Technical University of Denmark National Institute of Aquatic Resources The use of molecular markers for preserving genetic resources in wild fish populations Michael M. Hansen UBA 0.2 TAP2A UBA θ ST 0.1 TAP2A 0.0 -3.0 -2.0 -1.0 0.0 1.0 2.0 3.0 lnRH
Outline • Molecular markers • A number of cases illustrating the use of molecular markers in fish conservation genetics research: • Genetic monitoring of effective population size in the endangered North Sea houting ( Coregonus lavaretus ) • Genetic interactions between stocked and wild brown trout ( Salmo trutta ) • Local adaptation in brown trout populations • European eel ( Anguilla anguilla ) – one or several populations (preliminary results)
Molecular markers • Mitochondrial DNA: organelle DNA, haploid, maternally inherited • Microsatellite DNA: Short sequence motifs (repeat units) repeated a number of times, e.g.…TGTGTGTGT… • High mutation rate (10-2 - 10-4). High levels of variability (often > 10 alleles) • Currently most used genetic marker • Single nucleotide polymorphisms (SNPs): Single-base mutations in genomic DNA. Typically every 300-1000 bp. • Hundreds or thousands of loci can be screened using automated techniques
Genetic monitoring of effective population size in the North Sea houting ( Coregonus oxyrhynchus ) Hansen et al. (2006) Canadian Journal of Fisheries and Aquatic Sciences, 63, 780-787 • Anadromous salmonid fish • Previously distributed throughout the Wadden Sea area • Almost extinct • Only one indigenous population left – the Vidaa River N • Question: Risk of inbreeding and 60 o Norway loss of variation? Sweden • Effective population size, N e – Latitude Denmark measure of how much inbreeding North Sea and loss of variation that will take Vidaa R. The place in a given assemblage of Netherlands 54 o individuals Germany UK • Depends on sex ratio, variance in 100 km reproductive success, temporal 0 o 10 o fluctuations of N e Longitude
How high is the effective population size in Vidå North Sea houting? Temporal method – estimation of N e from genetic drift - random genetic changes - that has occurred over time (method by Beaumont (2003) used) Sample ⇒ Time 1 . . . Sample ⇒ Time 2 • Twelve microsatellite DNA loci analysed in samples from 1980, 1994 and 2002
How high is the effective population size in Vidå North Sea houting? • Effective population size: 577 (90% CI: 297 – 3720) • Clearly above 50, the critical value for avoiding inbreeding depression in the short term • Most likely also above 500, the critical value for preserving evolutionary potential (controversial) • No imminent danger of inbreeding and loss of evolutionary potential
Is the population stable, increasing or declining? • The method allows for estimating effective Expansion population size at the start Decline (1980) and end (2002) of 2002 the time interval • Wide confidence intervals • No evidence for expansion or decline Mode • If anything, effective population size appears stable 1980
Can this method be used for routine monitoring of population sizes? • Simulated bottleneck by ”allowing” only 25 males and 25 females to reproduce in Decline Expansion 2002 • 50 offspring of the simulated 2006 bottleneck ”sampled” in 2006 • Strong signal of population decline in simulated sample • Useful for routine screening of populations Mode • Can reveals strong population declines with minimal sampling effort 1980
Genetic interactions between stocked and wild brown trout ( Salmo trutta ) – experiences from Denmark • Habitat destruction for many decades – 97% of all rivers affected – Drastic declines of brown trout • The ”solution” – Stocking with brown trout from commercial hatchery strains – Strains kept in captivity for up to 120 years – Most originating from populations from eastern Jutland, Denmark • Are most populations descendants of stocked hatchery strain trout?
Genetic interactions between stocked and wild brown trout ( Salmo trutta ) – experiences from Denmark (Hansen 2002. Molecular Ecology, 11: 1003-1015 ) • Karup River – until the 1960s the best sea trout river in Denmark • Population declines in 1960-70’s • Intensive stocking with hatchery strain trout • At the same time supportive breeding of (supposedly) wild spawners caught in the river • Habitat restoration and regulation of net fisheries • The run has recovered since the early 1990s. • Hatchery strain or indigenous trout????
Genetic interactions between stocked and wild brown trout ( Salmo trutta ) – experiences from Denmark • How to obtain genetic data from the population prior to stocking? • Old scale samples – already used for analysis of Atlantic salmon (Nielsen et al. 1997. Molecular Ecology) • Karup River: – 1947-1956 – 1993-1996 • Hatchery strain: – 1992 • Microsatellites
Genetic interactions between stocked and wild brown trout ( Salmo trutta ) – experiences from Denmark • Admixture proportion analysis (”LEA” (Chikhi et al., 2001) Observed Wild Hat. (1950) strain Hatchery Wild How much? Con- temp. Admixture proportion of pop. hatchery strain: 0.06 (95% CI 0.00 – 0.24)
Genetic interactions between stocked and wild brown trout ( Salmo trutta ) – experiences from Denmark • Calculation of expected admixture proportion based on – estimates of natural reproduction – number of stocked trout - hatchery strain and supportive breeding – assuming equal fitness of indigenous and hatchery trout Observed Expected Hatchery Wild Hatchery Wild Admixture proportion of Expected admixture proportion of hatchery strain: 0.06 hatchery strain: 0.62
Genetic interactions between stocked and wild brown trout ( Salmo trutta ) – experiences from Denmark � Several other studies conducted (Hansen et al. 2000; 2001; 2006; Ruzzante et al. 2001; 2004) � Karup River results representative for most Danish rivers � In the best case stocking with hatchery strain trout is waste of money, in the worst case detrimental • Stocking with hatchery strain trout phased out in Denmark since 2005 • Only supportive breeding of local populations allowed • Most emphasis on habitat restoration
Local adaptation: juvenile life history traits in brown trout (Jensen, Hansen, Pertoldi, Holdensgaard, Mensberg & Loeschcke, in prep.) • Four Danish trout populations within a radius of 40 km • Lake Hald: Lake-dwelling – spawns in tributaries fed by ground- water. 6-8 degrees C during incubation • Norring Møllebæk: Resident – typically low temperatures during incubation, 2-5 degrees C • Lilleå River: Anadromous – typically low temperatures during incubation, 2-5 degrees C • Karup River: Anadromous – varying temperature regimes throughout the river system
Local adaptation.... • 14-21 families per population • Three batches of each family – incubated at 2, 5 and 8 degrees C • Early life history traits – Incubation time – Alevin length – Yolk sac volume – Growth rate – Length at swim-up • Does local adaptation exist for these traits? • 10 neutral microsatellite loci
Local adaptation.... • F ST – measure of genetic differentiation at molecular markers • Q ST – measure of genetic differentiation at quantitative traits • Q ST > F ST = directional selection (Merilä & Crnokrak, 2001) • Selection acts at alevin length and swim-up length Microsatellite F st 95% CI Incubation tim e Alevin length * Yolk-sac volum e * Swim -up length (very low power) G rowth rate 0 0.25 0.5 0.75 1 F st or Q st
Local adaptation.... • Temperature reaction norms (adjusted for egg size) • ”Cold” populations Lilleaa R. and Norring M. larger alevin and swim-up length at 5 degrees, smaller at 8 degrees • ”Warm” Lake Hald population performs well at 8 degrees • Local adaptation • Global warming? • Adaptation to current temperature regimes may be maladaptive if winter temperatures increase • Adaptation to increasing temperatures may occur if changes do not occur too fast Swim-up length Alevin Length Lilleaa R. 2.8 2 Lilleaa R. 2.7 1.9 2.6 1.8 2.5 1.7 Lake Hald 2.4 Lake Hald 1.6 2.3 1.5 Norring M. 2.2 Norring M. 1.4 2.1 1.3 2 0 2 4 6 8 0 2 4 6 8 Temperature Temperature
European eel ( Anguilla anguilla ) – one or several populations? (Hansen, Als, Bernatchez, Maes et al., preliminary results) • Both European ( A. anguilla ) and American ( A. rostrata ) eel spawn in the Sargasso Sea • Severe decline of European eel, IUCN Appendix II • Important question for management: is eel panmictic or do several genetically distinct populations exist? (Schmidt, 1922) ? OR
European eel ( Anguilla anguilla ) – one or several populations? • Classic textbook example of panmixia, but... Wirth & Bernatchez (2001) Nature, 409, 1037-1040 • Eels from nearly all the distributional range in Europe and North Africa, 7 microsatellite loci • Very low, but significant genetic differentiation (F ST = 0.0017, P = 0.0014) • Significant isolation by distance – conflicts with panmixia? • Dannewitz et al. (2005) Proc. Roy. Soc. Lond. B., 272, 1129–1137: It is all temporal variation!
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