BIOL2010, Semester 1, 2013
Practical 2: Community assembly: Priority effects and
environmental gradients
(Modified from Kingsolver 2006)
Background information
A natural community is an assemblage of coexisting species. Community assembly is
the set of ecological and evolutionary processes that combined determine which and
how many species can coexist in a given community. Understanding how communities
form and are maintained is key to understanding biodiversity patterns from local to
global scales. One of the main focuses of studies examining community assembly is on
determining which processes are actually driving particular outcomes in terms of
numbers and the identities of species. In this lab exercise we will use experimental
microbial communities to test the importance of two processes to the total species
richness and composition of species occurring in small aquatic communities.
The two processes that we are exploring are: priority effects and environmental
filtering (also called habitat or ecological filtering). “Priority effects” refers to the
importance of the order that species are introduced to an assembling community to the
outcome of the richness (number of species) and composition of species in that
community once it stabilises. Numerous studies of many different types of organisms
have found the order of introduction to be very important in assembling communities.
It is also clear, however, that this order is not always important and much research has
gone into trying to understand when priority effects are important and when they are
not.
Environmental filters are aspects of the abiotic environment that restrict which species
can survive in a given environment. Community assembly theory in general focuses on
the interplay between environmental constraints on organisms and competitive
dynamics among species and individuals in a community. The compatibility of a species
with a particular environment relates to the niche of that species. As species find their
environment more and more unsuitable (due to changes in environmental conditions) it
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becomes more stressed. This stress can reduce the fitness (population growth rates) of
the species, alter how effective they are as competitors for food or shelter and if the
incompatibility becomes too great, the species will be “filtered out” and no longer be
able to persist in the community. If you read more about environmental filters, you will
see that this concept is often discussed as it relates to the similarity of organisms
coexisting in a community. More specifically, it is generally believed that in communities
with very strong environmental filters, coexisting species will be very similar (reflecting a
restricted niche compatible with that environment) and closely related. On the flip side,
in communities were environmental filters are weak but competitive interactions are
strong, we expect to see more different species and less related species coexisting (as
competition drives difference the approach species take to persisting in a given
environment. The interplay between environmental filters and competition is NOT what
we are exploring in this practical exercise. All we are examining here is whether or not
the “harshness” (measured by saltiness in our case) of the environment is more or less
important to the diversity and composition of microbial communities than the origin of
the first colonizers of our experimental systems (priority effects). For this reason, what
you need to understand for the practical exercise is the concept of the ecological niche
and the idea that the environment can restrict which species are found in a community.
Key Questions:
1. How important are environmental gradients to community diversity and composition?
2. How important are priority effects to the diversity and final composition of
communities?
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Materials
Pond water from UQ lakes
Rain-water
Sodium chloride, reagent grade
24 large test-tubes (for each group)
Hay for infusions
Coloured tape (for marking test tube stands)
Marker pens for labelling test tubes
Eye droppers or pipettes
Compound microscope, magnification up to 400x
24 glass slides and cover slips
Paper and pencils for drawing protozoa and bacteria
Preparation
We will be using a simple hay infusion to culture a community of bacteria and
protozoans in the laboratory. These are the experimental communities we will be using
to test the importance of priority effects and environmental filtering. There are
protozoans both in the source water provided AND on the hay used in the infusions.
Although the cultures are quick to set up, you will need to let them develop for threefour weeks before you can assess the stabilized community in each solution. We will
also examine how communities change over this time period. Because we are looking at
priority effects, we will need to add solutions in both week 1 and week 2 of this prac
(detailed below). From weeks 2-4 you will need to make weekly assessments of the
composition of your communities (details below). These weekly measurements will let
you determine how your communities change over time.
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Although the vast majority of the bacteria and protozoan found in the hay and water
solutions are completely harmless, we cannot guarantee this so please be sure to wash
your hands after you hand water from your communities.
Overall Schedule:
Week 1 (Friday April 12):
1. Go over prac 2 in detail with your tutor
2. Set up your 24 microbial communities.
Week 2 (Friday April 19):
1. First assessment of microbial communities (composition and richness)
2. Top up infusions to allow for priority effect testing
Week 3 (Friday April 26):
1. Second assessment of microbial communities
2. Start process of creating a common species list with your larger group of 4 teams
Week 4 (Friday May 3):
1. Third and final assessment of microbial communities
2. Finalize common species list with larger group
3. Ensure that all small groups have the larger dataset for analysis
4. Discuss expected calculations for final report
5. Questions relating to final report
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Detailed instructions by Week
Week 1.
To test the importance of an environmental variable to community assembly, we will be
looking at the species richness and composition of microorganisms in six saline solutions.
To test for priority effects we will be using two sources of water introduced to our
infusions over two weeks. Each water source should have a distinct fauna living in it.
The two sources are: rainwater (R) and pond water (P). Rainwater is sourced from
Upper Brookfield and pond water has been collected on the day of use from UQ lakes.
1. The different salt concentrations detailed in Table 1 have already been prepared using
either pond water (P) or rainwater (R).
Table 1. Salt dilutions that have been prepared using pond water (P) and rainwater (R).
Label: description
Concentration (ppm)
g salt L-1
A: Fresh water
0 ppm
No added salt
B: Trace salt
1000 ppm
1 g L-1
C: Brackish
2000 ppm
2 g L-1
D: Saline
5000 ppm
5 g L-1
E: Highly saline
10,000 ppm
10 g L-1
F: Seawater
35,000 ppm
35 g L-1
2. In your groups, label 24 test tubes with the concentration (ppm) to go in each tube
(use the letters (A-F) listed in Table 1 for simplicity) and the source water that was used
to make up the solution (R or P). Place the labelled tubes in a test tube rack. There
should be two tubes for each ppm/source water combination (Table 2a).
Table 2a. List of tubes and how they should be initially labelled.
Pond
water
Rain
water
Set 1
Set 2
Set 3
Set 4
AP
AP
AR
AR
Bp
Bp
BR
BR
CP
CP
CR
CR
DP
DP
DR
DR
EP
EP
ER
ER
FP
FP
FR
FR
Select the tubes from Set 2 and label the with an additional “R” subscript (e.g. AP/R) to
indicate that the pond water will be followed by addition of rainwater. Then similarly
select the tubes from Set 4 and label with an additional “P” subscript (e.g. A R/P) to
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indicate that the rainwater will be followed by addition of pond water. When finished
you should have 24 tubes with the labels detailed in Table 2b.
Table 2b. List of tubes and how they should be finally labelled.
Pond
water
Rain
water
Set 1
Set 2
Set 3
Set 4
AP
AP/R
AR
AR/P
Bp
BP/R
BR
BR/P
CP
CP/R
CR
CR/P
DP
DP/R
DR
DR/P
EP
EP/R
ER
ER/P
FP
FP/R
FR
FR/P
3. In each of your 24 tubes place approximately 2 grams of hay (this does not need to be
weighed accurately) and insert it so that all the hay falls to the bottom the tube.
4. Fill all 24 tubes with the appropriate ppm/source water mixture (according to their
label) so that all tubes are half full. At this point Sets 1 and 2 should be identical and sets
3 and 4 should be identical. Once all tubes are properly filled cover the tubes with slipon caps to allow some air into the cultures. Incubate the tubes at room temperature for
1 week. Follow tutor instructions as to where to put your rack and make sure the RACK
is labelled using the coloured tape and marker pens provided so that your group can
identify your tubes from those in the rest of the class.
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Week 2.
In week 2 you will add a second solution to your tubes in order to test for priority effects.
NOTE, you must extract your samples for assessing community composition/richness
before you add the second solution so that you can assess the composition of your
communities before adding the second dilution.
1. Use a pipette or eyedropper to remove a small amount of fluid from near the
top of the each tube and place an individual drop on a microscope slide labelled
for the appropriate treatment (community). Place a cover slip over the drop on
each slide. Label each slide carefully so you know exactly which community you
are looking at. Examine the infusion under the compound microscope and using
a range of magnifications make a sketch and write a description of each
“morphospecies” (clearly distinct types of organisms) you see making sure you
record which organisms are in which treatment. At the end you should have a
species count and set of drawings for each of the 24 tubes (communities). You
do not need to record abundances (number of individuals of each species). Also
record which ones are common between different treatments. You should
design a data sheet in a way that easily allows you to do this (see example
below). One approach is to have a core set of drawings that you label
(number/letter or name) and then list the codes in each of your datasets, adding
to the drawings with each new species, rather than creating separate drawings
for at each tube’s community. You may want to assign one or two people to do
the drawings so they are consistent. However, make sure everyone in your
group has a chance to look at the species in the microscope. (Make sure there is
something on each of your 24 slides before adding new water into each tube.)
2. Once you have recorded the composition of all 24 of your communities, you
need to proceed to add the second infusion to each tube. To do this repeat
steps 1 and 4 from week 1. However, rather than adding each infusion to new
tubes you will be filling your existing tubes with additional liquid. Those marked
with a P or and or R alone (Sets 1 and 3) are those that will be given a top up of
the same dilution (A-F)/source water as went in the tube in week 1 (though the
actual solution will be freshly made in week 2). Fill each of these tubes to within
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1 cm of the top, place the cap back on the tubes and put them back in your rack.
The tubes marked with P/R or R/P will receive a top up of the same dilution (A-F)
but the other source water. So, for example, tubes in Set 2 had dilutions made
with pond water in week 1 and now you will be adding the same dilution but
made with rainwater. So your fresh water P/R tube (AP/R) should have had plain
pond water added in week 1 and will be topped up with plain rain water in week
2.
3. Once all tubes are topped up return them to your rack and store them as you did
in week 1.
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Week 3.
1. Bring you test tube rack to your bench.
2. Prepare you microscope slides so one is labelled for each of your 24
communities (tubes).
3. Use a pipette or eyedropper remove a small amount of fluid from near the top of
the each tube and place an individual drop on microscope slides laid out and
labelled for each treatment. Place a cover slip over the drop on each slide.
Examine the infusion under the compound microscope and using a range of
magnifications make a sketch and write a description of each organism you see.
Use the same recording method as you did in week 2 but make sure your records
from week 3 are clearly distinguishable from week 2).
4. Record the number of different “morphospecies” (clearly distinct types of
organisms) in each treatment and record which ones were found in each
community. You will want to match up your drawings from week 2 so that you
can determine which species were there in week 2 and week 3 and which ones
are new.
5. Team up with 3 other groups to make a larger group and spend some time
matching up drawings and descriptions so that you can decide on a single
common list of morphospecies across all groups. Once you have a common list
of drawings and “species numbers” start a NEW data sheet and transcribe your
data but using the set of common species names so that all 4 groups’ datasets
are completely compatible and there is no chance for error due to the need to
interpret drawings once you get to the analysis stage. It is recommended that
you use a distinct set of numbers, letters or names to designate your group
morphospecies list from those used by your individual groups so that you can
double check for mistakes when you transcribe your teammate’s datasets into 1
large dataset. (Never erase dataset and never assume you will remember
something later – record everything!)
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Week 4
1. Start lab by doing a final assessment of the communities in your 24 tubes
following the same procedures used in weeks 2 and 3. When you are sure you
have all the samples that you need (meaning wait until you are sure there is
something on each of your slides) you can dispose of your communities and
clean your tubes and caps.
2. Record your final communities as individual teams and then get back together
with the larger group to finish combining datasets with your common names.
3. Before you leave today, all datasets should be entered into excel and each group
should have a copy of the group excel file containing all of the larger group’s
data setup in a way that everyone understands.
4. We will go over the simple calculations that you need to make in this class
session and answer any questions about the final report.
5. Use any remaining time you have to make a work plan for your group. Make
sure that before you leave you have exchanged e-mail addresses as you will
need to do some calculations at home that you then share with your larger
group. Assigning 1 person per group to pass along their Jaccard matrix
(explained below) to the other groups would be a good idea.
Final report for practical 2:
The final report for Practical 2 should be submitted at the Biology Helpdesk by 4pm on
TUESDAY 21 May 2013. As for practical 1 ,each group should submit a single hard copy
of the report with the signed coversheet of each contributing group member attached at
the front. A single electronic copy should also be submitted through Turnitin.
The final report for this laboratory exercise should conform to the guidelines provided at
the beginning of semester and which you followed for practical report 1. These
guidelines are provided again in the Practical 2 folder on the Blackboard site.
There are many comparisons that you could make with the data you have collected in
this practical. In a 6-page report there is no way for you to make all comparisons and
trying to do so will not result in a good mark. There MUST be a clear reason for any
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graph or statistical analysis that you present – more is NOT always better. You may
include observational data on specific species or types of species as part of your analysis
as well as graphs and statistical results.
Below I have listed the analysis that I want to see you make in your report but you
should also include results on AT LEAST 2 additional comparisons/analysis of your choice.
Importantly, your report must focus on what evidence there is for priority effects
AND/OR the physical environment being important for determining species richness
and composition within your communities. The answer may be that there is no
evidence supporting the importance of one or both of these processes so don’t try to
guess the answer we want - use your data to come to your own conclusion.
Be sure to select your specific research questions (listed in the Introduction of your
report) carefully so that the results you present actually answer them.
For analysis, you are fundamentally comparing means of continuous data (counts)
among groups. This means that you will use t-tests or ANOVAs to analyse data. Though
we have provided details about some of the means you need to calculate, you may need
to group data in several different ways depending on the specific questions you want to
answer. For example, you can compare Dilution A and B or Community Set 1 and 2,
depending on your question. The first would mean averaging 4 values for dilution A and
4 for B, while the second would involve taking the mean of 24 data points for Set 1 and
24 for Set 2).
Because we have a factorial design including 2 treatment types (source water treatment
and dilution) the appropriate analysis for this dataset is an ANOVA, most properly
including an interaction effect between the two. If anyone in your group is currently
taking or has taken Biostats (BIOL2006), I encourage your group to use a more
appropriate analysis. However, if your group is not comfortable with this you can then
do a series of t-tests following the Excel-based methods explained and worked through
as part of Practical 1. Either approach can be used for a good mark on your final report.
An ANOVA cannot easily be done in excel but can be done in R or any number of other
stats packages. You will also be presenting at least two graphs showing change in
species richness and Jaccard’s over time (see above). In all graphs it is expected that you
will represent variances properly with error bars. As we only have 3 time points there is
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no need to statistically analyse time series data but you do need to present these graphs
(with error bars where appropriate) and explain what patterns they suggest in your
discussion.
For report include AT LEAST ONE the following (presented graphically):
1. Calculate the mean and standard deviation of species richness (number of
species) for each of the 24 treatments for each time period (week 2, 3, and 4)
using the data collected by the larger group. This will mean you have 4 data
points for each treatment in each week.
2. Create a plot in excel showing how the mean number of species in each
treatment changes over the three sampling weeks. Calculate means and
standard deviations for all P tubes and all R tubes and compare these across
time as well.
3. Using week 4 data only, calculate a Jaccard’s Index for each pair of tubes in your
small group’s samples (1 data point for each tube). This index provides an
estimate of how many species are in common between each pair of
communities. Gather each group’s set of values (most easily presented as a
matrix of values called a similarity matrix) and calculate the mean and standard
deviations for Jaccard’s index for each pair of communities (again using 4 values
for each pair). The Jaccard index equation is provided below. To statistically
compare dilutions and the four water source treatments use all four values for
each group. Present a Jaccard’s matrix of mean values in your paper.
4. Plot how the mean Jaccard’s Index for the different sets (means of at least 1
dilution using the larger dataset - your choice of which one, though your choice
should be justified) changes over time (6 lines). Does the mean number of
species in common increase or decrease for each?
Calculating Jaccard’s index. This is a pairwise calculation so you need to calculate a
Jaccard’s index for each pair of “communities” (test tubes) in your sample. You will
them average the indices for each pair using the values from all 4 groups.
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The calculation:
Sj =
a
,
a+b+c
where Sj is the Jaccard’s index, a is the number of morphospecies in sample A and
sample B (joint occurrences), b is the number of species in sample B but not in sample A
and c is the number of species in sample A but not in sample B.
Relevant readings: You may read these paper/ book chapters to help understand the
concepts explored in this lab and you may use these papers in your final report, but you
must have at least 5 references beyond those provided here for full referencing credit in
your final report.
Pages in Krebs Ecology that will help with relevant concepts.
Chapter 18 pages 353- 375. Talks about concepts relevant to Priority effects
Chapter 18 – page 368 – illustrates how Jaccard’s index is calculated.
Chapters 20 and 21. Cover some of the topics related to environmental filtering.
Chapter 19 pages 390-393 – ecological niche
Blaustein, L. and Margalit, J. 1996. Priority effects in temporary pools: nature and
outcome of mosquito larva – toad tadpole interactions depend on order of entrance.
Journal of Animal Ecology 65: 77-84.
Connell, J. H. and Ralph O. Slatyer. 1977. Mechanisms of succession in natural
communities and their role in community stability and organization. The American
Naturalist. 111: 1119-1144.
Emerson and Gillespie 2008. Phylogenetic analysis of community assembly and
structure over space and time. Trends in Ecology and Evolution. 23(11): 619-630.
Robinson, J. V., and M. A. Edgemon. 1988. An experimental evaluation of the effect of
invasion history on community structure. Ecology 69:1410-1417.
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Example data collection sheet and drawings:
Table 3. Example of how to structure your data sheets/drawings for data collection. You
need not use letters to distinguish among morphospecies, names or number are fine as
well.
Treatment
AP
BP
CP
DP
EP
FP
AP/R
BP/R
CP/R
DP/R
EP/R
FP/R
AR/P
BR/P
CR/P
DR/P
ER/P
FR/P
AR/P
BR/P
CR/P
DR/P
ER/P
FR/P
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Week 1
E,K,C,F
E, K,C,E,F
K,C,E
K,C,E
K,C
K
Week 2
E,D,K,C
Week 3
E,D,C