A Hitchhiker (Pollen)’s Guide to the Galaxy (Iowa Flora)

Pollen. It makes you sneeze, fertilizes plants, and transports you through time to ancient landscapes. Wait, what was that about time travel? Well, maybe we can’t quite hop onto a pollen grain like a microscopic version of Doc Brown’s DeLorean, but it can play a compelling role in paleobotany, the study of fossilized plant remains. With the help of two of our own enthusiastic Docs– Dr. Eckhart of the 2020s and Dr. Graham of the 1960s– and a very old chunk of partially decomposed plant matter (peat), we embark on an adventure through time in hopes of catching a glimpse of the flora of our college’s campus… 27,000 years ago.

If you haven’t explored the other entries on The Natural History of Grinnell College, allow us to catch you up. We are undergraduate students in Grinnell, IA taking a course entitled Evolution of the Iowa Flora under the instruction of Vince Eckhart. In the 1960s, a professor by the name of Ben Graham stashed away a sample of peat that was unearthed during the construction of Roberts Theater. Picking apart this peat, Graham and his students began to decipher the clues of Iowa of Old. Mysteriously, Graham didn’t seem to follow up as he proposed in his 1962 paper. Neither did anyone else, until 2018. Just a few years ago, the peat was unearthed once again, that time from a cardboard box in the science building basement. After a COVID hiatus from in-person classes, we (along with Team Macrofossil and Team Wood) pick up where the 2018 students left off, this time with revised methods. To behold this peat, to view its pollen, insect exoskeletons, papery plant tissue, and wood under the microscope is to face deep history. These fragments of Iowa flora lived long before climatic changes gave rise to the most well-known ecosystem in our area– the prairie– about 10,000 YBP. Today, as we walk over a lawn manicured by the College in a state where about 85% of the land is used for agriculture, we wonder what other remnants of the past might reside just a few meters below our feet. We begin to wonder what it was like here when the organic matter was laid down on the floor of a boreal sphagnum bog (like those in modern-day Canada) 27,000 years ago. Written here is our contribution to the tale of the Grinnell peat, a story barely known but slowly revealing itself to those who are curious enough to look closely.

As scientists in a biology laboratory, our portal to the past takes the form of microscope slides. We prepared 30 microscope slides as outlined in Figure 1. In November 2022, we examined and photographed the slides. We compared our photos taken at 200x magnification with the images on the Global Pollen Project website (https://globalpollenproject.org/Taxon). Using this technique, we identified pollen grains from the genera of Pinus (pine), Picea (spruce), and Quercus (oak). 

Previously, Ben Graham identified the pollen of “spruce, fir, pine, alder, maple, numerous 

‘betulaceous’ grains, and others characteristic of northern coniferous forest, or transition thereto”. Thus, our findings corroborate Graham’s identification of spruces but also evidence that there were oaks present on the land now referred to as the Grinnell College campus. Our identification time was limited, and thus suggest further examination of our slides to identify other pollen grains present– whether they corroborate Graham’s other findings or introduce new evidence. 

Figure 1. Picture collage of the steps in the preparation of pollen for analysis. 

We used modified and simplified methods based on a newly published protocol developed in 2022, by Santos and Lerdu. This procedure differs from previous methods of pollen extraction, as it doesn’t use corrosive acids. We created 26 microcentrifuge tubes, each filled with crushed peat dirt. Using a vortex mixer and microcentrifuge, we mixed the peat sample with potassium hydroxide to dissolve impurities, rinsed a number of times using distilled water, and used zinc chloride to create a density gradient that suspended the pollen in the liquid. Using distilled water again, we centrifuged the solutions, making the pollen clump into a pellet. We added glycerine to each tube to give the pollen something to stick to and made microscope slides for examination. For more detailed methods, see Santos and Lerdu 2022, or additional note at the end of this post.

A note on contamination: A classmate of ours on Team Macrofossil brought to our attention a grain of pollen reminiscent of a fidget spinner that he found while exploring macrofossils under the scope. We identified it as belonging to Oenothera, the evening primroses. However, this finding does not necessarily mean that there were evening primroses present in the Grinnell flora 27,000 years ago. It
could, rather, be evidence of present-day flora in the Grinnell College biology corridor: We suspect this pollen to be contamination brought into our lab from our professor’s Clarkia xantiana (an evening primrose) research laboratory just down the hall. 

During our analysis, we observed that the most common type of pollen in our sample was made of a single pollen grain with two air bladders, arranged similarly to Mickey Mouse ears. While the pollen of many conifers share this general shape, we determined that these grains were most likely from a pine tree based on its small size. Notably, spruce pollen, which we identified as being the second most abundant pollen in the sample, has a very similar grain shape to pine, with similar function, so a critical difference is their relative sizes; Picea grains are roughly 110 to 150 micrometers long compared to Pinus grains, which are 50 to 110 micrometers long (Figure 2 and 3). The quantity of the pollen suggests that pine trees in particular were abundant around Grinnell when this peat formed, though we may not be able to know exactly how close they were to the site of our peat. The air bladders we see in both Pinus and Picea suggest the pollen was mostly spread by the wind rather than animal pollinators, as research has shown that these structures improve the pollen’s ability to pollinate by wind dispersal, acting almost like a parachute, slowing the pollen’s fall and allowing to travel further. As a result, the pollen can travel massive distances under the right conditions, making it hard for us to assume where exactly the pine and spruce may have been in relation to the peat.

Figure 2: Pinus (pine) pollen sourced from Grinnell peat and photographed under 200x magnification. At right is a 2018 reference slide prepared by Professor Eckhart with a slightly different procedure, leading to the color variation between the two. The figure on the left, sourced from Leopold & Zaborac-Reed (2014), was critical in helping distinguish Picea and Pinus based on size and shape characteristics.

Figure 3: Picea (spruce) pollen photographed under 200x magnification. At left is the spruce reference slide prepared by Professor Eckhart in 2018. 

As we continued our analysis we found a less abundant pollen grain that we believe is Oak (Figure 4). The shape and size of the pollen convinced us that this is a match, and tells us that, similar to present-day Grinnell, oak trees were here 27,000 years ago.

Figure 4: Quercus (oak) pollen taken using a microscope set to 200x magnification. The image on the left is a reference slide made in 2018, showing contemporary oak pollen.

The image on the right is of oak pollen found in the peat.

Amidst the pine, spruce, and oak pollen grains, we saw a rather abundant pollen grain we didn’t recognize (Figure 5). As we worked on identifying this sample, we learned that as pollen dries, it might change shape. We suspect that the grains we photographed are on the drier side, which made identification more of a challenge. A 1972 paper by V. Sh. Vagababian describes the morphology of magnolia family pollen. Vagababian measured the length of these pollen grains to be around 50 μm, which is similar to the size of our samples. If this is a match, tulip poplar trees may have been present in Grinnell’s ancient landscape! 

Figure 5: The two images on the left are pulled from the page on PalDat, a palynological database, for Liriodendron tulipifera (commonly known as tulip poplar) showing a dry pollen grain on the top and a hydrated grain on the bottom. At right, the pollen we suspect to be tulip poplar under 200x magnification. Does it look like a match?

In collaboration with the wood and macrofossil group, we hope to increase our knowledge about Grinnell’s flora. However, due to time constraints and unknown finds, we know there is still so much left to do to uncover more knowledge about our college campus’ past. As you traverse our campus or wherever you are, remember that you are walking on a profound mass of history– what other stories does the land have to tell us? Stay curious. 

Authors:

Sonia Edassery, Joanie Fieser, Athena Frasca, Isabelle Jacqmotte-Parks, and Sam Takahashi

Notes: 

The following procedure describes the collage in Figure 1, stepwise: 

In a sterile environment, we created 26 microcentrifuge tubes with about 0.2 mL of mashed peat dirt and 1.5 mL distilled H2O (1). We then vortexed and spun the solution at 3000 rpm for 4 mins. We then added 1.5 mL 10% KOH to only the pellet and vortexed (2). Next, the tubes were put into an 85°C dry bath for 6 mins (3). We then vortexed the solution and spun it at 3000 rpm for 4 mins. The resulting solution looked dark and opaque, like coffee. We added 1.5 mL distilled water to only the pellet, vortexed it briefly, and centrifuged the tubes at 3000 rpm for 4 minutes (4). This step was done 6 times until the solution was light, transparent brown. We then removed the supernatant, or extra liquid, and air-dried the leftovers (containing pollen!) for 5 mins.1 mL of 1.9 g/mL zinc chloride was then added and vortexed (5) and spun at 1000 rpm for 4 min. We then transferred the supernatant to new microcentrifuge tubes with 1.5 mL distilled water, then vortexed and spun them at 3000 rpm for 4 minutes, twice (6). An equal amount of pellet, containing the pollen, and 80% glycerine was finally added to the tube. We made microscope slides by placing a drop of tacky glue on a clean microscope slide with some contents of the tube (7). We used microscopes set to 200x magnification, meaning every 10 units measured to be 45 micrometers (8).

References:

Graham Jr, B. F. (1962). A post-Kansan peat at Grinnell, Iowa: a preliminary report. In Proceedings of the Iowa Academy of Science (Vol. 69, No. 1, pp. 39-44).

Leopold, Estella & Zaborac-Reed, Stephanie. (2014). Biogeographic History of Abies bracteata (D. Don) Poit. in the Western United States. 

Rudney de Almeida Santos & Marie-Pierre Ledru (2022) Acid-free protocol for extracting pollen from Quaternary sediments, Palynology, 46:1, 1-8, DOI: 10.1080/01916122.2021.1960916

V. Sh. Agababian (1972) Pollen Morphology of the Family Magnoliaceae, Grana, 12:3, 166-176, DOI: 10.1080/00173137209429874