Population structure in the great reed
warbler at microsatellites
Study species and key questions:
You will explore population genetic parameters using GenAlEx.
Aim: to evaluate deviations from Hardy-Weinberg, population structure and isolation-bydistance.
The great reed warbler breeds in marshes in Eurasia and overwinters in Africa. Two
subspecies are recognized. There are slight plumage and morphometric differences
between populations and subspecies. The species colonized Sweden in the 1950s and
the current population size is approximately 500 pairs. During the colonization process
the Swedish population went through a (moderate) bottleneck.
Figure 1. Great reed warbler, breeding range and study populations.
Analyse the data in GenAlEx
1. Open GenAlEx and if Excel asks about macro, choose “Enable”. Check that the
GenAlEx add-in is activated.
2. Open “Warbler Population Structure -Genalex.xlsx” through Excel (>Archive, >Open).
a. Check that you have one spreadsheet with the genetic data for each
population and one spreadsheet with geographical distances.
b. Check that your genetic data is organised according to the example below,
and that you understand how the data is organised:
i. How many populations are there?
_________
ii. How many loci are there?
_________
iii. What is the name of the 4th locus?
_________
iv. Number of samples in Hungary?
_________
v. How many samples are there in total?
_________
vi. Which cell contains the first allele?
_________
vii. What is the geographical distance between the populations from
Sweden and Kazakhstan?
_________
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Figure 2. Example of a GenAlEx spreadsheet.
3. Visualise the allele frequencies by using >Frequency and then e.g. the options >Graph
by locus and >Graph by Pop for each Locus.
Browse through the output. Can you from the graphs already now get a feeling for
which populations are genetically quite similar/dissimilar? _______________
4. Evaluate the amount of genetic variation in the populations as measured by
heterozygosity and number of alleles, etc. (>Frequency, Options: > >Het, Fstat & and
Poly by Pop; look in HFP):
Sample size
Mean number
of alleles (Na)
Observed
heterozygosity
(Ho)
Expected
heterozygosity
(He)
Spain
Sweden
Latvia
Germany
Hungary
Belarus
Ukraine
Kazakhstan
Average
Which populations seem to contain particular low/high amount of genetic variation?
______________________________________________________________
5. Calculate the pairwise FST-values between populations using the AMOVA approach,
and also perform a permutation test to test whether the FST -values may differ from
zero (>AMOVA, Option A: >Codom-Allelic, >Analysis for Total Only, >Output As Tri Matrix,
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Option B: >Pie Graph, >Standard Permute, >999 permutations, >Output for Total Only,
>Output Pairwise Fst Matrix, >999 permutations).
a. Check results for the Global calculations (Fst):
What is the global FST-value in the data set?
What is the P-value for the FST-value?
What is the global FIS-value in the data set?
What is the P-value for the FIS-value?
________
________
________
________
b. Enter the pairwise FST-values and P-values between populations in the table
below (FstP or perhaps FstP (2))
Spain
Sweden
Latvia
Germany
Hungary
Belarus
Ukraine
Kazakhstan
Spa
*
Swe
Lat
Ger
Hun
Bel
Ukr
Kaz
*
*
*
*
*
*
*
Note. The Fst-values with the ‘frequency option’ and the ‘AMOVA option’ may differ somewhat
because they are calculated in slightly different ways.
6. Evaluate whether there is any Isolation-by-Distance in great reed warblers by
correlating genetic and geographical distances and testing the significance of the
relationship with a Mantel test (go to the spreadsheet called “Geographical
Distance”, choose >Mantel, Options: >Tri Distance Matrix, >999 permutations, >XY
Graph., >Y Matrix: FstP or FstP (2)). Check the results (MT) and make a rough plot
below:
Scatterplot: pairwise FST against geographical distance
Is this relationship statistically significant?
This exercise was put together by Bengt Hansson (March 19, 2014).
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p=
_____________