Biodiversity & Ecosystems

Measuring Genetic Diversity

Practical 6 Measuring Genetic Diversity

Step 1 To measure how genetic variation is spread within and between populations
you first need to determine allele frequencies in each population. The particular allozyme locus examined has two alternate forms. The identity of the two alleles in each individual is reflected directly by the banding patterns within each lane on the gel. For example, the first individual in the first lane of the first gel is heterozygous, that is, the two alleles it has are different and are indicated (+) by both a Fast and a Slow moving band on the gel. In contrast, the individual in the second lane is homozygous, as indicated by having a single band representing two Slow alleles.

Determine the allele frequencies in each population for the Fast-moving allele (p)
and the Slow-moving allele (q) by counting the number of alleles for individuals in each population (remember that a homozygote has two alleles the same so you have to count the + twice, and the total number of alleles for the 15 individuals is 2 x 15 = 30). The, divide that by the total number of alleles present in the population (always equal to two times the number of individuals).

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Measuring Genetic Diversity

Pterostylis isozymus, Slow Fast 1 2 3 4

Population 1 (individual 1 to 15 from left to right) 5 6 7 8 9 10 11 12 13 14 15 total 23 7

Pterostylis isozymus, Slow Fast 1 2 3 4

Population 2 (individual 1 to 15 from left to right) 5 6 7 8 9 10 11 12 13 14 15 total 12 18

Pterostylis isozymus, 1 Slow Fast 2 3 4

Population 3 (individual 1 to 15 from left to right) 5 6 7 8 9 10 11 12 13 14 15 total 4 26

Pterostylis polyzymus, Slow Fast 1 2 3 4

Population 1 (individual 1 to 15 from left to right) 5 6 7 8 9 10 11 12 13 14 15 total 12 18

Pterostylis polyzymus, Slow Fast 1 2 3 4

Population 2 (individual 1 to 15 from left to right) 5 6 7 8 9 10 11 12 13 14 15 total 14 16

Pterostylis polyzymus, Slow Fast 1 2 3 4

Population 3 (individual 1 to 15 from left to right) 5 6 7 8 9 10 11 12 13 14 15 total 8 22

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Measuring Genetic Diversity

Step 2. Next, you need a measure of genetic difference between populations. A commonly
used measure is Wrights fixation index, or Fst, which ranges from 0, indicationg no difference between populations, upwards, indicating increasing difference. To determine Fst, you need to calculate the

expected heterozygosity for each species (Hs).

Do this by

multiplying 2pq for each population and then averaging these values over all three populations within each species.

Allele frequencies for Pterostylis isozymus Fast allele

population 1 population 2 population 3 7/30 = 18/30 26/30

p
0.23 0.60 0.87

Slow allele
23/30 = 12/30 4/30

q
0.77 0.40 0.13

=2xpxq
2 x 0.23 x 0.77 = 0.35 0.48 0.23 1.06/3 = 1.06/3 = 0.35

average = (Hs) =

Allele frequencies for Pterostylis polyzymus Fast allele

population 1 population 2 population 3 18/30 16/30 22/30

p
0.60 0.53 0.73

Slow allele
12/30 14/30 8/30

q
0.40 0.47 0.27

=2xpxq
0.48 0.50 0.39 1.37/3 1.37/3=0.46

average = (Hs) =

Hs is heterozygosity if populations were isolated ie. not interbreeding

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Measuring Genetic Diversity

Step 3. Now, calculate the expected heterozygosity if all three populations were part of the same, extended breeding population (Ht). Do this by averaging p and q
over all three populations within each species, and then multiplying 2 x the average p x

the average q This would be the expected frequency of heterozygotes in the population if it
acted as one large breeding pool with no genetic differences at the local population level.

Expected heterozygosity (Ht) for Pterostylis isozymus Fast allele

population 1 population 2 population 3 average allele frequency 7/30 = 18/30 26/30

p
0.23 0.60 0.87 1.70/3 =0.57

Slow allele
23/30 = 12/30 4/30

q
0.77 0.40 0.13 1.30/3

=0.43 (Ht).= 2 x the average p x the average q = 2 x 0.57 x 0.43 =0.49

Expected heterozygosity (Ht) for Pterostylis polyzymus Fast allele

population 1 population 2 population 3 average allele 18/30 16/30 22/30

p
0.60 0.53 0.73 1.86/3

Slow allele
12/30 14/30 8/30

q
0.40 0.47 0.27 1.14/3

frequency =0.62 =0.38 (Ht).= 2 x the average p x the average q = 2 x 0.62 x 0.38 = 0.46

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Measuring Genetic Diversity

Step 4. OK, now you need to calculate the amount of local, within-population variation!

Deviations of the frequency of heterozygotes in separate populations (Hs) from what you would expect to find if they were all part of the same larger population (Ht) provide an index of the amount of
genetic variation that is found only in local populations. Thus, Fst = (Ht - Hs) / Ht, where values of Fst < 0.01 indicate little divergence between populations, and values Fst > 0.1 indicate great divergence between populations (that is, the populations are genetically different from each other). Values in-between indicate some genetic divergence

Follow the examples provided, calculate the fixation index for each species, and then compare the indices between the two species.

Pterostylis isozymus Fst = (Ht - Hs) / Ht = 0.49 0.35 / 0.49 = 0.29 Fst > 0.1 and indicates great divergence between populations of P. isozymus

almost none
Pterostylis polyzymus Fst = (Ht - Hs) / Ht = 0.02 Fst <0.1 and indicates no/some/great divergence between populations of P.polyzymous
Populations of Pterostylis isozymousare more divergent than populations of Pterostylis

polyzymous. That is, the populations of Pterostylsi polyzymous, are genetically most
similar to each other.

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Measuring Genetic Diversity

Your Report
In your report you should outline the original problem, and discuss the following questions in an essay style report (about 600 words). Use your calculations to support your discussion. Make sure you correctly reference your discussion

Are the populations of each species different from each other ? Does one species have more between population diversity than the other? Which one ? How will you allocate your scarce funds for wetland acquisition ? Justify your decision in terms of preserving the maximum amount of genetic diversity that characterizes these two species.

What considerations other than genetic ones might influence your choice ? Recall that our goal in this exercise is to capture as much of the genetic diversity that characterizes these species as is possible, given a limited budget. Note that both alleles at the locus surveyed are already found in each population of both species. Why does it matter that more than a single population of each species be protected ?

In the reading by Eldridge(1998) Trouble in Paradise, how did the scientists maximize the genetic diversity in the re-introduced population? Why didnt they just choose to use animals from just one of the islands ?