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Ecology – Pollen Core Lab The last part of the course focuses on ecology, the study of how organisms interact with each other and their non-living environment (e.g. water, soil). This two-part lab will touch on a few concepts covered in lecture (e.g. population growth and community diversity), but is primarily geared towards exposing you to a common technique used by ecologists to study past ecological changes. Paleoecology, ecology of the past, can help address questions such as: what was the climate like thousands of years ago? Are the plant and animal species living in an area today different from those that inhabited the area thousands of years ago? An understanding of past ecological changes can help us make predictions about future change. It can also help us understand the impact of human activities (e.g. industrialization) on ecosystems, and therefore information gathered by paleoecological studies is relevant to the field of conservation biology. This lab will focus on a technique commonly used by paleoecologists to reconstruct the vegetative history of an area. We’ll use pollen analysis to explore changes in the diversity of gymnosperms (a large group of seed-producing plants such as pine) near Lily Pad Pond in Colchester, VT. In the first lab, you’ll prepare slides containing pollen from a soil sample collected from deep under the shoreline of Lily Pad Pond, and learn to identify pollen grains of eight major gymnosperm genera. During the second lab period, you’ll count pollen grains to determine the relative percentage of each gymnosperm genera, and pool your data with your lab mates to generate a gymnosperm pollen profile – a graph depicting changes in gymnosperm diversity over time. You’ll then analyze the pollen profile to look for trends and think about the applications and limitations of pollen analyses.

IMPORTANT: You need this handout for BOTH parts of the lab (two lab periods). Please make sure you PRINT it and BRING it with you to both lab periods! OUTLINE OF ACTIVITIES • Part I: Prepare pollen slides and practice identifying pollen

• Part II: - count pollen to determine the relative abundance of gymnosperm genera from a particular time period

(pollen core depth) - pool your lab sections data to generate a complete pollen profile

LEARNING OBJECTIVES • Understand the applications and limitations of pollen analyses

• Learn how to identify pollen spores of eight gymnosperm genera

• Gain further practice gathering, organizing, analyzing, & presenting data • Interpret pollen data to develop hypotheses regarding the vegetation and climactic changes that occurred

over the past ~8,000 years in Colchester, VT INTRODUCTION Pollen Analyses Although fossils may be the first thing that comes to mind when you think about evidence that can give us clues to past events, many species’ pollen grains (a bunch of plant sperm encased in a hard shell) are preserved remarkably well over time. The physical structures of different species’ pollen grains are different,

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and therefore examining the composition of pollen from a soil sample can give ecologists clues as to the composition of species present in an area when that soil was deposited. Pollen analyses are most useful for assessing changes in the diversity of plant species whose pollen is dispersed by wind. These species generally produce a lot of pollen. For example, white pines produce pollen in mid-June and you’ve probably seen clouds of the yellow pollen being blown everywhere. Much of the pollen does not reach its intended destination, the egg of another plant, and instead settles wherever the wind takes it, including the surfaces of lakes and ponds. Pollen that lands on the surface of a lake or pond settles to the bottom. Gradually, years of sediment build up, so that an undisturbed lake bottom can contain an unbroken record of pollen fallout over many thousands of years. This is why pollen cores – literally a long “core” of soil that extends many meters below ground – are most often obtained from lake beds (see figure below). The soil at the top represents current day, and the soil at the bottom may be from many thousand years ago depending on the depth of the core. Plant’s that use pollinators to disperse their pollen generally produce less pollen, and therefore are not well-represented in pollen cores. Additionally, wind-pollinated plant species differ in how much pollen they produce and how far their pollen is carried by the wind. This is something that needs to be taken into consideration when analyzing pollen core data. Once a pollen core is obtained, pollen grains are isolated from soil samples of various depths and stained to make the pollen grains more visible. Scientists then prepare slides using the stained pollen grain samples and count the number of pollen grains present for a particular set of species. Identifying pollen grains accurately is a skill that takes quite a bit of practice! Typically, palynologists (scientists who study pollen) go through intensive training using “pollen identification keys,” and become experts in identifying pollen grains of the plant species in a particular geographical area. A palynologist will typically spend several hours analyzing a single sample, and count between 300 and 1,000 pollen grains. Due to the subjective (and challenging) nature of pollen grain identification, scientists are working to generate quantitative identification procedures that analyze images obtained using high-powered microscopes. Most of the eight gymnosperm genera we’ll focus on, described in a bit more detail in the next section, have fairly distinct pollen grain structures. However some of them, particularly Abies (fir), Picea (spruce), and Pinus (pine) are quite tricky to distinguish (see image on the next page). Another thing to keep in mind is that pollen grains are three-dimensional structures, so if you look at them from a different angle, they’ll look different! You won’t become an expert, but he practice you’ll get in the first lab period is VERY important! After counting pollen grains, scientists determine the relative proportions of each species’ pollen, and generate a graph known as a pollen profile. An example of a pollen profile obtained from Bugbee Bog in Caledonia County, VT is shown on the last page of the handout. Time is on the vertical axis, with older samples at the bottom and younger samples at the top. Each species has its own horizontal axis that indicates the relative proportion of that species’s pollen at a particular time. By assessing how the proportions of different species’ pollen changes over time, scientists can use what they know about the particular species to make inferences regarding past climate change, when certain species populated an area, how invasive species have altered the community composition, and more. For example, if the relative proportions of plants that thrive in colder climates diminished and the relative proportions of plants that grow better in warmer

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climates increased, you could infer from that result that the average temperature had increased during that time period. Because pollen profile graphs require special software to generate properly, our graphs will be a bit different, but will still provide a visual representation of how the relative proportions of gymnosperm genera have changed over time. In some cases, additional analyses are used to estimate the age of the soil samples at each depth. For your purposes, we’ll simply use a relative time scale. Based on the Bugbee Bog data, we can, very roughly, estimate that the oldest soil in our core is probably 8,000-9,000 years old. Gymnosperms Gymnosperms are a large group of seed-bearing plants that include familiar species, such as pines, and the majority are wind pollinated. The eight gymnosperm genera (groups of related species) listed below are common in the New England area. As you can see by the descriptions and images below the list, each genera has a unique pollen grain structure. Several of the genera’s pollen grains have bladders, which are air sacs that look like “ears” sticking out from the body of the grain. Other pollen grains have pores that look like little holes or bumps on the surface. Distinguishing between different species’ pollen grains within each group takes many months of training, so we’ll just focus on determining the relative proportion of pollen grains for each genera. As part of your first assignment for this lab series, you’ll gather some background data on each of the eight genera. In addition to these gymnosperm genera, you’ll see other species’ pollen grains in your samples. However, these species are relatively rare. Due to time limitations, we won’t be able to analyze a large enough number of pollen grains to obtain a sample size that would accurately reflect the true proportion.

• Abies (fir) = large-sized with relatively small bladders connected at an angle; distinct separation between body and bladder

• Picea (spruce) = medium-sized with relatively larger bladders connected at slightly less of an angle; “smooth transition” between body and bladder (not distinct separation)

• Pinus (pine) = small-sized with bladders connected at an angle; constricted where bladders connect to body (looks like it pinches in where they connect)

• Tsuga (hemlock) = large-sized sphere with bumpy surface • Larix (larch) = small-sized sphere; relatively smooth surface

• Betula (birch) = small-sized with three pores that appear to stick out from the surface; slightly triangular shape

• Fagus (beech) = medium-sized sphere with three pores and three grooves; pores do not stick out from surface; grooves may not be distinctly visible

• Quercus (oak) = small-sized pollen with three deep grooves IMPORTANT NOTE: These images are NOT to scale. You’ll be provided with additional images in lab.

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Part I: Pollen Slide Preparation Lab Activities Outline I. Quiz

To prepare for the quiz, read this handout carefully. Pay particular attention to the methods section. Although you are not expected to memorize specific details (e.g. how long you centrifuge your sample; volume of potassium hydroxide), you SHOULD be able to answer questions such as how many sides do you need to prepare, how many sections of the pollen core will you analyze, etc.

II. Lab Discussion: Pollen Core Lab overview III. Pollen Sample Preparation & Pollen Identification Practice IMPORTANT: Your Pollen Core Lab Background Information assignment (called “Bog Information Questions” in the lab schedule) is due at the BEGINNING of lab next week! The assignment is posted on BlackBoard. You should type your answers and bring a PRINTED copy of your assignment to lab next week. Part II: Pollen Analysis Lab Activities Outline I. Quiz

To prepare for the quiz, review this handout thoroughly. Pay particular attention to the overall goal of this analysis (i.e. why do a pollen analysis), and how you will collect/analyze your data.

II. Lab Discussion: pollen counting overview III. Data Collection – Pollen Counting IV. Pool Data and Generate & Interpret Pollen Profile IMPORTANT: Your Pollen Core Data Analysis is due BEFORE you leave lab TODAY. Make sure you give your lab TA the paper with your data tables and answers to the “Pollen Profile Generation & Analysis” questions!

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METHODS Below are the methods for preparing pollen grain slides, and collecting and analyzing data. You’ll complete parts A-E the first lab period, and part E during the second lab period as well as the Part II Questions. Because the process of purifying samples and counting pollen can take a while, your group will be assigned ONLY ONE section of the core, and then you will share your data with the rest of your lab. IMPORTANT: � You will be working with potassium hydroxide which can cause severe burns. Wear GLOVES at all times

and PUT THE CAPS ON all bottles immediately after use! � Your goal is to prepare FOUR good slides to count next lab period. The better your slides are the easier

your counting will be, so please take the time to prepare your slides carefully! Sections of the pollen core obtained from Lily Pad Pond are laid out on the bench. Each piece half-pipe contains 0.5 meters of core, and is marked at the top and bottom with its depth in meters. They’re wrapped in aluminum foil to prevent the soil from drying out, which could damage the samples. Your lab TA will assign you a particular depth. WRITE IT DOWN below.

depth (cm): ______________________ A. Obtain Your Soil Sample 1) Gently open up the interior of the core with a spatula: remember that the interior is pristine and the exterior

was most likely contaminated during collection. Be careful NOT to not to smudge material from the outside inward as you pry open the core.

2) Scrape off a pea-sized piece of soil from near the center of the core. Push the core back together and re-

wrap the foil. 3) Place the soil in a clean centrifuge tube. B. Clean Up Your Sample (Remove Excess Debris) 1) Using your spatula, break up the soil sample as much as possible. 2) Fill the tube one-third full with distilled water. 3) Wait ~1 minute for the soil to become thoroughly saturated. Use a vortex mixer to thoroughly mix the water

and soil. 4) Using a small piece of masking tape, label the top of a clean centrifuge tube with your initials. Place TWO

layers of cheesecloth in a funnel (it’s packaged in layers of four), and put the labeled tube under the funnel. 5) Filter your soil mixture through the cheesecloth to remove the largest impurities (e.g. small twigs). 6) Centrifuge (spin) your tube at 3200 rpm for 4 minutes. IMPORTANT: The centrifuge must be balanced, so

place your tube across from another group’s tube. Your lab TA will help facilitate using the centrifuge.

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7) Carefully pour off the liquid on top into the designated waste vessel. Resuspend the pellet (soil sample) at the bottom of the tube in 5 mL of 10% Potassium Hydroxide (KOH). Be careful with the KOH and make sure you are wearing gloves!!

8) Place your tube in a warm water bath for 20 minutes. The potassium hydroxide and heat will help break-up

excess debris, but will not damage the pollen grains. 9) Remove the tube from the warm water bath and fill to near the top with distilled water. Do NOT let it

overflow! 10) Centrifuge at 3200 rpm for 4 minutes. Again, be sure the centrifuge is balanced. 11) Carefully pour off the supernatant into the potassium hydroxide waste container. C. Stain Your Pollen 1) Add one drop of safranin stain to the pellet in the centrifuge tube. (Safranin is a stain used in many

biological applications.) 2) Clean a spatula using hot water, and then wipe it absolutely dry with a kimwipe to ensure that no

contaminating pollen is present. Stir the pellet up gently with the spatula to mix the stain in. 3) Let the stain soak into the pollen for 5 minutes. D. Prepare Slides 1) Obtain a clean slide. Using a permanent marker, label the edge of the slide carefully with the depth in cm

from which your sample came and your initials. 2) Put one SMALL drop (not more) of glycerol onto the center of the slide. 3) Remove a SMALL bit of the stained pellet from the centrifuge tube, and stir it slowly and thoroughly into the

glycerol. Remember that you need to make four good slides! 4) Carefully cover the pollen suspension with a cover slip. To reduce the number of bubbles trapped under

the cover slip, start by placing one edge of the cover slip on the slide near one edge of the glycerol suspension, then slowly lower the cover slip so that it contacts the suspension.

5) Check your slide to see whether there’s too much pollen (you can’t distinguish individual grains) or too little

(if they’re very sparse you won’t have enough to count). Check with your lab TA to see if you’ve got a good amount.

6) Repeat the steps above as many times as it takes to make FOUR good slides. If your last slide had too

much pollen, use less of your pollen sample. If it had too little, use more. You can use the same pollen sample over and over as long as it lasts. NOTE: Each partner should try to make two good slides!

7) Once you have FOUR good slides, label them A-D using a permanent marker.

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8) Rinse the stained sample from the centrifuge tube into the liquid waste container using a squirt bottle. After

that, use soap and a brush in the sink to clean the tube completely.

E. Pollen Identification Practice Use the pollen description and images in this handout, plus the additional identification “flow-chart” and images provided in lab to help you identify pollen from the eight common gymnosperm genera. During the first lab period, you’ll simply gain practice identifying pollen. For the second lab period, you’ll perform careful counts to collect data. When you collect data, you need to figure out a strategy for counting EACH pollen grain ONCE and ONLY ONCE. As you practice identifying the pollen grains, think about the best way to do this. A good way to double check your identification skills is to perform a count on a small group of pollen and then have your lab partner do the same. Compare your counts to see if you agree. IMPORTANT – Remember that there will be pollen from other species in the sample too! Focus ONLY on the eight gymnosperm genera. They should make up the majority of pollen grains in your sample. There may be other species’ pollen grains present in your sample, but they are too few in number to obtain an accurate count. Depending on your sample, you might not see pollen grains of all eight gymnosperm genera. F. Pollen Profile Generation & Analysis As noted in the introduction to this lab, you won’t be generating a “classic” pollen profile graph like the one from McDowell, et al. shown on the last page. Instead, we’ll use a couple different types of graphs in Excel that let you see the trends in different ways. You’ll pool the data for your section to generate the graph so that you have a much more complete view of changes in the abundance of particular species’ pollen over time. Although you are not responsible for generating the graphs, you ARE responsible for recording and analyzing your data, and answering questions (see below). 1) Take out a piece of lined paper and write your NAME and LAB SECTION on top. Write the depth (in cm)

from which you obtained your sample. IMPORTANT – Each person must turn in their OWN work! 2) Discuss with your lab partner exactly how you plan to collect the data for your sample. You need to count

AT LEAST 200 pollen grains from your four slides. If you’ve got good slides, counting more is better! You should take turns looking at the pollen and recording the data. Remember that you must:

- count each pollen grain ONCE and ONLY once - count ONLY pollen grains from the eight gymnosperm genera - count at least 200 total pollen grains

3) BOTH partners should generate well-labeled tables in which to record data. It’s probably best to record

everything in one person’s table and then copy it when you’re done. 4) Get the slides you prepared in the first week of this lab, and count your pollen. Again, each person should

spend some time counting and sometime recording. You can use one slide or multiple slides. If your pollen is sparse, you may need to use all four to be able to count 200 pollen grains.

5) When you’re done counting, make sure each person has a complete (and organized!) data table on their

sheet.

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6) Create a table to summarize your data analysis (see next step). Think about the best way to organize your results. Your table should include:

- count for each of the eight gymnosperm genera - total number of pollen grains counted - proportions of each of the eight gymnosperm genera in your sample

7) Now you need to analyze your data. It’s up to you whether you do the calculations in Excel or on a

calculator. To get the proportion of a particular genus in your sample, just divide the number of pollen grains counted for that genus by the total number of pollen grains counted. Your proportions should add up to one.

8) Enter your results in the table you created on your sheet of paper AND in your lab section’s Excel

spreadsheet. Your lab TA will project the graph of your section’s results on the screen so that you can use it to answer the questions below.

REFERENCES McDowell, et al. (1971) “Palynology and radiocarbon chronology of Bugbee Wildflower Sanctuary and Natural Area, Caledonia County, Vermont.” Pollen et Spores 13:73-91.

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PART II QUESTIONS Write your answers to these questions NEATLY on the same sheet of paper where you have your data tables. Be sure to LABEL each question clearly! Focusing on YOUR data, answer the questions below. 1) Which of the genera was/were most abundant in your sample? 2) Which of the genera was/were least abundant in your sample? 3) Were there any genera missing from your sample? If so, which ones? 4) Look at the pollen profile from McDowell, et al. 1971 (last page of this handout). Depth in meters is marked

along the right-hand axis. Roughly estimate the portion of the McDowell profile that corresponds to your depth (1000 cm = 1 meter). Are your most and/or least abundant genera similar to those reported by McDowell? If not, which genera were most/least abundant in the sample from that depth that McDowell et al. collected from Bugbee Bog? Note that the McDowell analysis did not include Larix (larch)

5) Search the web to find out some more information on the genera/genus that was MOST abundant in your

sample. List THREE pieces of information on your sheet and use at least TWO different reliable sources. Indicate the sources for your information (e.g. Wikipedia, USDA, US Forest Service, IUCN RedList). You do not need to write out the web address. Things you should focus on finding are listed below. (Hint – Remember the plant guides you used for your first Pollen Core Lab assignment!)

- the type of soil in which it grows best (e.g. acidic, sandy, silt) - “landscape” areas in which it is typically found (e.g. near lakes, on mountains) - particular species of the genus/genera that are common to Vermont and/or the New England area - description of the trees themselves (e.g. bark texture, height, type of needles) - locations other than North America in which the genus is found - uses – medicinal, agricultural (e.g. food, other products), etc. - any particularly unique/interesting fact that is relevant to this analysis

Focusing on your LAB SECTION’S results (graph on the screen), answer the questions below. 6) For EACH of the eight genera, BRIEFLY describe how their abundance changed over time based on the

data collected by your section. Remember that deeper is older and shallower is younger. 7) At which depth(s) is/are all eight genera present? 8) Is there one genus that sticks out as being particularly abundant at all depths (times)? 9) Is there one genus that appears to be particularly rare? 10) Are there depths (times) that have a similar distribution (i.e. the relative proportions of each of the genera

are similar)? What might that indicate about the climate during these two time periods?

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