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

“Don’t Drink the Dirty Chai” - An Examination of Macrofossils in the Grinnell College Peat Deposit

 Isabella Vergara, Hayden Bhavsar, Anna Lipari, Nate L’Esperance

Driving down Interstate 80, or almost anywhere else in Iowa, what do you see? It’s often fields, sometimes rolling, with sparse groups of deciduous trees poking out from the endless expanse of corn and soybeans. To most, the lack of, well, anything at all, creates a bland impression of the Hawkeye State. However, what if we were to tell you it wasn’t always this brown slab of corn? Would you ever assume that at some point, Iowa was a coniferous swampland akin to the vast forests of the United States’ Northwoods regions or Western Ontario? Grinnell Professor Ben Graham has helped to prove that Iowa indeed has an interesting, decidedly very different past.

Way back in the ancient, bygone, prehistoric era of 1960, Professor Ben Graham began to inspect the excavation site of what would eventually become a new Fine Arts center for Grinnell College in Grinnell, Iowa. After digging a mere 2.5 feet below the excavation floor, he began to find a 3 foot thick layer of dark brown, and loose organic matter. Graham found this layer to have an abnormally high water content of 82.4% at a depth of 16 feet, relative to 26% above to 28% below this layer (Graham, 1962.) Later, this layer was dated to 26-27,000 years before present. The layer consisted of peat, a type of organic soil resulting from prehistoric wetlands (Graham, 1962.) Peat originates from acidic freshwater bogs. Because of the acidic, low-oxygen conditions, most dead organic matter that fell into the water became preserved, as the waters would not decompose the debris (Xintu, 2009.) As time passed and more earth filled in over bogs, the resulting preserved organic material hardened into peat deposits. 

Due to the lack of decomposition, Ben Graham found that his excavated peat samples were full of preserved bits of leaves, seeds, stems, and pollen from many prehistoric plants. Formally, these preserved bits are called plant macrofossils. These macrofossils are classified as “remains large enough to be visible without a microscope” (NOAA.) We set out with the objective of indiscriminately extracting these plant macrofossils from the peat samples. What we find in the peat samples may give us more insight into what kind of plants may have existed in place of Grinnell College many tens of thousands of years ago. Similar extractions of the peat have yielded interesting results in 1962 and 2018, which we hope to build on with new techniques that may allow us to find more macrofossils.

As a class, we decided to pick one chunk of peat to share among team pollen, team wood, and us, team macrofossil. We placed small lumps of our peat into beakers with either purified water or KOH (an acidic solution intended to remove compounds that might interfere with imaging) and warmed them to dissolve the chunks into a coffee-colored sludge that we fondly described as a dirty chai. 

Figure 1: Left - the “Dirty Chai” consisting of broken up peat samples. We are testing deionized water and an acidic solution, KOH, for breaking up samples. Right - Anna and Nate searching through the samples for macrofossils with bronze fine mesh sieves (106 and 250um, shown in the bottom right corner). Macrofossils were obtained with tweezers and prepared on microscope slides.

Then, we gently sieved through the sludge under a microscope, to look for any little bits of plant material we could see. If we happened upon anything that looked plant-like, we used a pair of tweezers to gently place the plant bits on slides with a drop of tacky glue, so we could visualize them under our scopes later (Mauquoy et al., 2010). Once our tacky glue had dried, we took pictures of each of our slides and tried to describe and identify the plant material we were seeing based on published macrofossil keys (Lévesque et al., 1988). Then, we were able to compare our findings to the slides that students from previous years had made. 

In 2018, team macrofossil found what they thought was rhizome epidermis from the bog plant Scheuchzeria palustris. Our collection was much different from the 2018 team’s. We found a variety of plant parts amongst the peat. While we were unable to use these pieces to figure out with certainty what kinds of plants grew in the area since they were so small, we made informed guesses about the types of tissue we unearthed. Many of the macrofossils we found were wood fragments that required maceration for identification, a process which our group was not equipped to complete. Despite the abundance of unidentifiable wood, we found a higher volume of plant tissue compared to team Macrofossil of 2018. It’s possible that our fragment of peat had a higher concentration of plant material than the other, or that we tended to isolate smaller fragments than the other team, as there may be differences in our sampling and searching methods despite our efforts to replicate their work. 

In addition to wood, we found charcoal pieces (courtesy of Team Wood) which suggest the presence of fire in the peat bog. We also found significant amounts of epidermis tissue from unknown plants (figures 3 and 4). These samples tended to be very small, without distinguishing characteristics that pointed towards a specific species. We saw two main categories, tissue with short, square-ish cells and tissue with more elongated cells. The lack of stomata, donut shaped cellular holes that allow for gasses to move in and out of the plant, suggests that much of this tissue is bark or root tissue. We did find a few samples which we believe to be leaf tissue with stomata (see figure 4). Other interesting finds include a fossil resembling an amoeba (figure 5), a partial winged seed (figure 5), and what might be some sort of grass sheath (figure 1). 

Figure 2: A ring-shaped tissue fragment possibly from a grass sheath of Scheuchzeria palustris. The elongated cells and shape ring are vaguely similar to leaf scars on S. palustris. Left: 40x, Right: 400x magnification. 

Figure 3: Pieces of fibrous plant tissue with elongated cells. It is possible these come from some kind of marsh grass. 40x magnification. 

Figure 4: Plant tissue with short cells. The bottom right image shows tissue with stomata, donut shaped holes that allow for gasses to move in and out of the plant (shown in the bottom right corner at 400x). This piece probably came from a leaf or stem since these are the areas where stomata are present. Left image and top right at 40x magnification. 

Figure 5. Odds and ends. Left - Tissue fragment, possibly a piece of a winged or wind-dispersed seed. 40x magnification. Top right - a piece of charcoal found by Team Wood. 40x magnification  Bottom right - potentially a testate amoeba, evidenced by what appears to be a shell. 400x magnification.

Though we can’t draw any strong conclusions, our macrofossils hint at changes in prehistoric plant life. Our peat was formed around 27,000 years ago, but we don’t know the range of time covered. Team macrofossils in 2018 might have found different fragments because they extracted macrofossils from a piece of peat that formed when the area was a different kind of environment. Maybe they were looking at a piece from when it was a fen while our piece was from when it was a bog. In this case our findings might show environmental change. For instance, the presence of charcoal in our peat shows evidence of fire. And while we have been unable to make any genus-level identifications, this does say something about the diversity of plant life that once survived in this area– a far cry from the endless fields of corn that seem to dominate today’s Iowa, and a hint at the multiplicity of species that still exist today in the gaps between. 

Works Cited 

Lévesque, P.E.M., Dinel, H., Larouche, A. (1988). Guide to the identification of plant macrofossils in Canadian peatlands. Canadian Government Publishing Centre. 

Mauquoy, D., Hughes, P., van Geel, B. (2010). A protocol for plant macrofossil analysis of peat deposits. Mires and Peat 7(6), 1-5. 

National Oceanographic and Atmospheric Administration (NOAA). Plant Macrofossil. National Centers for Environmental Information.

Xintu, Liu. (2009). Conditions of Peat Formation. Coal, Oil, Shale, Natural Bitumen, Heavy Oil and Peat - Vol II. Encyclopedia of Life Support Systems (EOLSS)

Gettin Woody: How ancient peat wood can contribute to a vegetative history of Iowa

Image 1: Peat Bog from Sumava National Park in the Czech Republic. Kuttelvaserova Stuchelova/Shutterstock. Found in an article by Angela Nelson: Why You Should Care About Peat Bogs.

Peat bogs are often considered time capsules of the ecological history of an environment. Dead bugs and plant material freeze in time, partially decomposed, in the acidic layers of bogs. This is because as new peat accumulates, the older material underneath begins to rot. This rotting releases humic acid which preserves the organic material: the time machines that show us a vegetative past. Peat bogs originate from the incomplete decomposition of plant remains and other organic material growing in waterlogged conditions (usually in standing water of lakes, slow moving rivers, or areas of high precipitation). 

In 1960 a layer of peat deposit was discovered while excavating the foundations of the new Arts building at Grinnell College (Graham 1962). Radiocarbon dating of three pieces of wood found in the peat suggests that this section of Iowa was once a peat bog approximately 27,000 years ago. By identifying the organic material in the peat, we can construct a vegetative history. This vegetative history gives insight into the composition of the landscape, including the trees, plants and other organisms that used to live here. 

In an attempt to continue the study of the peat deposit, a group of Grinnell students in 2018, under the firm direction of Professor Vince Eckhart, took up the task. They found possible evidence of conifers (types of gymnosperms or seed producing plant) such as spruce (Picea), pine (Pinus), and larch (Larix), as well as a possible deciduous tree (a type of angiosperm or flowering plant). This corresponds with the current thoughts on the vegetative history of Iowa 27,000 years ago: mostly pine and spruce forest (Baker et al. 1989, 2009). This is consistent with what we know of the flora of bogs: lots of evergreen trees that can handle the acidic environment.

The work done in 2018 was a start, but as Grinnellians we had to ask the hard question: what else can we find out? This year we are continuing this vegetative endeavor by dividing into teams studying the macrofossils, pollen and wood samples. By studying the wood samples we collected from the peat we hoped to identify tree species comparable to what is currently known about the vegetative history of Iowa, and about the vegetative composition of ancient peat bogs. 

To begin we did exactly what anyone would, we played with some peat. With geology hammers, tweezers, assorted dentistry tools, and microscopes in hand we searched and searched for anything resembling wood. After hours of tedious work, we had eight pieces to work with. We also collected twigs from ten different tree species currently on Grinnell’s campus to compare to our peat wood. 

With our wood samples all together, we quickly found out that our work wasn’t over, as the reference wood we collected outside looked much different than the wood from 27,000 years ago. Our beautifully cut reference twigs made our dirt-covered peat fragments look alien. After trying to compare the wood by smell, we settled on a more comprehensive plan shown in Figure 1.

Figure 1. Wood slide preparation methods, including collecting, softening, maceration, and preparing slides. Methods adapted from Larter et al. (2017). 

After getting our precious wood samples onto our slides, we realized our identification was going to be trickier than we thought. Our peat wood looked 27,000 years old. Some slides had mangled cells, others had unrecognizable globs of mush, and two weren’t wood at all. Even our best peat wood samples looked very different from the orderly cells of our reference wood. But the key was in the pits.

Gymnosperm cells have rows of little pits, or holes that allow fluid exchange, along the tracheids. Angiosperms, on the other hand, have fibers and vessel elements. We realized none of our peat wood had vessel elements, so we knew we only had gymnosperms. 

We also realized many of our peat wood samples have helical thickenings. Helical thickenings are the extra layer of the cell wall that occur more often in softwoods and usually when the wood is under stress or compression, often at the joint of a branch. We wondered if these helical thickenings could have added structural integrity to the cells, allowing them to stick around for 27,000 years.

Based on the arrangement of the pits and the size of the tracheids, we think our samples are most likely Pinus or Picea. We agree with the wood group from 2018 in that Pinus has bigger pits than Picea, but our measurements for pit and tracheid diameter are a lot smaller. Despite the difference in measurements, our identification of our wood as Picea and Pinus is consistent with the pollen identification by Graham (1962) and with the pollen group’s analysis this year (A Hitchhiker (Pollen)’s Guide to the Universe (Iowa Flora)). We hope that this work will continue as there is much to consider, especially when we are limited by what we can mount on a slide.

Figure 2. Pictures of magnified (400x) wood cells. Left column: Picea references collected from Grinnell College Campus by 2022 Wood Team. Middle column: Peat samples, from PP2A and PP2B, prepared by Wood Team, and PP5C, prepared by Professor Vince Eckhart. Right column: Pinus references collected from Grinnell College Campus by 2022 Wood Team. 

Our findings match what is currently understood about the vegetative composition of Iowa 27,000 years ago and what we would expect to find in a bog: lots of spruce and pine. We found no further evidence of gymnosperms, and no indication of angiosperms. By learning what species were present 27,000 years ago, we can understand the ecological changes that Iowa experienced, which can help us predict how our environments will change in the future. Iowa 27,000 years ago is what Minnesota and Canada look like today, and as warmer temperatures reach latitudes further North, those environments and vegetation will begin to change. Knowing how species composition has changed will help us prepare for our changing future.

Authors:

    Elinor Arneson, Maria Eure, Elena Friedman, Noah Guyton, and Cicely Krutzsch

Acknowledgements:

    We would like to thank Vince Eckhart for his guidance and assistance, the BIO 305 class from 2018 for starting this journey for us, and the Professors Ben Graham and Andrew Graham for respectively collecting the peat in 1962 and then finding the box of peat in basement of the science building in 2018.

References:

Baker, R. G., Bettis III, E. A., Schwert, D. P., Horton, D. G., Chumbley, C. A., Gonzalez, L. A., & Reagan, M. K. (1996). Holocene Paleoenvironments of Northeast Iowa. Ecological Monographs. 66(2); 203--234. 

Baker, R. G., Bettis III, E. A., Mandel, R. D., Dorale, J. A., & Fredlund, G. G. (2009). Mid-Wisconsinan environments on the eastern Great Plains. Quaternary Science Reviews. 28; 873-889. doi:10.1016/j.quascirev.2008.12.021 

Graham Jr., B. F. (1962). A Post-Kansan Peat at Grinnell, Iowa: A Preliminary Report. Proceedings of the Iowa Academy of Science. 69(1); Article 7. 

Larter, M., Pfautsch, S., Domec, J., Trueba, S., Nagalingum, N., & Delzon, S. (2017). Aridity drove the evolution of extreme embolism resistance and the radiation of conifer genus Callitris. New Phytologist. 215; 97–112. doi: 10.1111/nph.14545.

Links:

https://www.treehugger.com/why-you-should-care-about-peat-bogs-4863716 

https://www.wwt.org.uk/discover-wetlands/wetlands/peat-bogs/#:~:text=Peat%20bogs%20are%20dense%20wetlands,peat%20can%20be%20metres%20deep. 

https://nph.onlinelibrary.wiley.com/doi/10.1111/j.1469-8137.2007.02317.x 

https://www.britannica.com/science/membrane-biology 

The Grinnell Peat, Part 2. Unboxing the past for a more complete picture.

For the first time since 2018, my BIO 305 class (Evolution of the Iowa Flora) was completely in-person. Equipped with the knowledge gained from that year's investigations of the Grinnell Peat and with a recently published protocol (Santos and Ledru, 2022) for visualizing pollen grains without extemely harsh chemicals, three student groups dived into peat (laid down 27,000 years ago; brought to light 60 years ago by Grnnell Professor Benjamin Graham), searching for fossil wood, other "large" plant fossils (macrofossils), and pollen grains. The students will reveal their findings in the next three posts to this blog. Meanwhile, I'll share something new I discovered in the peat this year: not a plant but a 1 mm long fossil springtail (six-legged arthropods that aren't insects). It's too bad Ben Graham's contemporary, Professor Kenneth Christiansen isn't alive to identify it for us. We need his book.

 

Santos, R. D. A., & Ledru, M. P. (2022). Acid-free protocol for extracting pollen from Quaternary sediments. Palynology, 46(1), 1-8.