Saturday, August 3, 2019

Oxygen Isotopes in Atmospheric Oxygen and the Dole Effect

Oxygen bubbles from algae

         One of the more intriguing intersections, to me, between botany and biogeochemistry was the “Dole Effect”. Malcolm Dole, a chemist at Northwestern University, had been measuring oxygen isotope fractionations in inorganic experimental systems for over 15 years starting in the 1940s. He and others, including Harold Urey, noted that the oxygen in the atmosphere had an excess of 18O relative to what it should be if atmospheric O2 was in equilibrium with water (Dole, 1935; Morita, 1935). In 1956, Lane and Dole (1956) published experiments that measured oxygen isotope fractionation during respiration by various organisms. Earlier experiments had shown that oxygen evolved from photosynthesis had the same isotopic composition as that of the water in the plant or the medium (Dole and Jenks, 1944). In the 1956 study, respiration was associated with an oxygen isotope fractionation between 10-25‰. The authors concluded that, “the O16/O18 ratio of atmospheric oxygen has risen to a point such that the ratios for photosynthetic oxygen delivered to the atmosphere and the oxygen extracted from the atmosphere by respiration are equal.” The problem of the Dole Effect persisted. Studies by Michael Bender, then at the University of Rhode Island, showed that oxygen isotope fractionation during respiration by marine phytoplankton and microbes in oceans was considerably less than what had been measured in the oxygen of the atmosphere (23.5‰) (Bender and Grande, 1987).
         In 1985, I was looking for additional challenges to my work as an isotope biogeochemist. I had spent eight years at the Geophysical Laboratory in the company of geochemists and earth scientists, far removed from biologists and botanists at the start of my career. I met Joe Berry at Carnegie’s Plant Biology Department at a Carnegie Institution of Washington meeting, and we immediately struck up a conversation about how we could weave together experiments on modern plant biology to tackle the potential factors that cause the Dole Effect. Berry, Chris Field, and Olle Bjorkman, all staff members at Carnegie’s Department of Plant Biology, were studying photorespiration in leaves, as well as conducting in situ work with Rubisco. If conditions are right, Rubisco has the potential to fix O2, rather than CO2. This can occur when temperatures are high, and the plant has made an excess of oxygen that might damage the plant via free radical production.
         At that time, we could not account for the factors that caused the Dole effect, which had implications for how biological processes were balanced globally.  Clearly, the measurements of isotope fractionation from respiration of O2 or from net oceanic or terrestrial processes could not resolve the problem Thus, I started on a major discovery endeavor to describe the oxygen isotope systematics of photosynthesis, photorespiration, and the other plant processes that could explain why the atmosphere has a δ18O of +23.5‰.
         Berry and I submitted a proposal to the Department of Energy and received the following reviews:
“The investigators underestimate the pitfalls of their experimental technique. The trapping of oxygen “on a molecular sieve column” (page 8) and the application of an oxygen electrode (pages 8,9) shift the ratio of the two oxygen isotopes...The hypothesis that photorespiration can account for part of the photosynthetic fractionation is interesting. Is there any reason (literature, preliminary data) to expect it to be so?”

Amazingly, the proposal was funded and on we went.
         To purify atmospheric oxygen from argon in air samples, Tom Hoering and I constructed our first vacuum line at the Geophysical Laboratory in Washington, DC. After oxygen was separated from argon, it was converted quantitatively to CO2. In late February 1985, we deconstructed the line piece by piece and shipped it to the Department of Plant Biology in Palo Alto, California. Working closely with postdoctoral fellow Robert Guy, we designed and built the “biological” part of the extraction line where enzyme reactions and cells would be incubated for experiments. Experiments began in earnest in 1986, when I spent a year’s sabbatical leave at Plant Biology.
         Our first experiments were with Anacystis nidulans, an easy-to-culture cyanobacterium. Cells were grown in the lab, then transferred to a collapsible bag that was sparged with helium to remove all traces of atmospheric oxygen. We had an oxygen electrode mounted in the bottom of the apparatus, which was constantly stirred. Light was turned on, and oxygen evolved. To kill the reaction, we added phosphoric and salicylic acids, then extracted the dissolved oxygen in a vacuum system. We confirmed earlier studies that showed there was little to no oxygen isotope fractionation during photosynthesis (e.g. Stevens et al., 1975). These first photosynthetic O2 measurements allowed us to refine our techniques and understand the various pitfalls. Typically, we would start on Monday to grow cells, get the chamber and line in good shape on Tuesday, run the first set of experiments on Wednesday, then repeat Thursday and Friday. Samples of CO2 were sealed into Pyrex ampules. Every two weeks or so, I traveled down to NASA’s Ames Center at Mountain View to work in David DesMarais’s lab, who kindly allowed me to use his isotope ratio mass spectrometer.
         Rubisco, which stands for ribulose 1,5 bisphosphate (Rubis) carboxylase (c) oxygenase (o), is an unusual enzyme in that it catalyzes the uptake of both CO2 and O2. Our second set of experiments was a “revisit” to my Ph.D. dissertation and the re-measurement of both carbon and oxygen isotope fractionation by Rubisco. Times had changed. Rubisco from spinach, cyanobacteria, and R. rubrum could now be expressed in E. coli; no need for growing massive amounts of cells or purifying enzymes. For these experiments, rather than the time-consuming purification and crystallization of PGA, we simply measured the isotopic composition of the remaining CO2. It turns out that the values I measured for my Ph.D. were several ‰ more negative than those I measured in California, because the δ13C of RuBP in my earlier experiments was influenced by unknown contamination. By measuring only CO2, we avoided that problem. We measured an isotope fractionation for carbon in CO2 of 29.4‰, similar to recent experiments by Roeske and O’Leary (1984).
         Oxygen isotope fractionations were calculated using Rayleigh equations that compared the oxygen isotope compositions of the O2 in our reaction chamber at the start of the experiment to the oxygen isotope compositions of O2 when we took a sample. Using our oxygen electrode, we could calculate the fraction of O2 that was consumed (Guy et al., 1993). We determined the isotope fractionation from the oxygenation part of the Rubisco reaction to be very similar to what was required to explain the Dole Effect. Approximately 30% of all O2 produced by photosynthesis is “fixed” by Rubisco prior to its being released. Another 20% is taken up by the Mehler reaction in which oxygen radicals are converted to peroxide, then converted via catalase to H2O. The isotope fractionation during this reaction yielded further isotope fractionation. We followed with measurements of oxygen isotope fractionations by glycolate oxidase in vitro and cytochrome oxidase. Experiments with cytochrome oxidase, the enzyme important for aerobic respiration, were another story. To perform these experiments, we needed large quantities of the enzyme cytochrome oxidase, which we could obtain from Sigma Chemical Company. Cytochrome c, on the other hand, was expensive--about $500 per sample---and it was destroyed at the end of each experiment. We managed to only have one or two successful runs, measuring an isotope fractionation of 6.6‰, hardly enough to explain the Dole Effect. 
         Rob Guy took the lead on respiration experiments (Guy et al., 1989) in which we investigated an alternative respiration pathway in plants that was cyanide-resistant. We used cells, isolated mitochondria, sub-mitochondrial particles, whole seedlings, as well as purified enzymes to show that whole plant respiration had a oxygen isotope fractionation slightly different from the cyanide resistant respiration, the type of respiration in plants like skunk cabbage. We measured oxygen isotope fractionation by almost all of the enzymes that could possible take up molecular oxygen. The work remains important—and the only studies to date—for understanding the biogeochemical cycles of oxygen on Earth.

Family Triumphs and other meaningful times

Alaska trip, Dana, Marilyn, Chris, and Evan, 2017
Sea Ranch Friends, from left Ron Benner, Sue Ziegler, Marilyn, Seth Newsome
            Positive experiences and good times are equally important in shaping a person’s personality. For example, although scientists rarely receive positive feedback, when we get a nice review or compliment from a student, it's a real rush. After my divorce, I spent a couple of years as a foot loose and fancy-free woman glad to be released from a difficult situation. Science was blooming and when I left for California to work on the oxygen isotope project, I met my future husband. People often ask how we met and it's a now “classic” story in our family.
            In 1985 during my first sabbatical trip to work at Carnegie’s Plant Biology lab, I signed up for a whale-watching trip that took place on a Sunday on St. Patrick’s Day, March 17th. Grey whales could be seen at Point Reyes National Seashore from the promontory where the lighthouse perches 100s of feet above the ocean. I didn’t have a car, so signed up to share a ride from Palo Alto up to the Visitor’s Center of Point Reyes with a Chinese grad student who had a new license and little familiarity with driving. The front passenger door of her car was inoperable, so I sat in the back seat with another woman. As we drove north through San Francisco’s hills, we clutched the doors in shear terror. When we assembled with the group, the leader of the trip asked if anyone wanted to ride with him in his VW bus. I leaped at the chance!
            The leader was a good-looking trim guy with curly brown hair from Berkeley wearing a t-shirt and jeans. On our way to the lighthouse, he spotted a bobcat in a meadow at the side of the road. Even before we reached our destination, he’d pointed out about a dozen coastal bird species. Throughout the day, we chatted on and off, spotted whales breaching and exhaling offshore. At lunchtime, we shared sandwiches on a bench overlooking Drake’s Lagoon, where I told him about my research with stable isotopes. As the end of the day drew near, I wrote down my phone number on a scrap of paper and handed it to him, hoping that he’d call. He looked pleased, but answered, “Great, we could talk about your research some more.”
            I called my friends in the following days and told them I was excited to meet this cool ecologist, Christopher Wood Swarth. By Wednesday of that week he called! We set a date for that weekend. On our first date, we ate beef tongue sandwiches at San Gregornio Beach and fried calamari for dinner. Why we chose those two weird foods, neither of us can explain, but we’ve never cooked them again. By the time my brief 6-week trip was ending, we had gotten to know each other and were ready to try out a long-distance relationship. We traveled back and forth from the DC area to California, culminating with a trip to Europe in August just prior to Chris’s fieldwork in Cameroon. It was an exciting day when he was able to call me all the way from the US embassy in Africa!
Chris, 1985!
            My second sabbatical trip started in January 1986. Chris returned from Africa, and we drove across the country with Sputnik the dog. We rented an apartment in Menlo Park and furnished it with Chris’s few belongings. That spring he proposed in a salt marsh—one of our favorite places—and I happily accepted. I returned to DC for the summer months, while wedding plans took shape. Back at the Lab, I told Tom Hoering about the engagement. He could tell I was excited and happy, but he wanted to make sure that this man was good enough for me. Tom and his wife Martha went for a vacation to Berkeley, ostensibly to visit a colleague at the Lawrence Berkeley Lab. One afternoon, Chris, who was working as a biology outreach teacher at the Lawrence Hall of Science, was notified that he had a visitor at the main desk. There was Tom Hoering, standing in the main lobby, with the sole intent of checking Chris out to see if he was suitable. Tom had a way of sniffing the air that meant he was thinking about a situation and sizing someone up. It was a brief meeting, but Chris passed muster apparently. In September 1986, we got married in my parents’ back yard in New Jersey with family and friends.
            After over 30 years of marriage, I can look back and see what was important for us as a couple, a family, and as a working woman. Before our kids were born, we both enjoyed the freedom to work as long as we wanted and travel wherever we needed to go. When Dana and Evan were very young, we hired the most remarkable young woman, recently emigrated from Nigeria to be their “daycare mother” for their first 5 years. Susan Agugua, bright, perky, and funny, arrived every morning at 9 am, every bit as compelling as Mary Poppins. Having someone you can trust to leave your children with allowed both of us to keep our careers going while raising a family.
Chris, Dana, Marilyn, and Evan: Christmas portrait, 2009
            When the kids were in elementary school, I took them to school and Chris picked them up from after-school care. When one of them was sick, we put our “cards on the table” and negotiated who had plans that could not be changed and who could shift things around and stay home. Being able to pursue a scientific career requires work on weekends, remote locations, and dedication. Whenever possible, we took them to work with us including overseas field trips in Australia and Belize. Chris worked for almost 25 years at an ecological park, a veritable treasure trove of fun places for the kids to explore and learn about nature. Both Dana and Evan also spent time in my lab, weighing samples and washing glassware. Evan and several of his buddies even learned to run the mass spectrometers during their senior year in high school. For several years, the whole family trekked out to Southern California to do field work re-examining the San Jacinto Mountain ecosystem first studied in 1908 by Chris’s grandfather, Harry S. Swarth. They helped trap mammals, pressed plants, assisted with collecting and preparing bird skins for museums, collected insects, and monitored bats.
Son Evan prepping samples GL 2009
            Early on I learned to take off from work when they had spring break or Christmas holidays. We usually took a trip to see a new national park or a new beach, or visited Chris’s parents in California. Sharing with them our love for nature and our dedication to things we found important were important lessons for them. Dana has become a talented organic farmer, an educator, and a natural born artist, hiking and camping whenever she can. Evan has chosen the medical field using his interest in the human body to start a career in nursing. They’re independent and resourceful. When I was diagnosed with ALS in 2016, they rallied around to help out.
            Key to not running yourself into the ground with marriage, family, and a career is to keeping your mind engaged on where you are at that moment. When I was at the lab, I got my analyses done, my writing completed, and my students and postdocs trained. When at home, I enjoyed cooking the nightly dinner, made sure our home was pleasant, if not completely clean, and delighted in hosting other families at our house. Our home was a mecca for the kids’ friends after we installed a half-court basketball platform and a hot tub in the backyard. As I matured as a scientist, I was more confident in my role as a leader and frequently hosted dinner parties at our house for colleagues from around the world. I morphed into the role of “Science Mother” making sure postdocs and students were getting what they needed in their careers, as well as their personal lives.
         In the past 25 years, I have made a point to mentor early career women as opportunities arise. I participate in programs with the Association for Women Geoscientists and the Geochemical Society. At the Carnegie and the University of California, I have been particularly outspoken about women’s rights as scientists and have spent many hours listening to and advising early career women. For most mentees, having a sympathetic ear to listen to their problems was enough. Other times, however, I needed to speak directly to Directors and Deans about abuses, in particular over sexual harassment and gender discrimination issues. These were not pleasant conversations and were outside of my scientific expertise, but in the few places where I have worked, it is essential that a woman in the profession provide guidance and advice.

Family, Friends, and Partners

Husband Chris Swarth at Point Reyes Seashore where we met

Suzanne O’Connell, 2015. “Success in the academy is a combination of many factors. Intelligence and hard work are essential but not sufficient by themselves. Help from mentors and advisors in learning how to navigate the complex corridors of the academy is also fundamental; it is unlikely that someone will master this process unaided. Unfortunately for the outsider, multiple studies have shown that workers in any field tend to mentor and advocate for people who are similar to themselves [e.g., Chesler and Chesler, 2002, McGuoire, 2002]. To break this pattern, mentors and mentees, students and faculty, insiders and outsiders, chairs and administrators need to examine the importance of passing information between groups and make sure this transmission occurs.”  

         Our personal partnerships and support mechanisms have been and will continue to be important to the success of underrepresented groups in science. One of the office staff at the Geophysical Lab, Marjorie Imlay, and I often discussed how women received the short end of the stick. She counseled me wisely, “By your work, shall ye’ be known.” Throughout, I stuck to scholarly research, published, and “kept my nose clean” for the first ten years of my career. While in graduate school, I married a local Texas guy who was not a scientist. At the time I was 21 and life was fun. By the time I was awarded my Ph.D. at the age of 24, I felt the impact of having a husband who did not understand the rigors of an academic life. At the Geophysical Lab, the marriage deteriorated as I became more successful. It ended in divorce.
         I had to learn the hard way that women in science need a sympathetic partner to succeed. The long work hours, the travel, and the fixation on seemingly small “problems” are things that academics are used to, but most others are not. It was a painful period for me, and I then awoke to the fact that women in science in the 1970s and 80s were either married to another scientist, and often subsumed by him, or unmarried. I was determined to be neither going forward. Today, 83% of academic women scientists are married to an academic, science partner (Schiebinger, 2008). The support and respect of our partners is key. Men in science have always had respect from their families.  Women need that as well, but sadly I suspect some women don’t get this. I didn’t until well into my career, so I feel keenly the importance of a stable family life.

Challenges we all face—my sister Barbara Anne Fogel Lis

My sister, Barbara Anne Fogel Lis and her son, Chris Rudolph, circa 2008
Marilyn, Barb, and my mother Florence Fogel, 2009
            Everyone—without exception—deals with personal or family trauma or drama sometime during her/his career. There is often a false sense that these things don’t happen to other people, just us. We then wonder why me? How did I end up with the wrong partner, difficult family, or life threatening illness? As I wrote earlier, I was no exception to this rule, even though after several years passed, no one remembered any more about my early trials. I am fortunate to have grown up in a loving family with parents who never divorced, a brother and sister who shared the family joys, and a nuclear family of husband and two children who get along completely most of the time. Although the big picture of family life was strong, there were constant challenges.
            Almost 54 years ago, I gained the role of Big Sister when Barbara Anne Fogel was born. It was Thanksgiving evening when my mother went into labor and that day we had hamburgers instead of turkey. Having a baby sister was a real thrill. We were very close during the years we overlapped at home. I both helped her and terrorized her. I told her there were alligators living under my bed named Theodore and Guinevere, and if she came in my room and messed around with my things, those alligators would come after her.
            Barb quickly became the Little Princess, charming my oft-grumpy father and busy mother. Barb followed me everywhere as a youngster, imitating everything she saw. She learned to count in German and Chinese, two languages I was learning in school. She listened to rock and roll music of the times, and “studied” by writing primitive letters in my high school textbooks. As a youngster, she was a character. When my brother would call home from college “collect”, she told the operator her name was Garfunkel, after the singers Simon and Garfunkel.  I started college when she was 6 years old. Essentially, she grew up as an only child in our house in Moorestown, New Jersey.
Fred, Marilyn, Barbie, my dad Art Fogel, 1972
            Around the age of 13, Barbie was suddenly ill. Her doctor checked her blood sugar, and she was sent immediately to the hospital for further tests. One of the unlucky few who have Type 1 Diabetes, Barb began the trial that colored the rest of her life. The diagnosis 40 years ago was tragic. Rounds of needles, insulin injections, meal restrictions, made her rebel as a teenager. She wanted to be a “normal” person, a popular girl, and step away from the daily grind of blood sugar testing etc.
            In her senior year of high school, I got a call from her.
            “Mar, promise you won’t tell, “she said between sobs.
            “Tell what?“ I answered.
            “Promise me!” she pleaded.
            “What?” I asked again with pain.
            Of course, I am not the first, nor the last, to learn of an unwanted pregnancy. I tried to calm her down and got off the phone. Because she was a diabetic and had not controlled her blood sugar during early pregnancy, she was a serious risk for problems. I needed to tell my parents the news. I phoned my folks that evening and said I needed to speak to them about something serious—in person. With husband Jack, we drove up the next afternoon. My sister hid out at one of her friends, while I gave them the news. My mother cried, and my father seethed with anger ready to shoot the boy who got her pregnant. It was one of the worst evenings of my life.
            Our peaceful family life was in tatters. Within a year, Barb, once she reached 18, married boyfriend David in a small private ceremony. I was not in attendance. My mother was the only one with the gumption to see the event. Barb and David’s life was a continuous roller coaster. Their first son Christopher was born within a year, and not long after that, David was sent to one of many prisons for petty crimes like driving without a license. After their second son Michael was born 13 months later after a troublesome pregnancy, Barb called me from the hospital.
            “What should I do?” she whispered.
            “Leave him. Go home to Mom and Dad.” I answered.
            “Do you think they’ll take me?”
            “Yes, Dad’s been waiting. Give them a call.”
Barbie moved in with my parents, Florence and Art Fogel, when Michael was born in 1985 and lived with them until she remarried in 1995. The separation was tough on everyone. David’s crimes became more serious—he harassed my sister and my parents when he wasn’t in jail. The family was on constant alert that Chris and Mike weren’t abducted. Fortunately, David never made good on his threats.
Family Birthday celebration, circa 2004
            This family ordeal was ongoing at the same time I was getting a divorce, working on my career, and then starting my own family. While thinking thoughts about stable isotope fractionation, bacteria in hot springs, oxygen in the atmosphere, I had to partition my brain to be sensitive and empathetic to family. I had become the family’s major problem solver, many times getting a call regarding a health or financial emergency in New Jersey, arranging for someone to cover me in the Lab, then bee lining up intestate 95 to Jersey. I handled heart attacks in both parents, strokes, cancers, pneumonia, broken bones, wounds infected with MRSA, alcoholism, drug addiction, and dementia. I worked through financial crises—college loans for Barb’s son Michael, foreclosures, business failures, within-family theft, and credit card fraud. Barb’s son Chris developed an addiction to prescription pain medication that brought a raft of sorrow and heartbreak to our close-knit family. It pretty much destroyed the happy holidays and birthdays we once shared.
Fogel Family: Barb, Tim, Mike, Chris, Fred, Linda, Dana, Chris S., Marilyn, Evan; Flo and Art seated, 1996
            Nephew Chris died in my sister’s arms on the evening of his 30th birthday from a heart infection. Although some stress was relieved by his passing, my sister’s problems became ever more clear.         Barb was beautiful, blonde, funny, caring, kind, loving, sweet and orderly. With the loss of her son, Chris, she and Tim got an enormous dog, Cooper, who as a dog, has many of the same qualities as Chris—good hearted, friendly, goofy, untrained. Their life reached a new level, where they no longer had to worry about Chris.
            Her second marriage to Tim Lis, a kind, hard-working man, had pulled her from poverty and disaster. But it wasn’t enough. She cycled in and out of periods of substance abuse, serious health complications from diabetes, and one step ahead of financial ruin. By this time, husband Chris and I had moved to California. I no longer hopped in the car for the white-knuckle ride to New Jersey. They were on their own. Tragically, her second husband Tim died at the age of 53 from a massive stroke, and Barb followed him eight months later dying of “despair”--despondent grief over his death and financial problems too large for anyone to solve.
Barb, son Mike Rudolph, Tim Lis, Graduation, 2010
            What makes a life turn hard? It doesn’t take much. We’re all one doctor’s visit away from a changed health status. For Barb, the diabetes starting taking its toll around the age of 40. Barb was proud of the fact that she had over 30 or 40 surgeries in her lifetime. How she withstood the constant onslaught of medical issues was a defining characteristic of her life. When you saw her, you could tell in an instant how she was feeling: if it was a good day or a bad day.
            There were many times when Barb was at her best. Entertaining on the back patio in summer: BBQ meats, potato salad, fresh Jersey corn, coleslaw, Jersey tomatoes, appetizers, clams, flounder---more food than anyone could possibly eat. Then there were the Christmas parties: the fried turkeys, the crisped prime rib roasts, kielbasa, sauerkraut, potatoes, succotash---all served hours after people were starving for food. Together with Tim, they built a business, worked together, and helped their families. The backyard at their home in Mount Laurel, New Jersey, contained a stunning garden that Barb and Tim were proud of. She loved puttering there, picking tomatoes, beans, and basil. In summer, clamming and fishing at the shore were favorite pastimes. Barb was most happy here.
Chris Rudolph and Barb, Christmas Party, circa 2007
            There were also those times when you found her exasperating. We could never get her to quit smoking. She never ate decent meals. Her sleep habits were troublesome. The “glass” was more often than not “half empty” rather than “half full”. She challenged everyone from her family to her friends to associates. She rarely sat down and kept still.
            Beyond the daily ups and downs, Barb was a loyal friend. We had long phone conversations, where she’d launch into a story, forget why she started it, then end it 15 minutes later. She followed everyone’s life. Your children, their birthdays, your anniversary, your birthday, your job, you name it. She was a friendly neighbor, offering up to those less fortunate to her, the last $20 even though she couldn’t afford it. She loved animals, hated spiders, and fought with ground hogs. There were times when you didn’t talk to her every day. For me, I always suspected something wrong. For most of you, you probably got texts out of the blue asking you “what’s wrong?”
            Her early and sudden death’s taught me that above all else, think about others and help them. Take care of yourself—your care is as important as the care you give to others. I wish that Barb had taken more care of herself. Love your family. You didn’t choose them, but they are the most important things in your life. Laugh as much as possible, and shed yourselves of “stuff” that can only weigh you down. Forgive those you might resent, and keep strong.
Florence Fogel, Mike Rudolph, his wife Sheri, and Barb, 2018

What kind of plant is that?

Marilyn chasing emu eating plants, 2009
C-3 and C-4 plants have distinct isotope compositions

C-3 and C-4 Plants
Another set of plants turn the carbon dioxide in air into a 4-carbon sugar and so we call them C-4 plants (C4). C4 plants evolved well after C-3 plants and this adaptation allows them to thrive in hot, dry environments, such as the outback of Australia. C4 plants absorb the lighter carbon-12 isotope significantly slower than the C3 plants, because the protein that uses the carbon dioxide works differently. Because these proteins in plants that take up carbon dioxide are different, their carbon isotopes are different as well. Scientists now use these differences to determine whether a plant is a C-3 or C-plant.
The shrubs in the photo--chenopods-have isotope signals between C-3 and C-4 plants. Emus eat their flowers and seeds.
Why is this important? Well, C-4 plants are usually grasses, especially in Australia. Grasses of the C-4 type mean that the climate was warmer and drier. If we know that an animal was eating C-4 plants we know that it lived at a time when the climate was warm and dry. This is important when we examine fossils from animals that went extinct years ago. When we measure their carbon isotopes, we can tell if they ate grass (usually C-4) or leaves of trees (usually C-3). Once we know this, we can predict what the climate must have been like many years ago.

How we Date an Eggshell - Amino Acid Racemization

Small eggshell fragments can be dated and studied for their isotope compositions

All living things use large molecules called proteins to do the work in a cell. These proteins are composed of building blocks known as amino acids. There are about 20 different amino acids that are found in proteins. Anywhere from 50 to several hundred amino acids are linked together to form a long chain-like protein. Almost every amino acid comes in two forms, levorotary (left-L) and dextrorotary (right-D). These forms of amino acids are mirror images of each other however cannot be overlaid onto each other, just like right and left hands. The words come from the fact that if you shine a light on left-handed molecules; they bend light to the left. The same is true for the right-handed variety. However, most living things only use the L amino acids. When a creature dies the L amino acids begin to convert to dextrorotary amino acids through amino acid racemization. Racemization is the process in which one mirror image molecule turns into another.
All organisms have 99.95% L-amino acids in their proteins. When a bird or mammal dies, the proteins in its bones and teeth start to degrade. As they degrade, proteins are decomposed by bacteria into the amino acid building blocks. When this happens, the L-amino acids are likely to flip over to their mirror image, the D-amino acids. This is called racemization. Because of this process, researchers can use the amino acids found in eggshell samples to approximately determine the time of death of a creature. Each amino acid has a different rate of racemization and this rate is affected by certain environmental factors. These factors include temperature, water concentration, and acidity in the environment. Typically, temperature is believed to have the greatest impact on the rate. In hotter temperatures, the rate of racemization is faster. Because temperature plays such a significant role, using amino acid racemization to date eggshells is not always completely accurate. However, because the more accurate radiocarbon dating is only effective for samples from 40,000 years ago and after, amino acid racemization dating must be used to age samples from before 40,000 years ago.
In the late 1960s it was discovered that there is a clear division in carbon-13 values among terrestrial plants. Some plants like trees, bushes, and some grasses that grow in colder climates have lower carbon-13 values than others such as corn, sugar, and dryland grasses. Plants with lower carbon-13 levels are now know as C3 plants while their higher counterparts are known as C4 plants. The isotopic division is created during photosynthesis. In most plants, during photosynthesis carbon dioxide is incorporated into either a 3-carbon compound (C3), and so we call it a C-3 plant. C3 plants are found in a much broader range of environments than C4 plants. All pine trees, most flowering plants, and most of the vegetable we eat are C-3 plants.

How we test for past climate

Marine isotope stages (MIS) are alternating warm and cool periods of climate in the Earth’s history. The stages are determined using oxygen isotope data from microscopic animals that live in the ocean at different depths. These animals, foraminifera, make calcium carbonate (CaCO3) shells from oxygen in H20 in the ocean. Scientists have learned that the oxygen isotopes in these small animals reveal the temperature of ocean thousands of years ago. Sediment cores from all over the ocean’s basins are analyzed for their oxygen isotopes by picking out the microscopic animals under a microscope and analyzing them on an isotope ratio mass spectrometer. Based on the oxygen isotope data that is measured, temperature records or curves are generated using these deep-sea core samples.
Earth’s surface was frozen or at least much colder than today. Warmer periods have lighter isotope levels (more 16O) and are known as interglacial periods, when the Earth was warmer, such as today. Each stage is numbered with a corresponding name. Even numbered stages are glacial and odd numbered stages are interglacial. These records provide researchers with a basis for understanding past climates and climate changes. Over the last 2.5 million years MIS data presents about 50 climate cycles! This data matches up with terrestrial evidence that shows corresponding cycles. Imagine how animals and plants have responded to this frequency of climate change.
Wolfe Creek Meteorite Crater (L): note change in vegetation; Northern Australian vegetation: dominated by grass, Acacia, and eucalyptus trees
This project uses samples ranging back to interglacial stage 7 which began about 200,000 years ago. One of the main project focuses is the extinction of the Genyornis that occurred between stages 3 and 4. After using amino acid racemization to date each eggshell sample, researchers matched each sample to a marine isotope stage. This provides important information concerning the climate in which each sample lived. Further, researchers can look for possible relationships between climatic fluctuations and extinctions events.

Big extinct Australian Bird

Giff and Marilyn at Geny Heaven (top); Megafauna: Geny is the large bird (bottom)

Genyornis newtonii
No one you’ve ever met has ever seen a Genyornis newtonii. That’s because around 45,000 years ago they went extinct. Like the emu, the Genyornis was a large, flightless bird. However, they were very heavily built and, as a result, were not the fastest movers. The large bird stood tall at around 2.2 meters with a weight of around 200-250 kg. With powerful legs, tiny wings, an enormous beak and hoof-like claws, the Genyornis has affectionately been nicknamed the ‘demon duck’.
Fossils of Genyornis bones and eggs have been found throughout the south, west, and east of Australia. During the time period that the Genyornis lived, these regions of Australia were probably more temperate grasslands. It is believed that the Genyornis ate mostly fruit and nuts with the occasional small prey, but these are guesses based on what their fossil bones look like. As in modern birds, the Genyornis had no teeth and therefore relied on small stones called ‘gizzard stones’ to help grind up and digest their food.
Riding trikes to get to remote field areas: Marilyn and Giff Miller, 2002
Because of their speedy extinction around 45,000 years ago, many believe persistent hunting and egg raiding by early aboriginal settlers led to the large bird’s disappearance. Others maintain that climate change and other natural factors also played a role. Scientists from this team found that Genyornis’ in south and eastern parts of Australia were probably subjected to extreme climate change due to early aboriginal burning. Based on their data, they found that Genyornis always needed fresh grass in its diet. When grasslands are burned, the edible tasty species of grass are damaged and no longer grow. Hard, spiny grasses, called Spinifex, are less tasty and may not have been able to support the large-bodied Genyornis.
We also don’t know if Genyornis ate small mammals, insects, and lizards like the emu does. Data from the ancient Genyornis reveals that it is highly likely that they did. How these large, flightless beasts were able to capture fast-moving prey is not known.

Rounding Third Base and Heading Home

Cards from Franny and Flowers the Rumbles   My daughter Dana is marrying George Goryan on June 25 at our home in Mariposa...