Edexcel A2 Bio

Appendix 2: Handling data for
A2 Biology
I by Neil C Millar
THE ISSUES
I Sampling and accuracy

I Data analysis software
I Types of data
I Handling very large and very small numbers
I Using software for data analysis
I Summarising data
I Displaying data
I Ecology statistics
I Making conclusions from data
I Comparative statistics
I Association statistics
I Statistics for nominal data
I Choosing a test
I Summary the six steps in data analysis
I Sampling and accuracy

In biology investigations, we are trying to find out something about the natural world, such as
how nitrates affect plant growth, or whether there is a link between atmospheric pollution and
species diversity, or if a drug reduces pain. To find out, we make measurements or observations,
but were never going to be able to observe every single plant or every species or every single
human. Instead, we make our measurements on a small sample of the total population, and hope
that our conclusion from the sample can be applied to the whole population.
How big should the sample be? Obviously a single measurement on one organism is not
enough since the organism could by chance have unusual characteristics, or the measurement
could simply go wrong. So we need several replicates (repeated measurements). Bigger samples
(more replicates) generally give more reliable conclusions, but too many replicates make an
investigation unwieldy and difficult to carry out. Indeed, one of the purposes of proper statistical
analysis is to generate as much reliable information as possible from as little data as possible. A
good rule of thumb is that for a reliable statistical analysis we should aim for at least 10 replicates
of each measurement. If its easy to do more, then do so, and if 10 is difficult, then smaller
samples are sometimes acceptable, but the bigger the sample the more reliable the conclusion.
With investigations on living organisms, choosing the sample is also important. Since no two
organisms are exactly alike, it is important to choose a random sample to even out individual
differences. If possible, individuals should also be chosen so that they are similar for example,
the same age, same sex, similar body mass, and so on. If the sample is random and big enough
then any measurements and conclusion we make about the sample should apply to the whole
population.
The aim is for our measurements to be accurate that is, close to the true value, which is the
value of a quantity if it could be measured perfectly, with no error. In biology, all measurements
have some error so we can never know a true value for certain, but we can try to get close to it
by minimising errors. There are two types of error.
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Random errors are due to mistakes, inaccuracies or just natural variation. Random errors are
always present, but can be minimised by taking many replicates. If the replicates of a
measurement are close together (so they have a small range) then the random error is small
and the measurements are said to be precise.
Systematic errors are errors in one direction only due to poor technique, or faulty or poorly
calibrated equipment (for example, a thermometer that always reads 1 C too high).
Systematic errors cannot be improved by taking more replicates, and can only be mitigated by
careful preparation and by using properly calibrated measuring devices. If similar results are
Edexcel Biology for A2 Dynamic Learning
Hodder Education 2009
APPENDIX 2: HANDLING DATA FOR A2 BIOLOGY
obtained independently by other techniques or by other people then the systematic error is
small and the results are said to be reliable.
Data that are both precise and reliable are presumed to be accurate.
I Types of data
The measurements and observations from an investigation are called data (singular: datum).
Data can be quantitative or qualitative.
Quantitative data comprise any measurements in numeric form. This is the most common
kind of scientific data, and includes measurements of quantities like length, mass, time,
temperature, pH, concentration or speed, as well as counts of things like legs, cells, species or
organisms. Quantitative data can be either continuous data (which can take any value), or
discontinuous or discrete data (which can only be whole numbers). In practice, the distinction
between continuous and discontinuous is rarely important, as all measurements are really
discontinuous at some level (for example, a length might be measured to the nearest whole mm
or m), and the same statistics and analyses can be applied to both.
Quantitative data can also be classified as either normally distributed or non-normally
distributed data. This distinction is more important since different techniques are used to
analyse data in the two groups. The majority of measurements in biology are normally distributed
(for example, height, mass, temperature, breathing rates, growth rate, blood cell counts, and so
on), and give the familiar symmetrical bell-shaped curve of the normal distribution when
replicates are plotted in a histogram (see Figure A2.12). But some kinds of data, such as arbitrary
scales (like 15) and calculated data (like IQ scores or diversity indices), are unlikely to be
normally distributed, and sometimes even simple measurements can deviate from a normal
distribution curve. These non-normal data must be treated as ordinal data, where only the ranks
(or order) of the values matter. While normally distributed data can be analysed using powerful
parametric tests, ordinal data must be analysed using the less-powerful non-parametric statistics.
How do you know whether your data are normally distributed or not? In practice, it often doesnt
matter very much, and it is usually safe to assume that your measurements are normally
distributed, unless youve been told otherwise. There are tests for normality, but they require a
large number of replicates and are not particularly reliable.
Qualitative (or categoric) data comprise observations using words rather than numbers.
Sometimes the words can be placed in rank order for example, observations such as
small/medium/large, the pain scores used by doctors and the ACFOR abundance scale used by
ecologists. These categories can therefore be assigned numerical ranks, and treated as ordinal
data, analysed by non-parametric tests. Categoric data that cannot be ranked are called nominal
data (for example, colours, shapes, species). Its a little surprising that we can do statistics at all
on such nominal data, but if a very large number of observations are made then the number of
observations of each category can be counted to give frequencies, and special frequency tests can
be used. Frequencies are often just called counts of things, but this can be very misleading as
counts are quantitative data, not qualitative.
The different kinds of data are summarised in Figure A2.1.
Figure A2.1
Different kinds of data.
data
quantitative data
(numerical)
normally
distributed
qualitative data
(categorical)
non-normally
distributed,
or ordinal
can be
ranked
cannot be
ranked
normally distributed data
ordinal data
nominal data
use parametric tests
use non-parametric tests
use frequency tests
I Handling very large and very small numbers

Sometimes in science we have to deal with very large or very small numbers. There are two
ways to deal with them.
1 Use prefixes. All SI units can take these prefixes in front of them to make them smaller or
larger:
103, milli, m
106, micro,
109, nano, n
1012, pico, p
103, kilo, k
106, mega, M
109, giga, G
1012, tera, T
The prefixes increase or decrease by factors of a thousand, so by choosing the right prefix, all
values can be in the range 1999. For example:
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10 mm instead of 0.01 m
2.56 MPa instead of 2 560 000 Pa
75 l instead of 0.075 ml
2 Use standard notation (or scientific notation) for example, 3.2 106 cells ml1. This is
useful for doing maths on large or small numbers.
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To multiply numbers together you add the powers for example, 103 106 109.
To divide numbers you subtract the powers for example, 103 106 103.
You should be able to convert between standard notation and prefix forms for example,
4 108 m 40 nm.
Notes on using units and prefixes

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Try to avoid centi (c, 102) or deci (d, 101) for example, cm, dm. Theyre not factors of a
thousand and cause confusion.
Names of units are always spelt with a small letter, even if theyre named after scientists (for
example, joule).
Symbols do not need a full stop (like an abbreviation) or an s (like a plural) for example,
3 min not 3 mins.
There should be a space between the value and its symbol (for example, 6 g not 6g).
There is no space between a prefix and a symbol (for example, 7 mN not 7 m N).
Use a space to indicate thousands, not a comma (for example, 72 000 not 72,000).
Use the index 1 for division in units, not a slash (for example, kJ m2 y1 not kJ/m2/y).
I Using software for data analysis

Until recently all data analysis and statistical tests had to be carried out by hand using a
calculator. But these days computer software can be used to carry out data analysis, more
quickly and more accurately than by hand. A search of the internet will reveal a large range of
statistical software available for biologists to use, from big professional packages like SPSS or
Minitab, to completely free software, and many of these will be able to do the analysis described
here. Many biologists also use Microsoft Excel for data analysis, which is an excellent generalpurpose spreadsheet, but lacks a number of useful statistical tests and charts.
Merlin is an add-in for Excel, which allows Excel to do most of the statistical tests and charts
a biology student might want to use. Functions like MEAN and ANOVA are simply typed into
cells like any other Excel function. There are no calculations and no look-up tables, so
hopefully no mistakes! Merlin can also be used for results tables and charts, which can be
plotted quickly, accurately and neatly. Merlin also includes a test chooser, which suggests the
correct test to apply following a series of simple questions. The Merlin package comes with an
introduction to statistics for biology students, and worksheets with practice questions. Merlin is
completely free, and is available from:
www.heckgrammar.kirklees.sch.uk/index.php?p10310
If this URL should fail, try a search for merlinstatistics.
I Summarising data
Having collected experimental data with a suitable number of replicates, the next step is to
summarise the replicate data with one or two values, which we can plot on a chart. In general,
we want the centre point of the replicates and an indication of the spread of the replicates (also
known as the error) around the centre. Although some of these summary values can be
calculated by hand, these days biologists usually use computer software for all their calculations.
The mean, standard deviation and confidence interval

Imagine an investigation into the effectiveness of a new fertiliser on the growth of potatoes. 20 equalsized plots are set up in a field with 10 similar potato plants growing in each of them. Ten plots are
chosen at random to be treated with the old fertiliser (A), while the other 10 plots are treated with
an equal concentration of the new fertiliser (B). So there are 10 replicates for each fertiliser. After a
fixed length of time the potato crop is harvested and the total mass of potatoes from each plot (the
yield) is measured. The yields for the 20 plots are shown in this Excel results table (Figure A2.2).
Figure A2.2
We need to summarise all these data. We can assume that the yields are normally distributed, so
the central value of the replicates is given by the mean (also called the arithmetic mean, or
average). To calculate the mean for fertiliser A using Merlin (see page 4), cell B13 contains the
formula MEAN(B3:B12). We really need to know how reliable this calculated mean is, so we
must also calculate a measure of the error of the mean. The most common measures of spread or
error are the standard deviation (SD) and the 95% confidence interval (CI). Both values are
calculated in this example: cell B14 contains the formula STDEV(B3:B12) and cell B15
contains the formula CI(B3:B12). These formulae are all dragged across to column C to
quickly make the same calculations for fertiliser B. The SD indicates the spread or scatter of the
replicates, while the CI indicates how accurate the calculated mean is. The CI has a simple
meaning: we can be 95% confident that the range mean CI encloses the true mean.
The confidence interval illustrates an important point about sampling. It is tempting to say
that the true mean yield for fertiliser A was 20.2 kg, but this would be wrong because weve only
measured a fairly small sample of 10 plots, not every potato. Instead we should say that, from our
sample, we are confident that the true mean yield for fertiliser A lies somewhere in the range
17.9 kg (20.2 2.3) to 22.5 kg (20.2 2.3). These values are called the lower confidence limit
and the upper confidence limit respectively. The error values are often quoted together with the
calculated mean, so we might say that the yield with fertiliser A was 20.2 2.3 kg. It is good
practice to calculate a confidence interval whenever a mean is calculated.
Error bars
Error values should also be shown on charts as error bars, to give a visual indication of the error
of the mean. There is no standard for what error bars should be, so the figure legend should state
whether the error bars are SD or CI (or something else). Again, the most useful is probably the
CI, since it shows graphically the likely range of the true mean. In Figure A2.2, Merlin has been
used to plot the mean yields on a bar chart, with the confidence intervals shown as error bars.
This makes the differences between the fertilisers easy to see. Although the bar for fertiliser B is
taller than that for A, the error bars show that there is a good deal of uncertainty in both means.
Since the confidence intervals overlap, we cant be sure that fertiliser B is significantly better
than fertiliser A. In the A2 Biology course, we shall learn about statistical tests that can properly
test for significant differences like this, but in the meantime, the confidence intervals give us a
pretty good idea.
The error bars can also be used to evaluate a conclusion, as this next example shows. It
concerns an investigation into the effect of light intensity on the rate of photosynthesis. Four
replicate measurements were taken at four different light intensities, and the means and 95%
confidence intervals were calculated using Merlin. A line graph is drawn of the mean rates
against light intensity, with error bars showing the 95% CI of the means (Figure A2.3). This
graph needs a smooth line of best fit, since there must be a smooth continuous relation between
light intensity and rate of photosynthesis. Now, we are confident that the true means lie
somewhere within the 95% CI error bars, so any line of best fit must pass through the errors bars.
In this case a curved line seems to fit the mean values best (solid line), but can we draw a
different line through the error bars? In fact, a straight line can also be drawn through the error
bars (broken line), so a linear relationship cant be ruled out.
Figure A2.3
The median
This next example shows an investigation into the abundance of brown algae on two rocky
shores. Eight quadrats were randomly placed on a sheltered shore and eight on an exposed shore,
and the abundance of algae in each quadrat was recorded. To make the data quicker and easier to
collect, abundance is measured on a five-point scale, with 5 being the most abundant. This kind
of scale is an example of ordinal data, because its not an even scale (for example, 4 is not
necessarily twice as abundant as 2). With ordinal data, its meaningless to calculate a mean or a
CI, and instead the best measure of the central value is the median, which is the value with
equal numbers of replicates above and below it. The spread of the data is given by the five-point
summary, which divides the data into four quartiles, each containing 25% of the data. Half of
the data points are located between the first and third quartiles, and this range (known as the
interquartile range) can be used as a measure of the spread.
Figure A2.4
In the results table for the rocky shore investigation, the median is calculated with the Excel
formula MEDIAN(B3:B10) in cell B11, and the interquartile range is calculated using the
Excel formula QUARTILE(B2:B10,3)- QUARTILE(B2:B10,1) in cell B12. These formulae
were then dragged across to column C to instantly make the same calculations for the sheltered
shore (Figure A2.4).
Figure A2.5
A box plot.
4th quartile (maximum)
3rd quartile
2nd quartile (median)
1st quartile
0th quartile (minimum)
The five-point summary is shown graphically as a box plot (or box and whisker plot, Figure
A2.5). This shows the median as a central line, the interquartile range as a box, and the
maximum and minimum values as whiskers. Unlike confidence intervals of the mean, the box
plot need not be symmetrical, and indeed an asymmetrical box plot is a good indicator that the
replicates are not normally distributed. A box plot for the rocky shore investigation has been
drawn using Merlin (see page 4) in Figure A2.4.
The mode
A third possible measure of the centre of a set of data is the mode, which is the most frequent
replicate value. There can be more than one mode, if more than one value is equally frequent.
The mode is rarely used in biology, except to indicate a bimodal distribution (one with two
modes or peaks).
I Displaying data
Almost all experimental data benefit from being displayed as some kind of chart. Humans are
visual animals, and we are more likely to understand a well-chosen chart than a table of
numbers. The choice of a suitable chart depends on the type of data and the type of
investigation.
Scatter graphs and line graphs plot two numeric variables against each other, allowing trends
between them to be observed. These graphs can be used for any kind of numeric data
continuous, discrete, normally distributed or ordinal. Note that the term line graph in business
and spreadsheet terminology refers to a different kind of graph where a variable is plotted at
equal intervals (usually in a time series).
When both variables are being measured, and you dont yet know whether there is a relation
between them, plot a scatter graph (or scattergram). In Figure A2.6, mass of seeds and number of
seeds produced were both measured for a number of plants to see if there was a link (or
correlation) between them. Neither variable depends on the other, so there is no independent or
dependent variable. The axes could be plotted either way round and no line is drawn.
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mean mass per seed/mg
Figure A2.6
A scatter graph.
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12
10
8
6
4
2
0
0
10
20
30
40
50
number of seeds
When you know (or suspect) that one variable affects the other one, then plot a line graph. You
usually perform a controlled experiment, varying one variable (the independent variable) while
measuring the other (the dependent variable). Now the orientation does matter: the
independent variable is always plotted on the horizontal (x) axis, while the dependent variable is
on the vertical (y) axis. Figure A2.7 shows the results of an enzyme investigation in which the
substrate concentration (independent variable) was varied while the rate of reaction (dependent
variable) was measured. Here we know that there is a continuous underlying relationship
between the independent and dependent variables, so we plot a smooth line of best fit through
the data (in this case a curved line).
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rate of reaction/units
Figure A2.7
A graph with a line of
best fit.
7
6
5
4
3
2
1
0
1
10
substrate concentration/mM
If were not sure that there is a smooth relation, and so the intermediate values between the
measured points are uncertain, we cant draw a smooth line of best fit. Instead, we join the points
with straight lines just to indicate that the points are linked in order, but were not making any
assumptions about intermediate values. In the example in Figure A2.8, a patients temperature
was measured every two hours throughout a day. The temperature could vary considerably
between measurements. Sometimes the points can be omitted completely, giving just the jagged
line.
Figure A2.8
A graph with no line of
best fit.
39.5
temperature/C
39.0
38.5
38.0
37.5
37.0
36.5
00:00
22:00
20:00
18:00
16:00
14:00
12:00
10:00
08:00
06:00
04:00
02:00
00:00
36.0
time of day
Bar charts plot a numeric dependent variable against a categoric independent variable. Bar
charts allow differences between the categories to be observed. The bars may be horizontal or
vertical (in which case it is sometimes called a column chart). There should be a gap between
the bars, and the bars can be in any order, though sometimes it can help understanding if the
bars are arranged in size order. In Figure A2.9, the first example is a column chart, with the
dependent variable plotted on the vertical axis. In the second example the categories have long
names, so it is easier to read when plotted with horizontal bars.
Figure A2.9
Vertical and horizontal
bar charts.
swamp and marsh

tropical rain forest
temperate forest
yield of potatoes/kg
25
agricultural land
20
coniferous forest
15
temperate grassland
10
tundra
5
desert
0
A
10
20
30
40
50
60
70
2 1
fertiliser
net primary productivity/MJ m
Box plots are used as an alternative to bar charts where you want to show the range of replicates,
or the five-point summary (see page 7). They are especially appropriate when the dependent
variable is not normally distributed. Like bar charts, box plots can be plotted horizontally or
vertically. The two box plots in Figure A2.10 show pH measurements from eight samples of pond
water in four different ponds shown as vertical and horizontal box plots.
Figure A2.10
Vertical and horizontal
box plots.
8.0
pond 1
7.9
pH
pond 2
7.8
pond 3
7.7
pond 4
7.6
pond 1
pond 2
pond 3
pond 4
7.6
7.7
7.8
7.9
8.0
pH
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Mosiac charts plot two nominal variables against each other, allowing associations between
them to be observed. This is the equivalent of the scatter graph for numeric data. In the example
in Figure A2.11, a number of moth traps were set up in three different habitats, and the number
of moths trapped of three species was recorded. The width of each bar is proportional to the
frequency of that species, and the height of each row is proportional to the frequency of that
habitat. Large boxes indicate an association between the two categories, so in this case moth A
prefers woodland, moth B prefers fields and moth C prefers mountains.
Figure A2.11
Mosaic chart.
woodland
field
mountain
species A
species B
species C
Histograms are used to show the distribution of many replicate measurements of a single
numeric variable (continuous or discrete). Histograms look like bar charts but the variable in
question is plotted on the horizontal axis and the vertical axis always shows frequency (so
histograms are sometimes called frequency histograms).
If the variable is continuous it is divided into classes or bins, and the number of replicates in
each class (the frequency) is tallied. The class size must be chosen to optimise the number of bars
and their height. The histogram has no gaps between the bars to indicate the continuous scale,
and the labels denote the boundaries between each class. In the example in Figure A2.12, the
body temperature of 30 individuals was measured. Body temperature is a continuous variable, and
the range has been divided into six classes each 0.3 C wide.
Figure A2.12
Histogram involving a
continuous variable
body temperature.
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frequency
12
10
8
6
4
2
0
35.8
36.1
36.4
36.7
37.0
37.3
37.6
body temperature/C
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If the variable is discrete, the histogram looks more like a normal bar chart, with gaps between
the bars and labels in the middle of each bar. The example in Figure A2.13 shows the number of
worms found in 40 randomly placed quadrats (a discrete variable).
Figure A2.13
Histogram involving a
discrete variable
number of worms per
quadrat.
12
10
frequency
8
6
4
2
0
1
worms per quadrat
A histogram can also be plotted as a frequency polygon to show the shape of the distribution
more clearly. Instead of bars this has points joined by straight lines. Figure A2.14 shows the
worm distribution as a frequency polygon.
Figure A2.14
Histogram plotted as a
frequency polygon.
12
10
frequency
8
6
4
2
0
1
worms per quadrat
Pie charts can be used to show the distribution of many replicate measurements of a single
categoric variable for example, the frequencies of different phenotypes in a genetic cross,
shown in Figure A2.15. The size of each segment is proportional to its frequency. Pie charts are
not very common in biology, although they are common in business.
Figure A2.15
Pie chart.
red
white
pink
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Kite charts (or kite diagrams) are used to display the abundance of different species along a
transect. Species abundances are shown as kites, with the width of the kites being proportional
to the abundance. Kite charts are often combined with line graphs and bar charts to display
abiotic data along the transect as well. A good kite diagram makes it easy to identify trends along
a transect and perhaps suggest which species are competing, or which species are adapted to
certain abiotic factors. The abundances can be measured on any scale, such as number of
individuals, percentage cover, or a 15 scale such as the ACFOR scale (Abundant, Common,
Frequent, Occasional, Rare). Its often helpful to arrange the order so that species abundant at
the start of the transect are plotted first and species abundant at the end of the transect are
plotted last, and some software can do this automatically. The kite graph in Figure A2.16 shows
the distribution of various marine animals and plants on a transect down a rocky shore.
Figure A2.16
Kite chart.
gut weed
bladder wrack
spiral wrack
coral weed
barnacles
ceramium
limpets
Irish moss
saw wrack
mussels
0
12 15 18 21 24 27 30
distance/m
I Ecology statistics
This section describes two special statistical techniques used by ecologists: estimating the size of
a population of animals, and measuring the diversity of a community.
Estimating populations
One of the most common measurements in ecology is measuring the population of a certain
species in a habitat. If the organisms are sessile (they dont move) then populations can easily be
measured by counting the individuals in a number of randomly placed quadrats and scaling up to
the whole area, but if the organisms are mobile the measurement is much more difficult. Several
techniques are available, but the simplest is the markreleaserecapture technique. The
population estimate using this technique is known as the Lincoln-Petersen Index.
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The basic procedure is very simple: a number of animals are captured using a suitable random
technique (nets or traps, for example), marked, released and some time later a second sample is
captured. The Lincoln-Petersen Index is:
N
MC
R
where:
N estimate of the population (the Lincoln-Petersen Index)
M number of individuals captured and marked in the first sample
C number of individuals captured in the second sample
R number of marked individuals recaptured in the second sample.
In fact, this simple formula is biased and tends to produce an overestimate of the true population,
especially for small samples, and a slightly different version is used by some software, including Merlin.
There are several assumptions in the markreleaserecapture technique if it is to be at all
accurate:
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the population is closed that is, there is no change in population between the first and
second sampling
the trapping is random that is, all animals have an equal chance of being caught
the marks must last until the second sampling, and must not affect the animals catchability
or survival
the marked animals must be randomly distributed in the population by the time of the second
sampling.
In practice, this means that the timing between the two samples is quite critical: long enough for
the marked animals to mix, but not long enough for the population to change or the marks to
come off. Suitable marks include rings on birds, tags on fish and mammals, and spots of paint on
invertebrates (marks with UV security pens are very good since they are invisible to predators
and investigators alike but show up under ultraviolet light).
Even if all the criteria are met, the Lincoln-Petersen Index is still a very inaccurate estimate of
the true population, so it is essential that an error value is also calculated, such as the 95%
confidence interval, which is analogous to the confidence interval of the mean. There is a 95%
chance that the true population size lies between the lower and upper confidence limits, where
the lower confidence limit index 95%CI and the upper confidence limit index
95%CI. It can be quite alarming to see just how wide the confidence range is!
Figure A2.17
Results of a mark
capturerecapture study.
The example in Figure A2.17 shows the number of woodlice captured in a markrelease
recapture study. The Merlin formula LINCOLN(B1:B3) is entered into cell B5 and the
formula LINCOLNCI(B1:B3) is entered into cell B6. This shows that the Lincoln-Petersen
population estimate is 217361 woodlice (289 72).
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Measuring diversity
An important and interesting property of an ecosystem is its species variety or diversity.
Communities with a large number of individuals of many different species are said to have high
diversity, while those with only small populations of a few species have a low diversity. The
concept of diversity is important to an understanding of habitats and conservation, and it can be
linked to stability and degree of human impact. There are several different measures of species
diversity, but one of the most common is the Simpson Diversity Index (D), which takes into
account both species richness and species evenness. The Simpson Index varies from 0 up to the
total number of species in the sample, with higher values indicating greater diversity.
Figure A2.18
Results of a study to
calculate Simpson
Diversity Index.
The example in Figure A2.18 shows the number of invertebrates captured in a three-minute
kick-sample in a stream. Site 1 is upstream of a small town and site 2 is downstream, below a
sewage outlet. Cell B17 contains the Excel formula SUM(B3:B16), cell B18 contains the
Excel formula COUNT(B3:B16), and cell B19 contains the Merlin formula
SIMPSON(B3:B16). These formulae were all dragged across to column C. In the example
here, site 1 has a higher diversity than site 2 by all the different measures.
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I Making conclusions from data

Having summarised experimental data and plotted a suitable chart, the next step is to make
conclusions from the data. Sometimes the data are so clear-cut that a firm conclusion can be
made easily without any further analysis. But often the conclusion is not obvious, and this is
where inferential statistics (or statistical tests) come in. They are called inferential because
they allow us to make inferences (or conclusions) from the data. Statisticians also call them
hypothesis-testing statistics because they test a mathematical statement called the null
hypothesis.
The null hypothesis is an important concept in data analysis and there is a specific null
hypothesis for each statistical test. In general, where you are comparing data the null hypothesis
states that there is no difference between the sets of data, and where you are looking for an
association it states that there is no association. A typical null hypothesis might state there is no
difference in plant heights in the two groups. Note that the word significant never appears in a
null hypothesis the null hypothesis is an absolute statement. Significance comes later in the
conclusion. Also, the null hypothesis has nothing to do with any scientific hypothesis that may
be made about the result of the experiment, so it may agree with the scientific hypothesis or it
may be quite different.
The outcome of an inferential statistical test is a probability (called the P-value) that the null
hypothesis is true. Like all probabilities, the P-value varies from 0 (impossible) to 1 (certain),
though in practice it never goes as low as 0 or as high as 1. Since the interesting P-values are so
small, it can be convenient to give P as a percentage (that is, 0100%) to avoid possible
confusion with very small numbers.
The lower the probability, the less likely it is that the null hypothesis is true. How low does P
have to be for us to reject the null hypothesis? In biology, the critical probability is usually taken
as 0.05 (or 5%). This may seem very low, but it reflects the fact that biology experiments are
expected to produce quite varied results. So if P < 0.05 (5%) then we reject the null hypothesis,
and conclude that there is a significant difference or association. If P 0.05 (5%) then we
accept the null hypothesis, and conclude that there is no significant difference or association. We
can also make stronger statements for very low P-values. If P < 0.01 (1%) we say that the
difference or association is very significant, and if P < 0.001 (0.1%) we say it is extremely
significant.
So, for example, the pulse rate of 20 subjects was measured before and after a meal to see if the
meal made a difference. A suitable statistical test would test the null hypothesis that there was
no difference in pulse rate before and after the meal. If the P-value from the test was 0.16 (16%)
we would accept the null hypothesis and conclude that there was no significant difference in
heart rate before and after the meal.
Inferential statistics can be subdivided into comparative statistics and association statistics.
I Comparative statistics
As the name suggests, these statistics compare groups and test for differences between them. (For
example, is this group bigger than that group?) The independent variable is usually qualitative
and the data should be presented as a bar chart (for normally distributed data) or a box plot (for
non-normally distributed data). The choice of comparative test depends on whether the
investigation is designed with independent samples or matched samples.
I
I
In an independent-sample experiment, the measurements in the different groups of data to be

compared do not match up in any particular way.
In a matched-sample experiment (also known as a paired sample experiment if there are just
two groups), each measurement from the first group matches up with a corresponding
measurement in the other group(s). This will be the case when measurements are made on the
same subject (for example, in a before and after experiment), or if the measurements were
matched in some other way, such as on siblings, or on the same day, or in the same habitat.
If it isnt obvious which value matches with which, then its probably not a matched
design.
16
Matched-sample experiments are often more powerful than independent-sample experiments, so

are more likely to detect significant differences. Tests for matched-sample experiments have
slightly different null hypotheses that those for independent-sample experiments, though the
difference isnt usually very important. However, the P-values can be very different, so it is
important to choose the right one.
The main statistical tests are ANOVA (analysis of variance) for independent-sample
experiments and Matched Samples ANOVA for matched data. For non-normally distributed
data the tests are the Kruskal-Wallace test for independent samples, and the Friedman test for
matched samples. They all test the null hypothesis that the sets have the same mean (or
median), so if P < 5% then the null hypothesis is rejected and we conclude that at least one of
the sets is significantly different from the rest. These tests can all be used to compare two or
more groups, but there are also tests designed specifically to compare just two groups. These twogroup tests include the paired and unpaired t-test, the Mann-Whitney U-test and the Wilcoxon
matched pairs test. They give exactly the same P-values as the corresponding tests above (for
example, the t-test gives the same result as ANOVA). So why bother with the two-group tests?
Partly because they were devised first, and partly because they are easier to calculate by hand,
without a computer. These days, when computer software makes all tests equally easy there is no
real reason to continue to use the two-group tests.
Figure A2.19
Figure A2.19 shows the results of an experiment comparing the effects of three different colours
of light on the rate of photosynthesis in Elodea, measured by length of oxygen bubble produced
in a given time. The measurements were replicated 10 times for each colour and the means and
95% confidence intervals were calculated for each set of replicates using the Merlin formulae
MEAN and CI. Cell B13 contains the formula MEAN(B3:B12) and cell B14 contains the
formula CI(B3:B12). The mean values were plotted on a bar chart with error bars
representing the 95% confidence intervals. Since the measurements are probably normally
distributed, we can use ANOVA to test the null hypothesis that there is no difference between
the mean bubble lengths. The Merlin formula ANOVA(B3:D12) is typed into cell B15 and
formatted as a percentage. The P-value is < 5% so we reject the null hypothesis and conclude
that the results from at least one of the colours are significantly different. From the means, green
light must give a significantly lower rate of photosynthesis than the other colours.
Figure A2.20
17
The data shown in Figure A2.20 are from a matched-sample experiment in which the mass of
food eaten by eight deer at four different times of year is compared. The same deer were
identified each time, so we use Matched Samples ANOVA to test the null hypothesis that there
is no difference in food eaten in the four months. The Merlin formula ANOVAM(B3:E10) is
typed into cell B13 and formatted as a percentage. The P-value is < 5% so we reject the null
hypothesis and conclude that the results for at least one of the months are significantly different.
From the means, significantly less food is eaten in May and August.
Figure A2.21
Figure A2.21 compares decay rates of three species of leaf, measured by percentage of leaf area
decayed after eight weeks burial. Percentages are not usually normally distributed, so we use
Merlin to plot a box plot of the results, and the Kruskal-Wallace test is used to test the null
hypothesis that the median decay is the same for the three species. The Merlin formula
KWTEST(B3:D11) is typed into cell B12 and formatted as a percentage. The P-value is <
1% so we reject the null hypothesis and conclude that there is a very significant difference
between at least two of the leaves. From the box plot, there has been significantly less decay of
the beech leaves.
Figure A2.22
The example in Figure A2.22 compares the symptoms of patients (on a score system) before and
after treatment with a drug. This is a matched-sample experiment with ordinal data, so is
analysed with the Friedman test with the null hypothesis that there is no difference in median
symptom scores for the three days. The Merlin formula FRIEDMAN(B3:D12) is typed into
cell B13 and formatted as a percentage. The P-value is < 5% so we reject the null hypothesis and
conclude that the result for at least one of the days is significantly different. From the medians,
there is a significant drop in symptoms after treatment, so the drug works.
I Association statistics
Association statistics investigate associations between two sets of data. There are two kinds of
association statistics: correlation and regression.
Use correlation if you dont already know whether there is a relation between the two
18
variables, so there isnt a dependent and independent variable. Typically you will be measuring
both variables on a series of individuals (for example, height and weight of people) or at a series
of times or locations (for example, depth and width of a stream). You certainly wont be varying
one variable while measuring another in a controlled experiment. A correlation test will tell you
if there is a correlation between the two variables and how strong it is.
Use regression if you already know or suspect that there is a relation between two variables
and you want to know more details about how exactly the two variables are related. Typically,
you design a controlled experiment in which you vary one variable (the independent variable)
and measure the other (the dependent variable). Linear regression will give you the slope of the
straight line describing the relationship.
Correlation statistics
Correlation statistics are used whenever you want to find out if there is a correlation between
two variables. (For example, if this variable increases, does that variable also increase?) This is a
very common biological problem you might investigate a correlation between blood pressure
and heart rate, or between smoking and heart disease, or between river flow rate and biodiversity.
The first step in investigating a correlation is to plot a scatter graph of one variable against the
other. The shape of the scatter graph indicates the type of correlation. If both variables increase
together then there is a positive correlation; if one variable decreases when the other increases
then there is a negative correlation; and if the scatter graph has apparently random points then
there is no correlation.
negative correlation
variable 2
variable 2
positive correlation
variable 1
no correlation
variable 2
Figure A2.23 Scatter

graphs showing different
kinds of correlation.
variable 1
variable 1
A correlation is described by two statistics. First there is a P-value, which tells you if there is a
significant correlation; then there is a correlation coefficient, which gives the strength of the
correlation. The correlation coefficient varies from 1 (perfect negative correlation) through 0
(no correlation) to 1 (perfect positive correlation). The larger the absolute value, the stronger
the correlation. The statistical tests used are the Pearson Linear Correlation test (also called the
product-moment correlation) for normally distributed data, and the Spearman Rank Correlation
test if either of the variables is not normally distributed.
Figure A2.24
Figure A2.24 shows the heights of 10 fathers compared with the heights of their grown sons.
Since height is normally distributed, we can use the Pearson test to test the null hypothesis that
there is no correlation between fathers and sons heights. A scatter graph is plotted. The P-value
is calculated using the Merlin formula PEARSONP(B2:B12,C2:C12) in cell C13, and the
19
correlation coefficient is calculated using the Merlin formula PEARSON(B2:B12,C2:C12)

in cell C14. The P-value is < 5% so the null hypothesis is rejected and we conclude that there is
a significant positive correlation with a strength of 0.72, so tall fathers do have tall sons.
Figure A2.25
In Figure A2.25, the social status of hens (measured by their pecking order, where 1 is the
highest rank) is compared with their mass. Pecking order is an ordinal variable, so we use the
Spearman test to test the null hypothesis that there is no correlation between mass and pecking
order. The P-value is calculated using the Merlin formula SPEARMANP(B2:B11,C2:C11)
in cell C12, and the correlation coefficient is calculated using the formula
SPEARMAN(B2:B11,C2:C11) in cell C13. The P-value is < 5% so the null hypothesis is
rejected and we conclude that there is a significant negative correlation with a strength of
0.77, so big hens are higher up the pecking order.
Correlation and causation

It is very important to distinguish between a correlation and a causal relationship. The fact that
two variables are correlated does not mean that the changes in one cause the changes in the
other. The changes may both be caused by a third factor, or the correlation could be just
coincidence. Further controlled studies would be needed to investigate cause and effect. For
example, the strong correlation between smoking and heart disease didnt in itself show that
smoking causes heart disease, but it suggested a line of further investigation that eventually did
demonstrate and explain the cause and effect.
Regression statistics
Regression is used to describe a relationship between two sets of data. It is used when you already
know that there is a correlation between two variables (usually, but not necessarily, a causal
relationship) and you want to quantify the relationship. Once again the data are plotted on a
scatter graph, but this time a line of best fit is added. The simplest type is linear regression,
which fits a straight line to the data, and gives the values of the intercept and slope (gradient) of
that line (a and b in the equation y a bx). It is also possible to do non-linear regression,
which fits a curve to the data, but this is complex and beyond the scope required here.
Figure A2.26
20
Figure A2.26 shows the absorbances of 10 yeast cell suspensions, as measured in a colorimeter,
and their cell concentrations, measured in a cell counter (haemocytometer). Merlin is used to
plot a scatter graph of the data pairs, with a straight line of best fit (or trend-line). The trendline is forced through the origin because the colorimeter is calibrated to read zero with water (no
cells), so this point is fixed. To find the slope of the line, the Merlin formula
REGRESS(A2:A12, B2:B12, 0) was typed into cells B15:C16 and entered as an array
formula. The labels are added by hand to make the numbers clear. The slope is this case is 0.152
and we can then use this to make quantitative predictions. For example, we could predict that a
sample with an absorbance of 1.37 has a cell concentration of 9 107 cells cm3.
I Statistics for nominal data
There are special statistics used to analyse nominal data: that is, data in categories that cannot
be ranked. So instead of recording quantitative measurements like this plant is 45 mm high or
45% of this quadrat is covered with bladder wrack, you would record observations like this
flower is red or bladder wrack is present in this quadrat. As a general rule, most biology
investigations should be designed to yield quantitative data, so nominal data are not very
common. But there are some cases where nominal results are desirable or the only practical
solution. Some examples of the categories that might be used in biology investigations are:
I
I
I
I
I
phenotypes in genetic cross experiments (such as male/female, round/wrinkled,

red/white/purple, and so on)
present/absent
yes/no
dead/alive
species names.
The categories used must be mutually exclusive, so an observation must clearly fall into one, and
only one category. For example, observing insects visiting different coloured wild flowers and
classifying them as red flower insects or yellow flower insects is not suitable, since the same
insect could visit two different flowers and so fall into two categories. But classifying the same
insects as male or female is suitable as an insect must be one or the other. The numbers of
observations of each category are counted to give frequencies. As we know, its best not to call
frequencies counts, as this can lead to confusion with quantitative data. To make the tests as
reliable as possible, the frequencies should be as large as possible, and one should aim for at least
100 observations in total, and preferably a lot more.
The most common frequency statistics are the chi-squared tests (2 test) and the G-tests.
Although the chi-squared tests are more famous, the G-tests (or log-likelihood ratios) are
considered by many statisticians to be better tests. They can be used whenever the chi-squared
tests can be used, and have the same null hypotheses. The G-tests give similar, though not
identical, P-values to the chi-squared tests. Both tests come in two forms: a goodness-of-fit test
(to test for differences) and an association test (to test for links). Unfortunately, the two forms
are sometimes confused and the term chi-squared test is used to refer to either test. But the
goodness-of-fit test and the association test have quite different uses and test different null
hypotheses.
Goodness-of-fit tests
What can we do with a table of frequencies? We certainly cant take averages or measure the
spread of nominal data, so the only thing we can do is compare the frequencies we obtain (the
observed frequencies) with some expected frequencies to see if there is a good fit between them.
This is the equivalent of a comparative test for quantitative data.
Where do we get the expected frequencies from? We must have some scientific hypothesis
that makes a quantitative prediction about the frequencies we would expect to find. The most
famous examples of these are Mendels laws of genetics, which predict certain ratios of
phenotypes from genetic crosses. Sometimes the expected frequencies can be calculated by
assuming that the frequencies in all the categories should be the same, but great care must be
taken to ensure that this is a sensible thing to say.
The best chart to use is a pie chart of the observed frequencies. This often shows intuitively
21
whether the observed ratio is similar to the expected ratio, and it is also a good test that the data
really are frequencies of nominal data. If the pie chart doesnt make sense, then it is possible that
the data are quantitative and should be analysed using a different test. Both the chi-squared and
G goodness-of-fit tests test the null hypothesis that there is no difference between the observed
and expected frequencies. If P < 5% then the null hypothesis is rejected and we conclude that
there is a significant difference between the frequencies.
Figure A2.27
In Figure A2.27, the frequencies of flower colours from a genetic cross are compared with an
expected 3:1 ratio using the chi-squared and G goodness-of-fit tests; 929 offspring from the same
genetic cross are observed and their flower colour noted. The observed frequencies of the two
colours are typed into cells B2 and B3, and the total is calculated in cell B4 using the formula
SUM(B2:B3). The expected frequencies can be calculated from the total number of
observations (929) using simple Excel formulae, so for example cell C2 contains the formula
B4*3/4. The formula CHITEST(B2:B3,C2:C3) is typed into cell C5 and
GTEST(B2:B3,C2:C3) into cell C6 and both are formatted as percentages. The P-values are
both > 5% so we accept the null hypothesis and conclude that the observed data are consistent
with Mendels law.
This example demonstrates a problem with large P-values in frequency tests. Remember that a
large P-value (>5%) allows us to accept the null hypothesis, but that doesnt provide any
evidence in support of the null hypothesis. So we cant conclude that these data support the
hypothesis that there is a 3:1 ratio; instead all we can honestly say is that these data dont
contradict the hypothesis that there is a 3:1 ratio.
Association tests
The association tests, which test for an association between two variables measured by nominal
data, are the equivalent of the correlation tests for two quantitative variables (but the term
correlation can only be used for numeric data). Remember that if both categoric variables can be
ranked, then you can and should use the Spearman Rank Correlation test rather than the lesspowerful association tests.
For example, you might investigate a possible association between blood group (A/B/AB/O)
and resistance to a certain disease (resistant/not resistant). The data are collected by sampling a
large number of individuals, measuring their blood group and disease resistance and so placing
each one of them in one of the eight possible categories. The frequencies of these categories (the
observed frequencies) are then presented in a table called a contingency table (because we are
investigating whether one variable is contingent or dependent on the other), as shown below.
Table A2.1 Contingency
table
Blood group
A
AB
resistant
not resistant
22
One variable is represented by the columns of the contingency table (blood group, in this case)
and the other by the rows (disease resistance, in this case). There can be two or more categories
of each variable (though its always a small number), and it doesnt matter which way round the
two variables go. The observed frequencies can also be plotted on a mosaic plot, which is
specially designed for contingency tables. The mosaic plot is just a graphical representation of
the contingency table, with each tile representing a cell in the table and the area of each tile
being proportional to the frequency.
The association is characterised by two statistics, just like a correlation. First there is a Pvalue, which tells you if there is a significant association, then there is an association coefficient,
which gives the strength of the association. The association coefficient ranges from 0 (no
association) to 1 (complete association), so the higher the value, the stronger the association.
The actual tests are the 2 (or G) test of association to give the P-value, and Cramrs V to give
the association coefficient.
Figure A2.28
This example investigates whether there are more nests in birch or oak trees in other words,
whether there is an association between tree species and presence of birds nests. A large number
of birch and oak trees are observed and the presence or absence of a birds next is noted, so each
tree can be placed into one of four categories. The observed frequencies of each category are
typed into a 2 2 contingency table (cells B2:C3). We dont have to produce expected
frequencies in this case as they are calculated automatically by the Merlin formulae. The Merlin
formula CHASSOC(B2:C3) is typed into cell B4 and the Merlin formula
GASSOC(B2:C3) is typed into cell B5 and formatted as percentages. The formula
CRAMER(B2:C3) is typed into cell B6. The P-value is < 5% from both the chi-squared test
and the G-test, so the null hypothesis is rejected and we conclude that there is a significant
association, though it is only weak, with a strength of 0.22. The mosaic plot shows us that the
presence of nests is associated with birch trees.
I Choosing a test
One of the most difficult tasks in data analysis is choosing the correct statistical test. This table
lists all the main statistical tests and shows that you can choose the right one by asking just two
questions. What kind of test? determines the correct row in the table, and What kind of data?
determines the correct column. Not all the tests listed here have been discussed in this appendix,
but they are available in Merlin. Merlin also includes a test chooser, where you answer a series
of simple questions to arrive at the best test.
23
Table A2.2 Choosing the

best statistical test
What kind of test?
What kind of data?

qualitative data
(words)
quantitative data
(numbers)
normally
distributed
summarise replicates
descriptive
statistics
comparative
statistics
cannot be
ranked
normally
distributed data
ordinal data
nominal data
parametric tests
non-parametric tests
frequency tests
mean, standard
deviation,
confidence interval
median, quartiles
frequencies, mode
histogram, normal-quartile plot,

DAgostino-Pearson test
chart
bar chart
box plot
independent samples
ANOVA
Kruskal-Wallace test
matched ANOVA
Friedman test
unpaired t-test
Mann-Whitney
U-test
paired t-test
Wilcoxon test
scatter graph
scatter graph
mosaic chart
Pearson correlation
Spearman
correlation, Kendall
correlation
G-test of
association, 2 test
of association
matched samples
independent
samples
matched
samples
chart
test for an association
details of a linear
relation
details of a non-linear
relation
ecology
statistics
can be
ranked
test replicates for

normality
2 groups
only
association
statistics
not normally
distributed
or ordinal
G goodness-of-fit
test, 2 goodnessof-fit test
linear regression
non-linear
regression
non-parametric
regression
analyse a transect
kite graph
estimate population size
Lincoln-Peterson index, Schnabel index
measure diversity
Simpson index, Shannon index
pie or bar graph
logistic regression
24
I Summary The six steps in data analysis

1 Plan a controlled
experiment
Start the plan by clearly stating the aim and deciding on the dependent and
independent variables (if appropriate). List all the other confounding variables
that might affect the outcome, and decide how to control them, if possible. If
some variables cant be controlled (like weather in a field investigation) then
at least be aware of them, and if necessary monitor them. Decide how many
replicates are feasible (more than 10, if possible) and what summary statistics
are appropriate (like mean and confidence interval, or median). Design the
results table with space to record all the raw data and the summary statistics.
Decide what chart is suitable and perhaps sketch what you think the chart
might look like.
Planning all these steps in advance helps to highlight problems and identify
mistakes in your thinking. Its too late to rectify mistakes after the experiment
is finished!
2 Choose a test and state the

null hypothesis
If appropriate, choose a suitable statistical test using Merlins test chooser

or Table A2.2 above. Not all investigations will need a statistical test. Look up
the chosen test in Merlins Help, or other reference, and state the null
hypothesis as precisely as you can for your experiment for example, There is
no difference between the means of the plant heights in the two areas. This
statement is what the statistical test will actually test. This step must be made
when the investigation is planned, not after the experiment has been carried
out.
3 Obtain results
Carry out the investigation and obtain results.
4 Present the data
Present your raw data in a neat results table. This can most conveniently be
done in Excel. Use Merlin to calculate descriptive statistics, such as the mean
and 95% confidence interval, and plot an appropriate chart.
5 Carry out the statistical test
Enter the Merlin formula for your chosen test into the spreadsheet. This will
return the P-value for that test.
6 Make a conclusion
State whether you accept or reject the null hypothesis, and write a sentence
explaining exactly what that means in this case. For example, if P < 5% then
you would reject the null hypothesis and say that the plants in group A are
significantly taller than the plants in group B. Or if P > 5% then you would
accept the null hypothesis and say that, as far as you can tell from the data,
there is no significant difference between the heights of the two groups of
plants.
I References
Ashcroft, S. and Pereira, C. (2003) Practical Statistics for the Biological Sciences, Palgrave
Macmillan
Ennos, R. (2000) Statistical and Data Handling Skills in Biology, Prentice Hall
Krebs, C.J. (1999) Ecological Methodology (second edition), Addison Wesley
Sokal, R.R. and Rohlf, F.J. (1995) Biometry: The Principles and Practice of Statistics in Biological
Research (third edition), Freeman
Zar, J.H. (1999) Biostatistical Analysis (fourth edition), Prentice Hall

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Appendix 2: Handling data for

I Sampling and accuracy

I Sampling and accuracy

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APPENDIX 2: HANDLING DATA FOR A2 BIOLOGY

normally distributed data

use parametric tests

use non-parametric tests

use frequency tests

Edexcel Biology for A2 Dynamic Learning

Hodder Education 2009

APPENDIX 2: HANDLING DATA FOR A2 BIOLOGY

I Handling very large and very small numbers

Notes on using units and prefixes

Edexcel Biology for A2 Dynamic Learning

Hodder Education 2009

APPENDIX 2: HANDLING DATA FOR A2 BIOLOGY

I Using software for data analysis

The mean, standard deviation and confidence interval

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Hodder Education 2009

APPENDIX 2: HANDLING DATA FOR A2 BIOLOGY

Edexcel Biology for A2 Dynamic Learning

Hodder Education 2009

APPENDIX 2: HANDLING DATA FOR A2 BIOLOGY

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APPENDIX 2: HANDLING DATA FOR A2 BIOLOGY

4th quartile (maximum)

2nd quartile (median)

0th quartile (minimum)

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APPENDIX 2: HANDLING DATA FOR A2 BIOLOGY

Edexcel Biology for A2 Dynamic Learning

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APPENDIX 2: HANDLING DATA FOR A2 BIOLOGY

swamp and marsh

net primary productivity/MJ m

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APPENDIX 2: HANDLING DATA FOR A2 BIOLOGY

Edexcel Biology for A2 Dynamic Learning

Hodder Education 2009

APPENDIX 2: HANDLING DATA FOR A2 BIOLOGY

worms per quadrat

worms per quadrat

Edexcel Biology for A2 Dynamic Learning

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APPENDIX 2: HANDLING DATA FOR A2 BIOLOGY

Edexcel Biology for A2 Dynamic Learning

Hodder Education 2009

APPENDIX 2: HANDLING DATA FOR A2 BIOLOGY

Edexcel Biology for A2 Dynamic Learning

Hodder Education 2009

APPENDIX 2: HANDLING DATA FOR A2 BIOLOGY

Edexcel Biology for A2 Dynamic Learning

Hodder Education 2009

APPENDIX 2: HANDLING DATA FOR A2 BIOLOGY

I Making conclusions from data

In an independent-sample experiment, the measurements in the different groups of data to be

Edexcel Biology for A2 Dynamic Learning