Ecosystems &Biodiversity 1

Measuring Genetic Diversity

Practical 6 Measuring Genetic Diversity Reading

Burgman, M.A. and Lindenmayer, D.B. (1998). Conservation Biology for the Australian Environment. Surrey Beatty & Sons, Sydney. Chapter 6 Loss of genetic diversity, populations and species. Eldridge M. (1998). Trouble in Paradise? Nature Australia 26, 24-31. Van Dyke F. ( 2003) Conservation Biology. McGraw-Hill. Chapter 6 Conservation of genetic diversity.

Introduction
If you look at any group of of individuals from the same species humans, cats, dogs you will quickly see that they are no all identical. Chromosomes are made up of a collection of genes, and the external expression of genes (the individuals phenotype) can vary considerably. This is because genes can have alternative forms called alleles, and the observed differences in appearance are caused by variations in which alleles are present in the genetic make-up an individuals. The gene pool of a species is the whole collection of all the possible alleles of all the genes in the species. Conservation ecologists cannot maintain all the genetic diversity in species, but they do try to maintain the genetic diversity found in individuals in local populations of plants and animals. For example, if the population of an orchid species in one valley has evolved adaptations that are different from the population of the same orchid species in the next valley, it is desirable to keep both populations to maintain the full evolutionary potential of the species. But, if both populations are genetically identical then conserving just one population will capture all the gene pool of the species. However, there are also problems which must be dealt with if only one population is conserved!

Ecosystems &Biodiversity 2

Measuring Genetic Diversity

Why does it matter ? Well, when collecting seed, or tissue samples, or animals for reintroduction programs, or animals for a zoo, how representative are these samples of the genetic variation of the whole species? What are the implications of using only a small proportion of the available genetic material? These founder populations may lead to bottlenecks, genetic drift, loss of fitness, and may limit the ability of a population to adapt to changes in its environment, and therefor to persist. Of course, to make the decisions about how much variation we need to conserve for the best chance of long term species survival, we must first be able to measure the genetic diversity within populations and secondly, determine how many populations we need to conserve. This practical is based on Gibbs et al. (1998). Problem-solving in Conservation Biology and Wildlife Management. Blackwell Science.

The task
Work slowly through the calculations for this task. You DO NOT need to remember how to carry out these calculations - the task aims to demonstrate one of the genetic tools available to conservationists to help make objective decisions about the genetic base of the populations they must manage. You are working for a conservation agency and are faced with a crisis decision: a piece of land with six different wetlands hosting the only known populations of two rare orchids is about to be developed as an industrial site. Your organization has only have enough funds to purchase and protect four of these wetlands. Which of the six populations should you protect ? The wetlands are of two types. Three are marshes with the worlds only known populations of Pterostylis isozymus. The other three are swamps where the only populations of the closely related but distinct species Pterostylis polyzymous lives. You send some leaf samples off to a colleague for genetic analysis, and receive back the data below, which are in the form of protein electrophoresis gels for one allozyme locus that is polymorphic in both these species. The locus has two alleles, Fast and Slow (because they move at

Ecosystems &Biodiversity 3

Measuring Genetic Diversity

different rates through the gel), that appear on the gel with the faster allele below the slower allele.

Divide into groups of two or three, and carry out the genetic assessment together, by following the four steps and filling in the worksheets as you go.

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). Read the page explaining gel electrophoresis and look at some of the web sites they have good explanations

Ecosystems &Biodiversity 4

Measuring Genetic Diversity

Ecosystems &Biodiversity 5

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

Ecosystems &Biodiversity 6

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 p

population 1 population 2 population 3 7/30 = 0.23

Slow allele
23/30 =

q
0.77

=2xpxq
2 x 0.23 x 0.77 = 0.35

average = (Hs) =

1.06/3 = 1.06/3 =

Allele frequencies for Pterostylis polyzymus Fast allele p

population 1 population 2 population 3

Slow allele

=2xpxq

average = (Hs) =

Ecosystems &Biodiversity 7

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 p

population 1 population 2 population 3 average allele 7/30 = 0.23 0.60 0.87 1.70/3

Slow allele
23/30 =

q
0.77

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

Expected heterozygosity (Ht) for Pterostylis polyzymus Fast allele p

population 1 population 2 population 3 average allele frequency (Ht).= 2 x the average p x the average q =

Slow allele

Ecosystems &Biodiversity 8

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.

Summary 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

Pterostylis polyzymus Fst = (Ht - Hs) / Ht = Fst _______ and indicates no/some/great divergence between populations of
P..polyzymous Populations of Pterostylis ____________ are more divergent than populations of

Pterostylis_____________. That is, the populations of Pterostylis___________,

are genetically most similar to each other.

Ecosystems &Biodiversity 9

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). Include the final summary section of Fst, and 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 choose to use animals from just one of the islands ?

Due Date Check the Assessment Details File for the date. This report is worth 10% of your total mark.

Ecosystems &Biodiversity

Measuring Genetic Diversity

10

gel electrophoresis
http://web.utk.edu/~khughes/GEL/index.htm http://www.bergen.org/AAST/Projects/Gel/index.html http://www.life.uiuc.edu/molbio/geldigest/electro.html

Gel electrophoresis makes it possible to determine the genetic difference and the evolutionary relationship among species of plants and animals. Using this technology it is possible to separate and identify protein molecules that differ by as little as a single amino acid. Because these differences are determined by genes, the differences in molecules indicates a difference in genetic make-up. Gel electrophoresis is a method that separates macromolecules-either nucleic acids or proteins-on the basis of size, electric charge, and other physical properties. The gel is a jelly-like substance, usually agrose, a substance derived from seaweed. The molecules have an electrical charge, and electrodes at either end of the gel provide the driving force. The gel acts like a sieve. Large molecules have difficulty getting through the holes in the gel, but small molecules move through easily. Because of this, large molecules will move more slowly than the smaller, faster molecules. As the separation process continues, the separation between the larger and the smaller fragments increases.
Each lane is an individual Large DNA or protein molecules will be Slow Small DNA or proetin molecules will be Fast

Each vertical lane belongs to a single individual, and by comparing the positions of the molecules across lanes it is possible to determine if they have the same combinations of molecules that is, if they are genetically similar or genetically different.