The use of molecular markers for preserving genetic resources in - - PowerPoint PPT Presentation

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The use of molecular markers for preserving genetic resources in wild fish populations

Michael M. Hansen

Technical University of Denmark

National Institute of Aquatic Resources

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

• Question: Risk of inbreeding and

loss of variation?

• Effective population size, Ne –

measure of how much inbreeding and loss of variation that will take place in a given assemblage of individuals

• Depends on sex ratio, variance in

reproductive success, temporal fluctuations of Ne

Germany The Netherlands Denmark

N

UK Norway Sweden North Sea Vidaa R. Longitude Latitude 0 o 10 o 54 o 60 o

100 km

How high is the effective population size in Vidå North Sea houting?

Temporal method – estimation of Ne from genetic drift - random genetic changes - that has occurred over time (method by Beaumont (2003) used)

• Twelve microsatellite DNA loci analysed in samples from

1980, 1994 and 2002

Sample ⇒ Time 1 . . . Sample ⇒ Time 2

• 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

How high is the effective population size in Vidå North Sea houting?

• The method allows for

estimating effective population size at the start (1980) and end (2002) of the time interval

• Wide confidence intervals
• No evidence for expansion
• r decline
• If anything, effective

population size appears stable

Is the population stable, increasing or declining?

1980 2002

Mode

Expansion Decline

• Simulated bottleneck by

”allowing” only 25 males and 25 females to reproduce in 2002

• 50 offspring of the simulated

bottleneck ”sampled” in 2006

• Strong signal of population

decline in simulated sample

• Useful for routine screening
• f populations
• Can reveals strong

population declines with minimal sampling effort

Can this method be used for routine monitoring of population sizes?

Expansion Decline Mode 1980 2006

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)

Wild (1950) Hat. strain Con- temp. pop.

How much?

Wild Hatchery

Observed

Admixture proportion of 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

Wild Hatchery

Observed

Admixture proportion of hatchery strain: 0.06

Expected

Wild Hatchery

Expected admixture proportion of 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

0.25 0.5 0.75 1 G rowth rate Swim

• up length

Yolk-sac volum e Alevin length Incubation tim e F st F st or Q st

Local adaptation....

• FST – measure of genetic differentiation at molecular markers
• QST – measure of genetic differentiation at quantitative traits
• QST > FST = directional selection (Merilä & Crnokrak, 2001)
• Selection acts at alevin length and swim-up length

* *

95% CI

(very low power)

Microsatellite

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

Alevin Length

1.3 1.4 1.5 1.6 1.7 1.8 1.9 2

2 4 6 8 Temperature

Swim-up length

2 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8

2 4 6 8 Temperature

Lake Hald Lake Hald Norring M. Norring M. Lilleaa R. Lilleaa R.

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 ?

• Classic textbook example of

panmixia, but...

• Eels from nearly all the

distributional range in Europe and North Africa, 7 microsatellite loci

• Very low, but significant genetic

differentiation (FST = 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!

Wirth & Bernatchez (2001) Nature, 409, 1037-1040

European eel (Anguilla anguilla) – one or several populations?

• The problem is that eels were not sampled at the spawning places in the

Sargasso Sea, but 5,000 km away in the foraging areas in Europe

• The Danish Galathea 3 Expedition: Samples of eel larvae at the

spawning places in the Sargasso Sea in March-April 2007

• Eels spawn in thermal fronts

European eel (Anguilla anguilla) – one or several populations?

• Species ID (European or American eel, or different Anguilliformes)

European eel (Anguilla anguilla) – one or several populations?

Technical University of Denmark

National Institute of Aquatic Resources

THANKS TO:

• The organizers for inviting me
• Collaborators:

– Dorte Bekkevold – Lasse Fast Jensen – Karen-Lise Mensberg – Volker Loeschcke – Cino Pertoldi – Louis Bernatchez, Greg Maes, Henrik Sparholt, Kim Aarestrup, Peter Munk and all other participants in the Galathea 3 Eel Project

• Funding:

– The Villum Kann Rasmussen Foundation – The Danish Natural Science Research Council – The Carlsberg Foundation – The Rod License Funds