the persistence of detectable DNA emitted from Northern pike carcasses. We then used eDNA sampling and traditional sampling (i.e., gillnets) to test for presence of Northern pike in four lakes subjected to a piscicide-treatment designed to eradicate this species. We found that our assay could detect an abundant, free-roaming population of Northern pike and could also detect low-densities of Northern pike held in cages. For these caged Northern pike, probability of detection decreased with distance from the cage. We then stocked three lakes with Northern pike carcasses and collected eDNA samples 7, 35 and 70 days post-stocking. We detected DNA at 7 and 35 days, but not at 70 days. Finally, we collected eDNA samples ~ 230 days after four lakes were subjected to piscicide-treatments and detected Northern pike DNA in 3 of 179 samples, with a single detection at each of three lakes, though we did not catch any Northern pike in gillnets. Taken together, we found that eDNA can help to inform eradication efforts if used in conjunction with multiple lines of inquiry and sampling is delayed long enough to allow full degradation of DNA in the water.

Spawning aggregations of Pacific herring (Clupea pallasi) often exhibit significant interannual variation in allele frequencies of neutral gene markers. We isolated 14 tetranucleotide microsatellites to examine hypothetical processes that may produce this unique genetic signal. We developed and tested primer pairs for each locus and then estimated locus variability in samples (n = 60) from two populations. The number of alleles per locus ranged from five to 49. The expected heterozygosity across loci and populations ranged from 0.20 to 0.96. These microsatellites will be useful for estimating genetic variation in herring on a fine geographical scale.

Mutation rate can vary widely among microsatellite loci. This variation may cause discordant single-locus and multi-locus estimates of FST, the commonly used measure of population divergence. We use 16 microsatellite and five allozyme loci from 14 sockeye salmon populations to address two questions about the affect of mutation rate on estimates of FST: (1) does mutation rate influence FST estimates from all microsatellites to a similar degree relative to allozymes?; (2) does the influence of mutation rate on FST estimates from microsatellites vary with geographic scale in spatially structured populations? For question one we find that discordant estimates of FST among microsatellites as well as between the two marker classes are correlated with mean within-population heterozygosity (HS ) and thus are likely due to differences in mutation rate. Highly polymorphic microsatellites (HS > 0.84) provide significantly lower estimates of FST than moderately polymorphic microsatellites and allozymes (HS < 0.60). Estimates of FST from binned allele frequency data and RST provide more accurate measures of population divergence for highly polymorphic but not for moderately polymorphic microsatellites. We conclude it is more important to pool loci of like HS rather than marker class when estimating FST. For question two we find the FST values for moderately and highly polymorphic loci, while significantly different, are positively correlated for geographically proximate but not geographically distant population pairs. These results are consistent with expectations from the equilibrium approximation of Wright’s infinite island model and confirm that the influence of mutation on estimates of FST can vary in spatially structured populations presumably because the rate of migration varies inversely with geographic scale.

Summary: WHICHLOCI is a program that determines the relative discriminatory power of alternate genetic loci and loci combinations for population assignment of individuals. Availability: http://www.oregonstate.edu/dept/comes/ genetics/software.htm Contact: Michael.Banks@oregonstate.edu Increased information content from highly polymorphic molecular marker types such as microsatellites has markedly improved resolving power for discrimination among closely related populations. This, together with increased automation of techniques for resolving genetic variation, results in an overall boon of new information.