Leaf Litter Lab (L3) Notes

A blog highlighting undergraduate research in the LeRoy Lab at Evergreen

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Chemical differences in Male vs Female leaf litter

A colorful willow leaf collected along on of the Pumice Plain streams at Mount St. Helens. Photo by Carri LeRoy 

Across the Pumice plain of Mount St. Helens, our research has focused mainly on understanding how plant sex differences influence ecosystem processes. Members of our lab are studying the leaf litter of male and female willows at a chemical level!

Evergreen undergraduate Iris Garthwaite standing in front of the TOF mass spectrometer at the Center for Urban Waters in Tacoma.  Photo by Joy Ramstack Hobbs 

To identify if male and female willows have unique chemical signatures, we measured condensed tannin, C and N, and a whole suite of compounds using Time of Flight Mass Spectrometry (TOFMS) in collaboration with the Center for Urban Waters at UW Tacoma. 

TOF Mass Spec readings give preliminary identification of the compounds in the willow samples. Of the 1,500 -1,600 individual compounds isolated in each sample, there were about 150 compounds that differentiate between the male and female willow samples at MSH! 

Cluster diagram showing over 100 compounds that vary between male and female willows (red = present, blue = absent). Unpublished data.

Published Undergraduate Co-authors!

Our National Science Foundation (NSF) grant provided funding for two full field seasons at Mount St. Helens. In the first year, we collected a dataset comparing the colonization patterns of willow males and females on the Pumice Plain and published a paper in Ecosphere with four undergraduate co-authors (LeRoy et al. 2020)! 

One of the first papers to be published from our work at Mount St. Helens. Four co-authors are undergraduates at The Evergreen State College. (LeRoy et al. 2020, Ecosphere)
Several figures from the paper showing male-female willow differences in initial leaf chemistry and chemistry throughout the decomposition process. (LeRoy et al. 2020, Ecosphere)

We found that the leaf chemistry of male and female willows differs, where males have significantly higher nitrogen and females have higher C:N ratios (LeRoy et al. 2020). These patterns persist through time in the stream, providing in-stream invertebrates with variation in food resources.

Getting started on data analysis and paper writing around the fire at our field camp at Mount St. Helens in summer of 2018! Photo by Shannon Claeson

On long field trips, we get right to work analyzing data in the field. Our team consists of Evergreen faculty, Forest Service collaborators, and lots of invaluable undergraduate research assistants. Nothing better than data-analysis by the fire-side!  

Our study made the cover of the issue in Ecosphere. This is a drone photo taken with a permit of willow colonization on the Pumice Plain. Photo by Carri LeRoy (Mavic 2 Pro)

Some of our research involves using drone technology to explore willow colonization patterns. We were asked to contribute cover photos to the journal Ecosphere and one of our drone images made the cover! Follow along to learn more about the awesome, NSF funded work, collaborative student-faculty research on aquatic-terrestrial interactions in early successional headwater streams of Mount St. Helens! 

Willows at MSH!

Healthy willow growth alongside a young stream at Mount St. Helens. Photo by Carri LeRoy

Willows, Salix species, are a common riparian plant across the globe and are a key early successional species. Willows are known to increase water quality and stabilize banks, so it is not hard to believe they are a dominant riparian plant on the Pumice Plain of Mount St. Helens.

Willows on the Pumice Plain (Salix sitchensis) are dioecious – meaning they have male and female individuals. Here you can see them tagged with blue and pink flagging. Photo by Carri LeRoy

Willows are a dioecious species (they have both male and female individual shrubs) that play a vital role in understanding ecological interactions across the Pumice Plain. Our lab has documented plant sex ratios, colonization locations, and chemical differences among willow populations to understand primary succession.

Female Sitka willow need to produce costly flowers and fruits. This might be one reason they colonize closer to streamsides. Photo by Angie Froedin-Morgensen

Interestingly, a high proportion of riparian plants are dioecious. We are working to understand what advantages there are to having male and female separation along streamsides in particular. In our system, females colonize closer to streams – maybe higher resource availability for producing flowers? Stay tuned for tomorrow’s post about our latest paper regarding willow sex differences at Mount St. Helens!

Condensed Tannins!

Prepped samples of ground leaves (left) and tannin samples mid-extraction process (right). Photos by Iris Garthwaite

Across the Pumice Plain of Mount St. Helens, Sitka willows (Salix sitchensis) make up a major percentage of the riparian (streamside) vegetation. Parts of our research focuses on identifying phenolic compounds called condensed tannins within willow leaves!

Evergreen undergraduates, Lauren Thompson (left) and Maddie Thompson (right) running the condensed tannin extraction and assay. Photo by Iris Garthwaite

Condensed tannins of willow leaves may act as anti-herbivore compounds and make willow leaves less palatable based on their concentrations. The processes of condensed tannin extraction and assay are very long but filled with numerous colorful steps!  

The colorimetric condensed tannin assay results in an awesome gradient of pink! Photo by Madeline Thompson

Phenolic compounds may differ across the Pumice Plain or by plant sex (willow is dioecious!). Leaf chemistry can influence which insects utilize the leaves and rates of decomposition for leaf litter!

Benthic Macroinvertebrates of MSH

Caddisflies of all shapes and sizes have colonized Mount St. Helens. Photo by Angie Froedin-Morgensen

The eruption of Mount St Helens created pyroclastic flows, mudflows, and ash fallout that covered the Pumice Plain in over 100 ft of sterile material. Since then, new watersheds have formed, diverse fauna has colonized MSH streams, and our lab team has been there to document!

Our benthic macroinvertebrate sorting station. Photo by Angie Froedin-Morgensen

The benthic macroinvertebrates of MSH colonize, eat and utilize leaf litter that falls into streams. With the use of leaf litter bags, Undergraduates Angie Froedin-Morgensen and Brandy Ku’ualoha Kamakawiwoole spend hours under a microscope sorting these aquatic insects!

Stoneflies are particularly common inhabitants of Mount St. Helens streams. Photo by Angie Froedin-Morgensen

Aquatic insects like Ephemeroptera, Trichoptera, and Plecoptera all play a critical role in the aquatic food webs! Benthic macroinvertebrate communities provide amazing insight as indicators of biological conditions, and communities differ across MSH streams!  

Interview with Evergreen Undergraduate Angie Froedin-Morgensen

What is one of those mind-blowing facts about MSH that you can’t un-know?“I love how Mount St Helens was a completely sterile environment, then lupines showed up. The process of natural regeneration is amazing. It reminds me of the Big Bang Theory, life happening on its own.”  

What attracted you to research at Mount St Helens? “Being outside, learning the fundamentals of stream ecology, and the roles of rivers and streams in larger landscape processes.” 

What is your role in the L3 lab? “Bug master!” Angie has been leading the processing of 2 seasons x 3 harvests x 32 #litterbags = 192 litterbag communities. Aq. Insect abundances range from 25-100 per sample, so lots of bugs! Stay tuned for a blog highlighting pictures of some of the aquatic insects Angie’s documented! 

Fav aquatic insect? “Caddisfly (Trichoptera). I like the variation in their cases. Caddisfly silk is being studied as a potential waterproof adhesive. That this natural polymer can inspire product engineering is really cool!”  

Greatest challenge/area of growth? “Writing has been my greatest challenge. But I’ve learned a lot about keying insects and getting through peer-reviewed papers, getting through them faster, understanding terminology.”  

Quirky facts about the L3 lab? “How we aren’t intimidated by each other. We get to troubleshoot things together. We have a lot of independence in our lab, so we get to ask each other all the silly questions!”  

What are your future goals? “Finish sorting and identifying all the MSH 2019 litterbag samples, co-author a peer-reviewed paper on the leaf litter study, work with Dr. LeRoy to become a better scientific writer.”  

Snacks: What lab member’s lunch are you most envious of in lab meetings? “Iris or Joy!” Who has the best field snacks? “Deb has the best junk food, but Carri’s sesame sticks are also a win.”   

Do you identify as a Greener? “I do. I created my own path at Evergreen. It allowed me room to grow. I came to school for one thing, then my interests changed after working with science faculty. Now I’m weeks away from a BS Degree!

DNA Extractions!

Litterbag showing willow leaf litter and a unique metal tag for identification!   

Across the Pumice Plain, willow leaf litter bags are placed into streams to conduct a variety of assays. After the leaves are colonized by bacteria and fungi and begin to decompose, we remove the leaves and use DNA extractions to identify microbial communities!

Various stages in the DNA extraction process: Macerated leaf material, intermediate stages of DNA extraction, and final extracts for analysis. Photos by Lauren Thompson

The DNA extraction process entails numerous lengthy, yet exciting steps. Whether its shaking samples up vigorously with a vortexer or lysing cell matter with solutions, Evergreen Undergraduates Lauren and Maddie Thompson are always up for the challenge!

Undergraduate Maddie Thompson testing for DNA concentrations on the nanodrop! Photo by Lauren Thompson

Extracted DNA from willow samples is then sent off to characterize microbes present! This identifies what microbes colonize first, starts decomposition, provides nutrients to aquatic macroinvertebrates, and may be influenced by the sex of willows!

Organic Matter Processing – Canvas Strips!

Evergreen undergraduate Maya Nabipoor getting ready to install canvas strips in Geo-West Creek on the Pumice Plain of Mount St. Helens. Photo by Carri LeRoy 

To assess organic matter processing (OMP) within streams across the Pumice Plain, a subset of our research focuses on the use of canvas strips! Canvas strips are a standardized method to accurately examine in-stream ecosystem function. (Tiegs et al. 2013). @ScottTiegs 

Canvas strips with metal ID tags are placed across the streams and riparian zones of the Pumice Plain. This is done to measure how OMP rates vary across environmental differences in early successional streams of Mount St. Helens.

Canvas strips are used to estimate organic matter processing rates in streams and in riparian zones on the Pumice Plain of Mount St. Helens. Photo by Carri LeRoy 

In order to measure OMP, canvas strip assays rely on the loss of tensile strength that corresponds to cellulose degradation while in-stream. Through collaborations with the Olympic College, our team measured this using a giant machine called a tensiometer!

Insects on Willows: Weevils, Beetles, Galling Herbivores

Chrysomelid beetle larvae chew on willow leaves – leaving these patterns of skeletonization behind. Photo by Carri LeRoy 

Willows across the Pumice Plain of Mount St. Helens provide more than just riparian vegetation! They provide shelter, habitat and food to a variety of insects. Insects like Chrysomelid beetle larvae chew their way through willow leaves!

The poplar & willow stem-boring weevil, Cryptorhynchus lapathi, was introduced to Mount St. Helens in 1989. Photo by Carri LeRoy 

Another insect that utilizes the willows is the stem-boring weevil, which is native to Europe but was introduced to Mount St Helens in 1989! The tiny long-nosed guys, show up a lot in our studies as they cause branch death and mortality of willows.    

Galling herbivores like this likely galling midge show variation in activity across the Pumice Plain of Mount St. Helens. Photo by Carri LeRoy

Insects like galling midges have specialized feeding behaviors that require willow leaves as a host. They create new microhabitats for their young via galling. Stay tuned to learn more about the aquatic macroinvertebrates that colonize willow leaves that fall into streams!

New Road Across the Pumice Plain at Mount St. Helens

The Truman Trail at Mount St. Helens is beautiful! Help stop the USFS from building a road here! Photo by Carri LeRoy.

Since the eruption of Mount St Helens, the landscape has turned into a living laboratory for ecological research. It’s the most studied volcano in the world. It allows scientists to track how ecosystems and species respond to major disturbances.


USFS already built this new road to access Spirit Lake in 2018. This was supposed to be the alternative to the Pumice Plain road. Photos by Carri LeRoy

Despite MSH being protected for natural recovery and research, for the past three years the USFS has been trying to build a road across the face of Mount St Helens. The creation of this road would destroy the Truman Trail and alter the ecosystem completely. Learn more about the threatened Truman Trail here: https://www.mshinstitute.org/about_us/rumblings-newsletter/romanos-rumblings-summer-2018.html

The Truman Trail provides excellent views of both Spirit Lake and the crater of Mount St Helens from the Pumice Plain. Photos by Carri LeRoy. 

Our research on newly developed streams across the Pumice Plain studies would be destroyed. Our lab and many others have been able to fight this off the last two years. To help get our voices heard, we need to actively fight this planned action: https://www.fs.usda.gov/project/?project=57259

You can find out more about the project and read several scientists’ objections to the new proposed road here: https://tdn.com/news/local/forest-service-confirms-proposal-for-spirit-lake-access-road/article_bfa7f34f-4efc-5d80-9588-bda6549a75d7.html

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