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Biology 103 Assessing Biological Diversity An Exercise in Community Ecology Note: A calculator will be needed for this lab exercise. Objectives: 1. 2. 3. 4. Learn two methods of quantifying biological diversity. Assess biological diversity in communities. Examine and identify protistans and microscopic animals. Learn to use a compound light microscope. Introduction: Biodiversity may be defined as “The full range of natural variety and variability within and among living organisms, and the ecological and environmental complexes in which they occur. It encompasses multiple levels of organization, including genes, species, communities and ecosystems” (Nature Conservancy, 2005). Loss of biodiversity is occurring at a rapid pace. It is estimated that thousands of organisms go extinct every year. Conservation International has identified a number of ‘biodiversity hotspots’ around the world that they think should be the focus of conservation efforts (CI, 2005). Even though the term ‘biodiversity’ is often used in the context of rain forests, coral reefs and other charismatic environments, biodiversity is a concept that knows no boundaries. Weedy fields have more biodiversity than manicured lawns. Quantifying biodiversity is one method of reporting on and assessing the quality of a resource. For instance, lakes, ponds, streams, and rivers are valuable resources, providing drinking water, recreation, and habitat for a plethora of organisms. Assuring high quality of these resources is of great concern and great importance. One way environmental biologists check the quality of such resources is to assess their biodiversity. All else being equal, high biodiversity is a sign of a high quality, unpolluted resource, while low biodiversity may indicate a polluted resource. Being able to determine whether a body of water is polluted by assessing biodiversity is valuable. For instance, it is much easier to check for the presence of aquatic invertebrates in a stream than to do the myriad physical and chemical tests that would be required to check for every possible pollutant. The term “pollutant” is defined by the Virginia Department of Environmental Quality as “Dredged spoil, solid waste, incinerator residue, sewage, garbage, sewage sludge, munitions, chemical wastes, biological materials, radioactive materials, heat, wrecked or discarded equipment, rock, sand, cellar dirt, and industrial, municipal, and agricultural waste discharged into water.” (Va. DEQ, 2005). From the DEQ’s definition it is obvious that many things can be pollutants; checking for all of these in a water sample would be costly and time-prohibitive. But what all these pollutants have in common is that they have an undesired effect on living organisms. Thus checking for impacts on a community of living organisms is one way to check to see whether any pollutants are present. Once a resource has been determined to contain a pollutant, the time and effort to determine what that pollutant is can then begin. There is a national project called “Save Our Streams,” in which citizens can adopt a portion of a stream, and survey it several times a year for aquatic insects. If the insect diversity in the stream drops drastically, it may indicate a problem which warrants the more extensive, and more expensive, chemical and physical tests that could determine what the pollutant is. A program like this allows citizens without technical training and expensive monitoring equipment to nonetheless check on the quality of an aquatic resource (Izaak Walton League of America, 2005). Go to Appendix 1 to learn two methods of quantifying biodiversity. Do the problems there to familiarize yourself with these two methods. Methods: In this lab we’ll use an artificial aquatic ecosystem, and compare biodiversity between systems, and see whether we can identify a polluted system. Our model ecosystem is a freshwater “hay infusion,” which consists of grass, soil, spring water, and a community of bacteria, protistans, algae, and microscopic animals. The algae may be bacterial or protistan. The heterotrophs in the community may be herbivores, carnivores, or detrivores. The taxa present in this community may include the following: I. Kingdom Eubacteria Blue-green algae – (Cyanobacteria) – these photosynthetic bacteria are much smaller than the eukaryotic organisms listed at II and III. The cells may exist as individuals, or as clumps, or as filaments. II. Kingdom Protista Mobile ciliates (Phylum Ciliophora) -- these move relatively quickly across the field of view. cells are covered with, though the cilia may not always be visible. They are heterotrophs. The surface of their Stalked ciliates – (Phylum Ciliophora) -- these are attached to the substrate, and may be funnel-shaped. These heterotrophs have cilia which they use to produce water currents that bring food into their oral groove where they ingest it. Amoeba (Phylum Sarcodina) -- these relatively slow-moving cells move with pseudopodia, which are formed when the plasma membrane changes shape and allows the cell to flow from one location to another. They feed by wrapping pseudopodia around their prey or carrion. Flagellates (Phylum Mastigophora) -- these move with the aid of a flagellum, which is often too small to be seen even at 400x magnification. Some of them (Euglena spp.) are green and photosynthetic. III. Kingdom Animalia Rotifers (Phylum Rotifera) – members of this phylum are often called “wheel animals” because at their anterior end, they have a ring of constantly whirling cilia, as the rotifer produces currents that move food into its mouth. At the posterior end, they often have one or two pointed “toes.” These multicellular organisms have a complete digestive system that is sometimes full of algae; through the transparent body wall, the digestive system may appears as a green tube running the length of the body. Annelids (Phylum Annelida, Class Oligochaeta) This phylum includes the earthworms, which you are unlikely to see in a hay infusion. However, smaller relatives of the earthworms, which can be seen as segmented and often covered with bristles may be seen. Roundworms (Phylum Nematoda) – a living roundworm is easily identifiable by its method of movement: they move by thrashing back-and-forth rapidly. These worms are tapered to a point at both the anterior and posterior end, and are unsegmented. Water mites (Phylum Arthropoda, Class Arachnida) though these are arthropods, there is no evidence of segmentation visible. These relatives of terrestrial mites and ticks have eight jointed legs and a spherical body. Methods: 1. Use a compound light microscope to observe the hay infusions. Refer to Appendix 2 for directions on using the microscope. 2. Learn to recognize the organisms in your aquatic ecosystem. Use the pictorial guides to identify the organisms. You won’t be able to identify these organisms to species, but, as a class, decide at which taxonomic level you wish to identify them. For example, do you want to attempt to classify them all to the proper Genus, or to the appropriate Phylum? For example, if to phylum, you might give them names like “Ciliophoran #1”, or “Rotifer #1”, and so on. 3. Decide what variables you need to control and how you will control them. About Controlling variables: When doing the survey, an equal amount of effort must be spent surveying each of the treatments, if the comparison of the diversity indexes are to be meaningful. Before you start your surveys of the communities, you should decide on the amount of time you will spend, what magnification you will use, whether you look at just one field of view or scan back and forth several times, and whether you use just one observer or multiple observers. Any other factors you can think of that might effect how many organisms should be controlled as well. All of these factors are your controlled variables. 4. Conduct your survey. List your results in Table 1. 5. Quantify “taxon richness” and “taxon heterogeneity.” (“Taxon” is used here rather than ‘species,’ since identification of these organisms to species is highly technical.) Results: List the taxa seen, and the number of individuals of each taxa seen, and calculate Simpson’s Index. (There are tables in Appendix 1 to help you in your calculations.) Treatment name Taxon name Number of individuals Treatment name Taxon name Number of individuals Table 1: Taxa seen in two hay infusions Taxon richness Taxon richness Simpson’s index Simpson’s index Conclusions and Discussion: Compare the measures of biodiversity between the communities. Answer the assigned questions on a separate piece of paper. 1. Is there any evidence that one of the ecosystems contains a pollutant? Explain. 2. Does species richness differ between the two treatments you surveyed ? 3. Does species heterogeneity differ? 4. Does biodiversity differ? 5. If biodiversity differs in your two treatments, what are the components of the Simpson’s index that make the index different? 6. Is the total number of individuals in both groups the same? 7. Is there any evidence that the available niches are different in the two treatments ? 8. Do the communities differ in the two treatments? 9. Do you think there’s any difference in the two ecosystems you surveyed? What evidence do you have for that? 10. How could you go about identifying the pollutant, if any, in this model ecosystem? 11. If an environmental biologist surveyed a mountain stream for aquatic insects, and found relatively low diversity, compared to the other streams he’d surveyed in his life, how could he figure out whether or not the water was polluted? 12. Can a pollutant eliminate a niche? Explain. 13. Draw a food web of the taxa in one of the hay infusion communities you surveyed. 14. Besides pollution, as modeled here, what other factors might affect biodiversity? Literature Cited Conservation International. (2005). Biodiversity Hotspots Conservation International. [Online]. Available: http://www.biodiversityhotspots.org/xp/Hotspots Izaak Walton League of America. (2005). Save Our Streams. IWLA. [Online]. Available: http://www. iwla.org/sos [October 10, 2005]. Nature Conservancy. (2005). How We Work; Conservation By Design. The Nature Conservancy. [Online]. Available: http://nature.org/aboutus/howwework/cbd/science/art14307.html. [October 10, 2005]. University of Delaware, Department of Biological Sciences. (2005). Virtual Microscope. U. of Delaware. [Online]. Available: http:\\www.udel.edu/scope [October 10, 2005]. Virginia Department of Environmental Quality. (August 31, 2005). Glossary of TMDL Terms. Va. DEQ. [Online]. Available: http:\\www.deq.state.va.us/tmdl/glossary.html [October 10, 2005]. Appendix 1 Methods of Quantifying Biodiversity Introduction: A community is a group of different species occupying a specific area. Communities are often described briefly by their dominant vegetation, as with the terms "grasslands," "deciduous forest," or "rain forest." However, communities can be more precisely described and quantified. One property of a community that is quantifiable is its structure. Community structure includes characteristics such as the number of species, and the relative abundance of each species. The number of species present in a community is the Species richness; it is simply the number of different species of organisms in the community. Just count the species in your sample to calculate it. Measuring the relative abundance of species or the ‘Species heterogeneity’ is a bit more complex. Species heterogeneity takes into account the relative numbers of individuals of the various species in a community. 'Hetero' is from a Greek word meaning ‘different’; ‘homo,’ is from a Greek word meaning ‘same.’ A lawn with only one species of grass in it would be homogeneous. If it had a few dandelions in it, it would be less homogeneous since now it has two different species. A meadow with several species of wildflowers in it would be even less homogeneous and more heterogeneous than the weedy lawn. For an example, compare the data from the two communities below. The numbers represent the number of individual organisms of each species. I.e., in community 1 there are 18 of species A, 1 of species B, and 1 of Species C. Species A Species B Species C Community 1 18 1 1 Community 2 7 8 5 The Species richness is 3 in both Community 1 and 2; it is the same in both communities, but the communities are quite different. By measuring species heterogeneity biologists can place a numerical value on the diversity in communities and then use that value when describing or comparing communities. We will measure species heterogeneity by calculating a number known as Simpson’s index. Simpson’s index is a scale ranging from 0 (no heterogeneity and no diversity) to a maximum close to 1 (high heterogeneity or lots of diversity). This index is a measure of the probability that two organisms picked at random from the community will be different species. If there is only one species in the community, then the probability of picking two different organisms will be 0, and Simpson’s index would be 0. If there are two species in the community, then the chance of obtaining two species in a random pick of two individuals will be higher, i.e. some number above 0 but less than 1. This is how to calculate Simpson’s index: Let pi stand for the proportion (a fraction of the number of species to total number of individuals in your sample The probability of picking two individuals of this species at random is their joint probability, that is: [(p i)(pi )], or (pi)2. If we add up the joint probabilities for each species, and subtract it from 1, we get Simpson’s index. ). [The subscript 'i' means to calculate the proportion for each species.] s D = 1 - ∑ (pi)2 i=1 where D = Simpson’s index pi = the proportion of individuals of species 'i' in the community s = number of species in the community ∑ means sum of all the (pi)2 , one for each species in the community. Example 1: Using the data from Communities 1 and 2 that were mentioned in the Introduction. For Community 1: For Species A, (pi)2 For Species B, (pi)2 For Species C, (pi)2 = (18/20)2 = (1/20)2 = (1/20)2 = 0.81 18 of 20 individuals are Species A = 0.025 1 of 20 individuals are Species B = 0.025 1 of 20 individuals are Species C ∑ = 0.815 Simpson's Index is 1 - 0.815, or 0.185 for Community 1 For Community 2, more briefly: Simpson's Index = 1 - [ (7/20)2 + (8/20)2 + (5/20)2] Simpson's Index = 1 - [0 .1225 + 0.16 + 0.0625 ] Simpson's Index = 1 - [0.345] Simpson's Index = 0.655 Practice Problems: Tables to organize the data needed to calculate Simpson's Index are found on the last 2 pages of this exercise. 1. Calculate Simpson's index for the community pictured below: . 2. Two bags of Mixed Nuts” were purchased. One was called “Mixed Nuts” and the other “Deluxe Mixed Nuts.” Which bag has the greater diversity? Compare the diversity of these two kinds of mixed nuts. Species Brazil Nut Cashew Pecan Almond Hazel Nut Peanut Number in "Mixed Nuts" 1 8 1 15 5 85 Species Richness for Mixed Nuts = Diversity Index for Mixed nuts = Species Richness for Deluxe Mixed Nuts = Number in "Deluxe Mixed Nuts" 8 15 7 18 30 0 Diversity Index for Deluxe Mixed Nuts = 3. Table 1: Species Number pi = Number /Total Number pi2 Σpi2: Total Number: D = 1 - Σpi2 = Table 2: Species Total Number: D = 1 - Σpi2 = _________ Number pi = Number /Total Number Σpi2: pi2 Table 3: Species Number pi = Number /Total Number pi2 Σpi2: Total Number: D = 1 - Σpi2 = Table 4: Species Total Number: D = 1 - Σpi2 = _________ Number pi = Number /Total Number Σpi2: pi2 Appendix 2 Use of the compound light microscope A virtual microscope, with directions for use, is available at: http://www.udel.edu/scope. The Virtual Scope at the site is very similar to the scopes used in lab. Figure 1: the NikonTM Alphaphot-2 microscope, one of the kinds of microscopes used in the General Education Biology labs at Radford University. I. Before looking through the microscope: 1. The microscope should be plugged in and turned on. 2. Rotate the revolving turret containing the objective lenses so that the 4x objective lens is in a vertical position (The 4x lens is the shortest objective lens on the microscope). 3. Put the slide on the stage in the slide holder. 4. Center the object over hole in stage using the Y-stage and X-stage control knobs. 5. The stage should be all the way up; move it all the way up using the coarse focus knobs – the largest knobs on the side of the scope. II. When looking through the oculars on the scope 1. If you can’t see a perfect circle of light (the field of view) something is wrong. Make sure you’ve done 1 – 5 above. (With the stage all the way up, the object you are viewing will not be in focus.) 2. Using the coarse focus knobs, slowly move the stage down until the object is focused. 3. Use the fine focus knobs to adjust your focus 4. Adjust the light, either with the brightness control knob (like a dimmer switch, it goes from 1 to 10), or use the substage diaphragm lever (a lever below the stage that moves back and forth) or raise and lower the condenser (a knob below the stage moves the condenser; condenser so-called because it can “condense’ the light into a sharp beam, if desired.) 6. Center the image using the stage control knobs before you go to the next higher power if you should desire greater magnification. Proper Care for your Compound Light Microscope 1. Always carry the microscope in an upright position with both hands, one supporting the base and the other grasping the arm. 2. Never clean the ocular and objective lenses with anything but the lens paper supplied. 3. Notify your instructor if your microscope is out of order. Do not attempt to repair it yourself. 4. Use only the knurled ring to change objective lenses, NOT the lenses themselves. 5. Always have the LOW power lens in place before inserting a slide. Always return to LOW before removing a slide. 6. Always focus and center the object and adjust the light before turning to the next higher power lens. 7. Use the coarse focusing knobs only when the LOW power objective is in place. 8. Clean your microscope slides with lens paper before examining. 9. When you are finished using the microscope: a. Make sure that the stage manipulator bar is not extended out to the left. b. Unplug the microscope and coil the cord beside the microscope. c. Put the dust cover back on the microscope. d. Carefully slide the microscope back into the cabinet, unless instructed otherwise.