Proceedings of the South Dakota Academy of Science

Volume 79 2000

Published by the South Dakota Academy of Science Academy Founded November 22, 1915

Editor Kenneth F. Higgins

Terri Symens, Wildlife & Fisheries, SDSU provided secretarial assistance

Tom Holmlund, Graphic Designer

TABLE OF CONTENTS

Consolidated Minutes of the Eighty-Fifth Annual Meeting of the South Dakota Academy of Science...... 1 Presidential Address: Understanding Change. Lenore Koczon...... 5

Complete Senior Research Papers presented at The 85th Annual Meeting of the South Dakota Academy of Science Angler Opinions Regarding Fishing Success and Crappie Regulations in a Small South Dakota Impoundment. Timothy J. Bister, Bradley M. Baker and David W. Willis...... 11 Biological Control of Canada Thistle (Cirsium Arvense) in South Dakota. Z. Liu, S.A. Clay and M. Brinkman...... 21 Identification of ESTs that are possibly related to the FHB-resistance of wheat (Triticum aestivum L.) cultivar Sumai 3. D.H. Xing, Y. Yen and J.C. Rudd ...... 35 An Ichthyosaur Bone From the Jurassic of South Dakota. David C. Parris and Barbara Smith Grandstaff...... 41 A Bison Skull from Lyman County, South Dakota. B.S. Grandstaff, D.C. Parris and J.E. Martin...... 45 Using the Ocean Optics R-2000 Raman Spectrometer in the Undergraduate Laboratory. Deanna L. Donohoue, Gary W. Earl and Arlen Viste...... 63 13C NMR Spectra and Quantum Chemistry Calculations for Benzonitrile and 4-Aminobenzonitrile. Steven R. Moeckly, Gary W. Earl and Arlen Viste ...... 71 Synthesis and Analysis of Quaternary Compounds Containing Phosphate and Borate Anions. David Stone and Gary Earl ...... 75 The Synthesis of Methyl Carbonate Quats and Their Anion Conversions. Drew J. Paulson and Gary W. Earl ...... 79 Molecular Modeling of Selected Antibiotics and Antibiotic Resistant Enzymes. Kari Lunder, Gary W. Earl and Arlen Viste ...... 83 Amino Acid Concentration of Soybean With Protein Variation in Different Environments. R.A. Scott and K.D. Kephart...... 89 Misadventures in Soybean Transformation: Particle Bombardment vs. Agrobacterium and Cotyledonary Nodes. S.A. Hansen and C.D. Carter ...... 97 Summer Habitat Associations of Rare Fishes in South Dakota Tributaries to the Minnesota River. Douglas J. Dieterman and Charles R. Berry...... 103

Abstracts of Senior Research Papers presented at The 85th Annual Meeting of the South Dakota Academy of Science Use of Shelterbelts as Breeding Habitat by Birds in Eastern South Dakota. Dale L. Droge and Jeffrey S. Palmer ...... 113 Investigating Leatherback Turtle (Dermochelys Coriacea) Egg Laying and Egg Survival. Valerie E. Wilson and Donna Hazelwood ...... 115 Water Quality and Phytoplankton Dynamics of the Ottertail Power Plant Cooling Pond, Milbank, SD. Amy L. Gronke and Nels H. Troelstrup, Jr...... 117 Characterization of a Rural Stream to Define Critical Reaches Influenced by Non-Point Source Pollution. A.M. Larson and N.H. Troelstrup, Jr...... 119 Distribution, Breeding Patterns, and Developmental Physiology of Some South Dakota Terrestrial Isopods. J. Likness and L.G. Johnson ...... 121 Invertebrate Community Characteristics in Relation to Ecoregion, Shoreline Management and Habitat in Eastern South Dakota Lakes. C.W. Braskamp and N.H. Troelstrup Jr...... 123 Impacts of Land Use on Aquatic Invertebrate Abundances and Their Correlation to Nutrient Concentrations in Prairie Pothole Wetlands. Brandon Jahnke and Diane Rickerl...... 125 Detecting Indicators of Poor Absorption of Colostral Antibody in South Dakota Beef Calves. Amanda K. Weaver, William B. Epperson, Timothy A. Wittig, Richard J. Pruitt, Donald M. Marshall, and Alan R. Bender...... 127 Losses Associated with Gastrointestinal Nematodes in Yearling, Stocker, Grass Calves. Keith Mertz, Bill Epperson, and Mike Hildreth...... 129 Effects of Melanocortins (MSH) and Agouti Protein on Melanogenesis in B16 Melanoma Cells. Partha Ramasastry and Nels H. Granholm ...... 131 Measurement of Estrone and Progesterone in Mouse Serum Using Enzyme Linked Immunoassays. Maureen Diggins, Julia Spiry and Nels H. Granholm...... 133 Comparison of Native and Introduced Plants in Selected Fields and Roadsides in Tall Grass Prairie in Ottertail County, MN. Donna Hazelwood ...... 135 Seed Biology of Honey Locust in the Northern Great Plains. Soja Sekharan, Arvid Boe, Paul Johnson and Shannon Puckett ...... 137 Evaluating Site-Specific Post Emergence Weed Control Strategies. C.M. Cole, S.A. Clay, D.E. Clay, K.J. Dalsted, C.L. Reese and C.G. Carlson...... 139 Using Spectral Reflectance and Remote Sensing to Compare Crops and Weeds. C. Poppen, S.A. Clay, D. Clay, K. Dalsted, C.G. Carlson, and M.M. Ellsbury ...... 141 Determining Conditions for Growth and Maintenance of Ustilago Maydis Under Laboratory Conditions. Kazmann J. Foster and Donna Hazelwood ...... 143 Evaluating Yield Differences of Phytopthora Resistant and Non-Resistant Soybean Varieties by Soil Electro-Conductivity Zones. C.L. Reese, D.E. Clay and C.G. Carlson ...... 145 The Influence of Habitat on Species Richness and Abundance of Wetland Birds at Ordway Prairie, South Dakota. Mary C. Miller and Lester D. Flake ...... 147 Chromosome Arm Dosage Effects on the Expression of the High Molecular Weight Glutenin Proteins of Wheat. Jill D. Kruse, Jesse D. Munkvold, Erin E. Lee, Elizabeth A. Hamann, Ellen J. Hamann, and Michael K. Wanous...... 149 Carbon Isotope Discrimination as a Marker for Heat-Tolerance in Wheat. Kara J. Fuehrer, Steven R. Moeckly, Michael E. Chapman, Michael K. Wanous and Patrick F. Byrne ...... 151 A Mathematical Model of Two Competing Species in a Simple Chemostat When the Growth Function of Each Species is of Continued Fraction Form. Melissa A. Wittgenstein and A.S. Elkhader ...... 153 EDB Contaminant Plume at Nemo, South Dakota. Perry H. Rahn and Christopher S. Johnson ...... 155 Direct Electrochemical Oxidation of Amino Acids at Diamond Thin-Film Electrodes. Eric Hammond, Miles Koppang and Greg M. Swain...... 157 Experiences in Creating an Internet Chemistry Course and Students’ Attitudes Towards the Course. Dwayne W. Rehfeld ...... 159 Working with Dihydrogenatedtallow Dimethyl Ammonium Methylcarbonate. Jenny Bjergaard and Dr. Gary Earl ...... 161 Synthesis of Dimethyl Dihydrogenatedtallow Ammonium Compounds Exhibiting Aromatic Anions. Steven R. Moeckly and Gary W. Earl ...... 163 Synthesis of a Biodegradable Surfactant from Starch. Levi M. Stanley and Gary W. Earl...... 165 Synthesis of Quaternary Ammonium Compounds from 2-Aminoglucose. John D. Gilbertson and Gary Earl ...... 167 Virtual Geology Field Trips of South Dakota. Howard J. Woodard and Mike Hildreth ...... 169 Isolation, Cloning, and Sequencing of the Porcine Agouti-Related Protein Gene (agrp). Michael Halverson, Nels Granholm, Carl Westby and Donald Marshall...... 171 Biosystematic Evaluation of South Dakota Populations of Beckwith’s Clover Using RAPDs. Jeffrey D. Noll, Gary E. Larson and Melvin R. Duvall...... 173 Native Plants of the Northern Great Plains: Ethylene Induction of Asteraceae Seed Germination. April Stahnke and R. Neil Reese...... 175 Inheritance of Glume Length in Oats. Mei Shen and Dale Reeves...... 177 Production of Cell Wall Deficient Forms (L-Forms) From Selected Bacillus Strains. Nichole Baye, YongMei Luo, and Bruce Bleakley ...... 179 Electroanalysis of Biogenic Amines and Amino Acids at Diamond Thin-Film Electrodes. Miles Koppang, Eric Hammond, Malgorzata A. Witek and Greg M. Swain ...... 181 Avifauna of Agricultural Wetlands on Three Farms in Eastern South Dakota. Thomas R. Kirschenmann, Daniel E. Hubbard and Diane H. Rickerl ...... 183 Abstracts of Invited Symposium Papers presented at The 85th Annual Meeting of the South Dakota Academy of Science Electrochemistry and Characterization of Complex Surfaces Royce Engstrom, Organizer ...... 187 Nutrition Symposium. Bonny Specker, Organizer ...... 193 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 1

CONSOLIDATED MINUTES OF THE EIGHTY-FIFTH ANNUAL MEETING OF THE SOUTH DAKOTA ACADEMY OF SCIENCE APRIL 28-29, 2000 MOORHEAD, MN (TRI-STATE MEETING)

FRIDAY, 28 APRIL 2000—EXECUTIVE COUNCIL MEETING—4:30 P.M.

Proceedings Editor K. Higgins provided an update of progress on past and current Academy Proceedings. With help (Tom Holmlund, SDSU—for- matting and layout), Ken plans to have all past 1996-1999 and this year’s (2000) proceedings caught up and published by December, 2000. He pre- sented handouts to the Executive Council members describing how he has been corresponding with authors and invoicing for page and reprint charges. Page charges have been set at $25/page to cover costs for formatting, mail- ings, etc. Ken has temporarily set p an Academy account at SDSU and he explained how this should lighten Secretary/Treasurer Bill Soeffing’s work- load also. This will be voted on tomorrow. Ken mentioned he has never been provided with any guidelines or de- scription of his actual duties nor a copy of the Academy by-laws, etc. Some members believed the last update of the by-laws was published in the 1987 proceedings. Ken also suggested that a $100 honorarium by provided to Terri Symens for her able assistance beyond her normal job duties. Neil Reese volunteered to write a letter of thanks and Bill will provide Neil with a check. Neil also offered to search for a means to put the Acade- my’s proceedings on a web site in 2000 or 2001. Ken mentioned that Tom Holmlund might be able to do this as well. Neil also mentioned that action to have the 2001 annual meeting in Pierre fell through when no lodging could be found to host the meeting along with state rates. Therefore, Royce proposed that USD would host the 2001 Academy meeting, likely in the first or second weekend in April. Considerable discussion occurred relative to the status and future of the Junior Academy. Problem with getting High School sponsors and participa- tion. Last year Lenore Koczon and Ken Higgins lead the organization of the Junior Academy at the DSU-Madison meeting. Someone suggested that perhaps this (Jr. Academy) should be the re- sponsibility of the First and Second Vice Presidents of our Academy. Royce pointed out that checks needed to be sent to last year’s and maybe even the 1998 Jr. Academy winning presenters. Bill Soeffing pointed out the need to renew affiliation/application with AAS and NAS; meetings usually in January-February. Also, Academy CD renewed in August at 4.5% and it may go to 6.5% in- terest. 2 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000)

Three names submitted for life membership (25+ years member of Academy): Robert Stoner, Arlen Viste, and Lewis Kaurer. No nominations received for fellows award in 2000. Ken Higgins requested past and present lists of memberships (1996-2000) so that he could distribute printed volumes of proceedings as they become available. Neil Reese would like the same to help target new membership recruit- ment activities among institutions and agencies. Bill Soeffing passed out copies of the 1998 and 1999 budgets. Chuck Es- tee and Royce Engstrom will perform audits review. Executive Council meeting ended at 6:00 p.m.

SATURDAY, 29 APRIL 2000 SDAS ANNUAL BUSINESS MEETING—2:00 P.M.

President Neil Reese called the meeting to order and recognized a Quo- rum of members were present. Bill Soeffing moved that an account should be established at SDSU to fa- cilitate collection of monies for Academy Proceedings production, distribu- tion, and other associated costs and that a letter of appreciation with a $100 honorarium be approved for Terri Symen’s assistance to the Academy. Mo- tion passed. Chuck Estee and Royce Engstrom reported that their audit found the Sec- retary/Treasurer books in good order for the 1998 and 1999 budgets. Royce moved these be approved. Motion carried. Gary Larson gave the Awards and Resolution committee report (see fol- lowing report). Members present voted on new Academy Executive nominations and winners were: Second Vice President: Miles Koppang Members at Large: Donna Hazelwood and Mike Wanons Neil Reese led a discussion on the possibility of a more user-friendly web site and potential annual costs, etc., including a web site domain name. Estee moved, Maureen Diggins seconded a motion to support. Motion car- ried. Neil Reese proposed that we all attempt a conserted effort to advertise the S.D. Academy and to initiate recruitment on each campus. He proposed establishment of an ad hoc member promotion and recruitment committee with a representative on each campus. Neil announced that next year’s (2001) annual meeting will be hosted and held at USD in Vermillion. He thanked all that helped him with Academy business during the past year with special mention to Ken Higgins and Bill Soeffing. He then passed the President’s duties and gavel to Lenore Koczon of NSU after which Lenore gave her Presidential Address entitled “Understand- ing Change.” The Annual Business meeting closed at 3:00p.m. following Lenore’s ad- dress. Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 3

STATEMENT OF RECEIPTS, DISBURSEMENTS AND CHANGES IN CASH BALANCES FOR FISCAL YEAR 1999

Cash Balance on 1 January 1999 Certificate of Deposit $ 5750.94 Savings Account $ 9.15 Checking Account $ 9275.19 TOTAL BEGINNING CASH $ 15035.28

Receipts Membership Dues (Life-32/Regular-80+2/Associate-53) $ 1905.00 SDAS Proceedings Sales $ 84.55 Page and Reprint Charges $ 800.00 Annual Meeting Registration $ 430.00 Luncheon and Banquet Tickets $ 611.00 Other Income $ 1.00 Interest on Investments $ 422.80 TOTAL RECEIPTS $ 4254.35

Disbursements Corporate Filing Fee $ 10.00 Annual Meeting Dining $ 1245.16 SDAS Proceedings Printing $ 2380.71 Stale Warrant $ 35.52 TOTAL DISBURSEMENT $ 3761.32

Cash Balance on 31 December 1999 Certificate of Deposit $ 6173.74 Savings Account $ 9.15 Dakota State University Activity Account $ 586.00 Checking Account $ 8759.42 TOTAL ENDING CASH $ 15528.31

REPORT OF THE RESOLUTIONS COMMITTEE

The membership of the South Dakota Academy of Science thanks Dr. Row- land Barden, President of Moorhead State University, for making the MSU cam- pus available for our joint meeting with the Minnesota and North Dakota academies. We also wish to thank Drs. Vijendra “VJ” Agarwal, Joseph Provost, and Raymond Sicard of MSU for local arrangements and meeting coordination. 4 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000)

Thanks to President-Elect Lenore Koczon for her illuminating address “Un- derstanding Change” presented at our annual business meeting. Neil Reese is thanked and commended for his able leadership over the past year and for his efforts in establishing and maintaining the SDAS web site. We are greatly appreciative of the time and effort contributed by Secretary- Treasurer Bill Soeffing and Editor Kenneth Higgins. The Academy congratulates Sandra Dyson of Kennebec who was recog- nized as the Physical Science Teacher of the Year. Congratulations also to Janet Jacoway Simmons of Brandon Valley High School who was distinguished as Bi- ology Teacher of the Year.

Respectfully submitted, Gary Larson, Resolutions Committee Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 5

PRESIDENTIAL ADDRESS Understanding Change

Address to the South Dakota Academy of Science April 28-29, 2000

Presented by Lenore Koczon Northern State University Aberdeen, SD 57401

Despite what the media would like to have us think this year marks the end of a milestone in civilization—the last year in a millenium. The second millenium is going to start in just about 7 months from today on January 1, 2001. And as everyone else did in December of 1999 I have been thinking about what has happened and what changes I can expect to see in the next few decades. If we start by looking back… One thousand years ago life was very different than it is today. I for one would not have the opportunity to stand before a gathering of scientists, as women simply did not do that in 1000 AD. I would not have been listened to if I insisted on speaking and who knows, if my work had survived, at best it might be published today as a diary of a medieval madwoman! The civilizations of Egypt, Rome and Greece were already shadows of what they once where. Much of what the people of these times learned was fading from the collective memory of the people or already lost. But each of us today possesses the rediscovered knowledge of these philosophers and teachers from these ages as the foundation of what we know today. In ancient times, the heavens were well observed and charted in part because these an- cient peoples believed that they could presage natural disasters but more im- portantly they were the homes of the their gods. Astronomy and mathematics thrived and texts dating back to 1700 BC show that the Babylonians knew the Pythagorean relationship and could solve the quadratic equation.

In 400 BC the followers of Democritus and Plato argued whether or not matter could be continuously divided or if there was a fundamental stopping point. Our word “atom” is derived from the classical Greek language and means uncuttable. That idea did not resurface again until the 1800s when Dal- ton tried to explain the behavior of matter and proposed that there was a fun- damental stopping point to matter, which he called the atom. The contribu- tion of the Greeks to medicine is unquestionable. It was the Greek, Hip- pocrates who first proposed that disease was a natural phenomenon and as such needed to be treated with something other than prayer and ritual. 6 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000)

When Rome conquered the Greeks they were impressed by their level of knowledge but wondered why they were such intellectuals as common sense is what kept the Romans in power. And after all, they did conquer the Greeks… The process of trying to explain an observation—of doing research if you will—was quite foreign to the Romans and new arguments as to WHY and HOW the things they saw happening around them in the occurred were few and far between. From what I remember of a classics course in my un- dergraduate years at my alma mater, the writings of Pliny the Elder in the first century AD were a collection of natural phenomena, odd tales, mystical crea- tures and some science all rolled into many volumes, which seem more de- signed to entertain rather than to educate. As Rome gave way to the rise of Christianity the followers of this new re- ligion faced a dilemma, as they were part of this world but not really part of this world. The world they were living in couldn’t be ignored but to be over- ly concerned with worldly things was dangerous to the soul. None-the-less the early Christians soon found themselves involved in discussions, in philosophy and metaphysical arguments about Christ and their beliefs. A friend of mine who is a Benedictine scholar would tell you that you have to understand this world to be able to see the plan of God and the monks of his order and of other orders therefore could not allow the learning of the ancients to be for- gotten. So meticulously they copied the old manuscripts and had them wait- ing for those who would seek to rediscover them. At the beginning of this millennium some of the scientific discoveries that where made include the use of the first magnetic compass in 1117. Explosives were used in warfare in the West for the first time in 1151 and eyeglasses came onto the scene in 1288. An automatic striking clock dates back to 1334. Things were starting to change and the pace of technological innovation and scientif- ic discovery were quickening. Then the bubonic plague deluged Europe from 1348 to 1361 and the Ottomans were moving into Europe with the Battle of Kosovo in 1389. In 1492 Columbus sailed the ocean blue and discovered the new world. This shook up man’s belief in the ancient philosophies. For years—from the works of Ptolemy through Augustine—it had been held that there could only be three continents. Suddenly there was a forth one that offered fame and for- tune to those who could turn navigation into a more trustworthy venture. This in turned spurred the study of mathematics. The Renaissance was a time of feverish intellectual activity that led to re- discovery of the ancient knowledge and laid the foundation for the Scientific Revolution. The discoveries from this age challenged much of what man be- lieved and its effects on societies across the globe is still discussed and chal- lenged in many scholarly circles. One of the more dramatic and at that time even heretical discoveries was that of the act of placing the sun at the center of the solar system by a Polish astronomer and mathematician, Nicolas Coper- nicus. Despite the mathematics that substantiated his claims of a sun-centered universe, his theories were challenged and debated. Contemporaries of Coper- nicus could not understand why God would have placed man and Earth where they are lost in the immense space. After all, they believed that God did noth- Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 7 ing without a purpose and was not man his greatest creation? I have long been fascinated by the works of Copernicus and a few years ago a friend of mine shared a quote with me from this great scientist:

What I know is not approved and what is approved is not correct. that must sum up some of the frustration that he felt. Shortly after his death three more astronomers made observations and discoveries that affirmed the work of Copernicus and shattered the Aristotelian view of heavenly perfection. Tycho discovered a moon and a new star that where not where Aristotelian doctrine predicted and there was mounting evidence from his observations that the earth was not motionless. Galileo’s telescope proved even more stars ex- isted in the universe than had been imagined. Kepler discovered Mars. Yet despite the work of these men no one could propose an alternative explana- tion to the universe from Aristotle’s theory that made sense. No one could ex- plain why objects did not fly off the face of the Earth. No one that is, until Isaac Newton. In the beginning of the 1600s almost all that was known was being questioned and new answers were not easily forthcoming. In this era Rene Descartes proposes a simple explanation for the motion of an object. He states that bodies once in motion will remain in mo- tion in a straight line unless and until they are deflected from this line by the impact of another body. Hence by his theory, all changes of motion are the re- sult of impacts. Descartes theory did not involve a god or god-like being who intervened and controlled motion. But the 17th century was still a time of deep religious feelings. Isaac Newton set out to explain natural phenomena in such a way that God would be preserved and revealed to those who read his work. Newton’s simplistic genius into the insight of natural phenomena and his math- ematical ability—attested with his invention of calculus—regulated the universe once again with the help of God. He formulated three laws to explain the mo- tion of objects in the heavens and on earth. He saw gravity as the interven- tion of God in the physical world. His Principa (Philosophiae Naturalis Prin- cipia Mathematica or the Mathematical Principles of Natural Philosophy) which was published in 1687 is still one of the more revered scientific works of the millennium. Unfortunately my own discipline, chemistry, was stilled mired in “mystical argot” in the 17th century. It really is not until the early part of the 19th cen- tury that it makes a break form the search for the philosophers stone to be- come a discipline that it is based on scientific law. Meanwhile advances keep happening in science. Some of these were not great discoveries but where driven by the need to get information out to all sci- entists—not just those who could afford a priceless book. Scientific Societies— many still in existence toady – formed in England and in France. The printing press soon ushered in scientific journals. Some of the advances in technology that occurred from the advances in science in the 17th and 18th century include the mechanical calculator (1671), the piston pump (1690), the improved steam engine (1769), a vaccine against small pox (1795), a differential calculating machine (1833), Mendel’s Laws of Heredity (1866) and the invention of the light bulb (1879). 8 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000)

In the 19th century we see the birth of the modern ages of chemistry, bi- ology and physics. Chemistry finally comes of age with Dalton’s Atomic The- ory. One point he makes is that under ordinary physical circumstances chem- ical change, or if you prefer a chemical reaction, cannot transmute one type of an element to another type of element. The search for the secret reaction that will turn lead into gold had finally come to an end. Other events that help to turn chemistry into a real science include the ordering of the elements on the first periodic table and the realization that structure and reactivity are connect- ed. Of course understanding structure will take a few more years and even to- day sometimes puzzles or eludes us! Modern biology advances with Linnaeus’ classification system and Lamark’s suggestion that species evolve and Darwin’s evidence for evolution. The work of Pasteur, Koch and others revolutionizes thinking about the caus- es and eventually the cures for diseases that have plagued human beings since long before the start of this millennium and in some cases still cause untold suffering throughout the world. Physics continues to change and to tackle questions that could not be an- swered all that long ago. The discovery of X-Rays, the work of the thermody- namist Max Plank and of Albert Einstein shook the very foundations of this branch of science. Since the time of Newton light was treated as a wave. Work in the latter part of the 19th century and early 20th century had evidence mounting that it can and did behave as though it consisted of particles. And then there was the Theory of Relativity. While many an elementary school stu- dent can quote E = mc2 how many of us in this room can really comprehend what it means and what implications it will have on the future. Science has helped people at the end of the second millennium of the Common Era look and act and believe differently than our ancestors did at the start of it. The question of where it will take us next has many answers, most of which are not even thoughts in our imagination today. We have a contri- bution to make to the new millennium for not only are we scientists seeking to advance the knowledge base and the teachings of all of its disciplines but we are also educators. We are educating tomorrow’s scientists. The verb "to educate" has it roots in Latin and means to lead forth. Likewise, science means knowledge. It is interesting to think about where our students will lead us with the knowledge we have given them.

REFERENCES CITED

Bridson, Gavin (1994), The History of Natural History. Brock, William H. (1993), The Norton History of Chemistry. Also published as The Fontana History of Chemistry, 1992. Ferris, Timothy (1988), Coming of Age in the Milky Way. Kuhn, Thomas (1996), The Structure of Scientific Revolutions, 3rd Ed. Struik, Dirk J. (1987) A Concise History of Mathematics, 4th Rev. Ed. Complete Senior Research Papers

presented at The 85th Annual Meeting

of the South Dakota Academy of Science

Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 11

ANGLER OPINIONS REGARDING FISHING SUCCESS AND CRAPPIE REGULATIONS IN A SMALL SOUTH DAKOTA IMPOUNDMENT

Timothy J. Bister, Bradley M. Baker and David W. Willis Wildlife and Fisheries Sciences South Dakota State University Brookings, SD 57007

ABSTRACT

Anglers at Lake Alvin, South Dakota were interviewed during random-strat- ified creel surveys as part of a study designed to asses the effects of a 23-cm minimum length limit for black Pomoxis nigromaculatus and white P. annu- laris crappies. Anglers were asked what type of fish they were seeking, to rate their fishing success for that day (i.e., terrible, poor, average, good, or excel- lent), and their opinion regarding the crappie minimum length limit (i.e., in fa- vor or opposed). In both winter and open-water seasons, the majority of an- glers stated that they were fishing for anything, with crappies always being the second most common response. Angler type was divided into crappie and non-crappie anglers for within-group and between-group comparisons. Catch- ing and harvesting at least one crappie influenced crappie angler success rat- ings. However, neither catch nor harvest affected crappie or non-crappie an- gler opinion regarding the length limit during the winter and open-water sea- sons. The success rating from non-crappie anglers was not dependent on catching or harvesting a crappie during the open-water season. However, non- crappie and crappie angler success ratings were similar during winter seasons. Anglers at Lake Alvin thus favored the regulation even when positive results were not evident. Therefore, anglers appear to trust the decisions of conser- vation agency biologists when it comes to placing regulations on South Dako- ta fish populations.

Keywords

Black crappie, white crappie, angler opinion, fishing success, minimum length limit

INTRODUCTION

The opinion of anglers regarding restrictive fishing regulations is important to fisheries managers. The public’s perception of regulations may play an im- portant role in whether or not a proposed regulation will be supported by an- glers. An evaluation of a 254-mm minimum length limit on crappies Pomoxis spp. in Delaware Reservoir, Ohio found that 51% of anglers were in favor be- fore the regulation was enacted and 74% of anglers were in favor four years 12 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) after implementation of the minimum length limit (Hale et al. 1999). During the same study, 14 to 46% of other Ohio anglers were in favor of minimum length limits. Anglers complained about the small sizes of black crappies P. nigromacu- latus and white crappies P. annularis they were catching in Lake Alvin, South Dakota. Population assessments indicated truncated size and age structures (Guy and Willis 1993). However, growth rates were moderate compared to other South Dakota crappie populations (Guy and Willis 1995). Thus, state bi- ologists suspected that these populations were being overharvested (Knapp and St. Sauver 1995). Prior to any regulation changes, South Dakota Department of Game, Fish, and Parks (SDGFP) biologists conducted an angler opinion survey to evaluate how receptive anglers might be to restrictive harvest regulations on crappies in Lake Alvin. Initially, anglers were asked their opinion on a 20-cm (8-in) min- imum length limit. Of the anglers who responded, 91% were in favor. These initial respondents were resurveyed to assess their opinions on a 23-cm (9-in ) minimum length limit; 93% of those who responded were in favor. With pub- lic support, SDGFP instituted a 23-cm (9-in) minimum length limit for black and white crappies in Lake Alvin beginning January 1, 1996. Regulation signs were posted at lake access points and the new regulation was listed in the state fish- ing handbook. The objectives of this study were to determine the types of fish sought by anglers, if catching or harvesting at least one crappie had an influ- ence on how crappie and non-crappie anglers rated their fishing success, and if catching or harvesting at least one crappie had an influence on the opinions of crappie and non-crappie anglers regarding the minimum length limit.

STUDY AREA

Lake Alvin is a permanent warmwater impoundment located in southeast- ern South Dakota (Lincoln County) and is owned and managed by SDGFP. The lake has a surface area of 36 ha, maximum depth of 7.9 m, and mean depth of 3.4 m. Lake Alvin is a eutrophic impoundment, has a moderate lev- el of turbidity, and contains few submerged aquatic macrophytes (Stueven and Stewart 1996). There are very few prey fish species present in the lake. Thus, the prey base available to crappies primarily consists of zooplankton and macroinvertebrates.

METHODS

Random-stratified creel surveys were conducted at Lake Alvin during both the winter season (December through February) and the open-water season (April through September). From December 1997 through September 1999, an- glers were asked the following questions. What type of fish are you fishing for today? How would you rate your fishing success today: terrible, poor, average, good, or excellent? What is your opinion of the minimum length limit for crap- pies in this lake: in favor, highly in favor, opposed, highly opposed, or no opin- ion? Success and opinion questions were closed-ended to allow for easier in- Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 13 terpretation of data (Pollock et al. 1994). Responses were used to determine the percentage of anglers seeking various fish species during each season, how anglers rated their fishing success, and their opinion toward the crappie mini- mum length limit. Anglers were divided into either “crappie anglers” or “non-crappie anglers” for the purpose of this analysis. Chi-square analysis (SAS 1996) was used to determine if catching or harvesting at least one crappie had an influence on angler success rating or angler opinion regarding the minimum length limit. Comparisons were made for crappie anglers between years and between win- ter and open-water seasons. Comparisons were also made between crappie anglers and non-crappie anglers. Length limit opinion responses were com- bined into favorable and unfavorable categories because the number of anglers who responded either “highly in favor” or “highly opposed” was minimal. An- glers who had “no opinion” concerning the regulation were not included in the analyses.

RESULTS

Crappies represented the most sought fish during winter seasons (Table 1). The majority of open-water anglers stated they were fishing for “anything,” with crappies being the second most common response. The open-water sea- son included more diversity in angler type and in opinion regarding the length limit (Table 2). Harvesting and/or catching a crappie influenced how crappie anglers rat- ed their fishing success in both open-water and winter seasons. For example,

Table 1. Types of fish sought by anglers at Lake Alvin, South Dakota during winter and open-water seasons from 1997 to 1999. The number of anglers re- sponding (% is in parentheses) is listed for each season.

WINTER SEASON OPEN-WATER SEASON Fish sought 1997/1998 1998/1999 1998 1999

Anything 23 (28.75) 38 (40.86) 251 (44.82) 270 (63.08) Crappie 33 (41.25) 42 (45.16) 161 (28.75) 89 (20.79) Panfish 15 (18.75) 2 (2.15) 43 (7.67) 5 (1.16) Game fish 9 (11.25) 8 (8.60) 30 (5.35) 0 Bass 0 0 26 (4.64) 10 (2.33) Walleye 0 3 (3.22) 12 (2.14) 17 (3.97) Carp 0 0 11 (1.96) 0 Bluegill 0 0 9 (1.60) 15 (3.50) Northern pike 0 0 8 (1.42) 3 (0.70) Catfish 0 0 4 (0.71) 10 (2.33) Bullhead 0 0 3 (0.53) 2 (0.46) Yellow perch 0 0 2 (0.35) 7 (1.63) 14 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 12345 LENGTH LIMIT OPINION ANGLING SUCCESS RATING -- Y N- 0- 8 Y 0 0 01240 1 - N- 275 82042 3109124 N Y- 114 0 - Y 49 6 10 N 18 1229106 0 2 40 64 0 8 0 49 20000 2738139 22 15 0 6 3 5 14 0 N -N 2 - 0 69100 N 487 2822602 -N - 28 420 2226300 6 8 26 0 0 0 Winter 1998/1999 Y - 39 0 4 6 15 20 0 Open-water 1998 Y -Open-water 1999 93 Y 8 - 15 39 24 25 27 8 8 7 12 10 9 0 Angler type Season Year Caught Harvested In favor Opposed Crappie Winter 1997/1998 Y - 6 0 2 10 5 6 11 Table 2.Table their opinion of a crappie mini- (crappie and non-crappie) responding to questions asked regarding Number of anglers at Lake Alvin, length limit and their rating of angling success during creel surveys mum season, separated by South Dakota year, whether or not the angler caught a crappie, and whether or not the angler harvested a crappie. Angling success rating respons- es were 1 = terrible, 2 = poor, 3 = average, 4 = good, and 5 = excellent. Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 15 28720 12345 LENGTH LIMIT OPINION ANGLING SUCCESS RATING -- Y- N 0- Y 0 00520 - N 8 6- Y 0 N 0 05110 - 310 8- 686 Y 0 13 34 15477160 04810 12 N 2 15 139 0 0 118 91 00013 0 197 28 5 24 83 125 71 23 3 N -N 8 0 -N 1241300 - 234N 34 - 42 132 90 4 68 144 26 6 5 16 34 2 80 0 83 0 42 14 8 Winter 1998/1999 Y - 39 2 4 14 11 14 0 Open-water 1998 Y -Open-water 1999 75 Y 0 - 6 33 58 24 3 9 0 7 46 26 71 3 Angler type Season Year Caught Harvested In favor Opposed Non-crappie Winter 1997/1998 Y - 0 0 Table 2 continued. Table 16 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) during the open-water season, crappie anglers rated their fishing success sig- nificantly higher when they harvested at least one crappie (χ2=20.2, P=0.001) (Table 3). Similarly, winter crappie anglers rated their fishing success signifi- cantly higher when they caught at least one crappie (χ2=52.8, P=0.001). There was no significant difference between winter crappie and non-crap- pie angler success rating when they did not harvest a crappie (χ2=2.75, P=0.60), likely because most winter non-crappie anglers were seeking crappies as well as other panfish. While there was a significant difference in fishing success rat- ing between open-water crappie and non-crappie anglers who did not catch a crappie (χ2=20.5, P=0.001), these anglers did not necessarily have to catch a crappie to rate their success as “average” or better. Crappie angler opinions regarding the minimum length limit were not in- fluenced by whether or not they caught or harvested a crappie during either the open-water or winter seasons. Most anglers of all types in all seasons were in favor of the regulation (Table 4). For example, there was no significant dif- ference in length limit opinions between crappie anglers who did or did not catch a crappie during open-water seasons (χ2=1.44, P=0.23). There was also no significant difference in crappie angler opinions regarding the crappie min- imum length limit between the 1998 and 1999 open-water seasons (χ2=0.955, P=0.33), even though benefits of the regulation were not evident (Bister 2000). In addition, there was no significant difference between the opinions of win- ter and open-water crappie anglers (χ2=0.476, P=0.49). Finally, there were no significant differences between the opinion of crappie and non-crappie anglers in either the winter (χ2=0.08, P=0.78) or open-water (χ2=1.16, P=0.28) seasons when asked if they favored the crappie minimum length limit.

CONCLUSIONS

Crappies were the fish species most sought by anglers at Lake Alvin. Most anglers of all types in all seasons were in favor of the crappie minimum length limit. Catching and/or harvesting a crappie influenced crappie angler success ratings. Anglers rated their fishing success higher when they either caught or harvested a crappie. Winter non-crappie anglers appeared to be similar to crappie anglers. However, open-water non-crappie anglers did not necessari- ly have to catch or harvest a crappie to rate their success as average or good. Winter crappie and non-crappie anglers were probably similar angler types, as evidenced by their similarity in success ratings based on catching or harvesting a crappie. Angler opinions regarding the crappie minimum length limit were not de- pendent on catching or harvesting a crappie. There were no opinion differ- ences for crappie anglers between seasons or between years. Anglers still fa- vored the regulation even though the size structure of crappie population in the lake was not improving (Bister 2000). Therefore, anglers appear to trust the decisions of state conservation agency biologists regarding restrictive regu- lations on state fish populations. Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 17 P 2 χ GROUP 1 GROUP 2 CC OC OC OC 98 OC 98 OC 98 W NoC 98 O Yes 98 & 99C 97/98 & 98/99 O -C - No O 98 & 99 - -C - W 98 & 99C No - W Yes 98 & 99C 97/98 & 98/99 No Yes C W - 97/98 & 98/99 C No W - O - 97/98 & 98/99 C Yes O C 97/98 & 98/99 No C C - - O Yes - W 99 - O O NC 99 No 97/98 & 98/99 NC O 99 NC Yes NC 98 & 99 No Yes 99 O NC Yes W O 98 & 99 NC W - - - - NC 98 & 99 97/98 & 98/99 - - W Yes 98 & 99 97/98 & 98/99 No W Yes No 97/98 & 98/99 3.62 52.80 Yes - Yes 3.51 - 97/98 & 98/99 0.001 - 0.306 - - 20.20 0.477 4.68 - No - 0.001 14.74 0.322 Yes 14.77 0.005 No 9.52 0.005 Yes 10.59 4.07 0.009 5.70 0.032 0.396 2.75 0.222 11.24 0.601 0.004 Type Season Year Caught Harvested Type Season Year Caught Harvested Table 3.Table angler success rating comparisons at Lake Alvin, Chi-square results from conducted during creel surveys South Dakota September 1999. December 1997 through from season, were delineated by Comparison groups year, and whether or not they harvested a crappie.caught a crappie or whether not they C = crappie anglers, NC = non-crappie anglers, O = open-water, and W = winter. 18 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) P 2 χ 0.96 0.330 0.71 0.390 0.47 0.490 1.16 0.280 0.08 0.780 aa aa aa aa aa CO 99 CC W O 98/99 98 & 99 NCNC O W 98 & 99 97/98 & 98/99 aa aa aa aa aa GROUP 1 GROUP 2 CC OC WC W 98 & 99C W 97/98C W No 97/98C O 97/98 97/98 & 98/99 No W - - 98 & 99 97/98 & 98/99 - No C C O C W 98 & 99 W 98/99 Yes 98/99 No - - - 1.44 No 0.230 0.42 0.515 0.97 0.326 CC OC OC OCO 98 O 98 98 98 No 98 Yes - - - - No Yes C C O C O C O 99 O 99 99 No 99 Yes - - - - No 5.76 Yes 6.69 0.016 0.010 0.50 0.37 0.476 0.545 Type Season Year Caught Harvested Type Season Year Caught Harvested Catching or harvesting a crappie was not considered for comparison. Table 4.Table angler opinion rating comparisons at Lake Alvin, Chi-square results from conducted during creel surveys South Dakota September 1999. December 1997 through from season, were delineated by Comparison groups year, and whether or not they harvested a crappie.caught a crappie or whether not they C = crappie anglers, NC = non-crappie anglers, O = open water, and W = winter. a Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 19

ACKNOWLEDGMENTS

We would like to thank Todd St. Sauver, Allen Knapp, and David Lucchesi of the South Dakota Department of Game, Fish and Parks for their assistance. We would also like to thank the following individuals for their help in con- ducting creel surveys: J. Harrington, B. Harrison, E. Wald, D. Walter, P. Chvala, and N. Harris. Dr. Mike Brown and Craig Paukert provided statistical assis- tance. Partial funding for this project was provided by Federal Aid in Sportfish Restoration Project F-15-R, Study Number 1568 administered through South Dakota Department of Game, Fish and Parks. This manuscript was approved for publication by the South Dakota Agricultural Experiment Station as Journal Series No. 3185.

LITERATURE CITED

Bister, T. J. 2000. Evaluation of a 23-cm (9-in) minimum length limit for black and white crappies in Lake Alvin, South Dakota. Master’s thesis. South Dakota State University, Brookings. Guy, C. S., and D. W. Willis. 1993. Statewide summary of sampling data for black and white crappies collected from South Dakota waters. South Dakota Department of Game, Fish and Parks, Fisheries Completion Report 93-12, Pierre. Guy, C. S., and D. W. Willis. 1995. Growth of crappies in South Dakota wa- ters. J. Freshwater Ecology 10:151-161. Hale, R. S., M. E. Lundquist, R. L. Miller, and R. W. Petering. 1999. Evaluation of a 254-mm minimum length limit on crappies in Delaware Reservoir, Ohio. N. Am. J. Fish. Manage. 19:804-814. Knapp, A. and T. St. Sauver. 1995. Experimental nine inch crappie length lim- it proposal at Lake Alvin, Lincoln County. South Dakota Department of Game, Fish and Parks, Pierre. Pollock, K. H., C. M. Jones, and T. L. Brown. 1994. Angler survey methods and their applications in fisheries management. American Fisheries Soci- ety, Special Publication 25, Bethesda, Maryland. SAS Institute. 1996. SAS/STAT user’s guide. Release 6.12 edition. SAS Insti- tute, Cary, North Carolina. Stueven, E. and W. C. Stewart. 1996. 1995 South Dakota lakes assessment. South Dakota Department of Environment and Natural Resources, Pierre.

Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 21

BIOLOGICAL CONTROL OF CANADA THISTLE (CIRSIUM ARVENSE) IN SOUTH DAKOTA

Z. Liu, S.A. Clay and M. Brinkman Plant Science Department South Dakota State University Brookings, SD 57007

ABSTRACT

Canada thistle, Cirsium arvense L., is an aggressive invasive species that is found in the northern United States and not easily controlled by chemical and mechanical methods. Integrating biological control agents with other methods may improve control. This study investigated the effects of Canada thistle gall fly (Urophora cardui Diptera: Tephritidae); Canada thistle stem or crown wee- vil (Ceutorhynchus litura Coleoptera: Curculionidae); Canada thistle bud wee- vil (Larinus planus Coleoptera: Curculionidae); and Canada thistle tortoise bee- tle (Cassida rubiginosa Coleoptera: Chysomelidae) on Canada thistle at three SD sites 1997 and 1998, about three years after initial release. Active stem wee- vil larvae were found in all release sites early in the season (May, June). Bud weevils were found in a limited number of plants in July. Galls and gall fly larvae were found late in the season. Defoliation from the tortoise beetle was not noted at the one site of release. Total nonstructural carbohydrate (TNC) contents of roots were 50% lower in roots from release sites compared to TNC levels in roots from control sites at two locations in 1998. Since Canada this- tle infestations rely on roots for survival, reduction of root TNC may be an im- portant mechanism to reduce patch vigor, and ultimately, Canada thistle’s im- portance in the SD landscape.

Keywords

Canada thistle gall fly, Urophora cardui, Canada thistle stem weevil, Ceu- torhynchus litura, Canada thistle bud weevil, Larinus planus, Canada thistle tortoise beetle, Cassida rubiginosa

INTRODUCTION

Canada thistle, Cirsium arvense L., is a perennial weed that colonizes dis- turbed and undisturbed sites with rapid growth occurring through production of multiple shoots from lateral roots. Chemical and mechanical methods used to control Canada thistle have been moderately successful. Integrating other management techniques with these more traditional control methods may im- prove Canada thistle control. Nichols (1993) reported that integrated use of bi- ological and chemical control reduced Canada thistle infestations by 90% in 10 yrs in eastern Colorado. 22 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000)

Biological control of weeds is the use of living organisms (insects, pathogens, bacteria, and other organisms) for control of a certain weed. The weeds chosen for biological control are usually non-native in origin and one that has natural predators in it’s native home range. Before introducing the predator into the new environment, it must be screened to ensure that it will not attack desirable plants in the new area. In order to be successful, the con- trol agent must establish in the new environment and then attack the weed with enough vigor to do harm to the weed. Therefore, the control agent must already be adapted or adaptive to the new environment in order to be suc- cessful. Success in a biocontrol project is defined as: 1) having the control agent continuing to perpetuate itself in the new environment; and 2) reducing the weed population to levels below an economic threshold. Complete erad- ication of weed will not occur because the control agent will not eat itself out of “house and home”. There are several positive effects of using biological control agents for Canada thistle management. These positive effects include: 1) biocontrol agents that are approved for release are selective and will not move to eco- nomically important crops; 2) the agents can infest plants in inaccessible ar- eas; 3) once established in the habitat, the control agents are self-perpetuating and may migrate to other Canada thistle infestations; and 4) once established, the method is cost effective, less expensive, and less labor intensive than chem- ical and mechanical methods. The ultimate goal of Canada thistle biocontrol is to reduce plant vigor and its dominance in the landscape. Several Canada thistle biological control agents have been approved for re- lease in SD by USDA-APHIS-PPQ within the past decade. The first insect, Canada thistle gall fly (Urophora cardui Diptera: Tephritidae), was released in 1993 at Custer State Park (Helbig, USDA-APHIS-PPQ, Pierre, SD personnel communication). Other biological control agents approved for release are: the Canada thistle stem or crown weevil (Ceutorhynchus litura Coleoptera: Cur- culionidae) and the Canada thistle bud weevil (Larinus planus Coleoptera: Cur- culionidae), released in 1994; and the Canada thistle tortoise beetle (Cassida rubiginosa Coleoptera: Chysomelidae), released in 1995. Release sites are now located from the Black Hills to eastern SD with numbers released at one time ranging from 100 to 20,000. In order to assess the impact of the insect on Canada thistle the biology of the insect must be understood. Each one of these insects attacks the Canada thistle plant in a unique way.

Canada thistle gall fly (Urophora cardui)

This insect was the first biological control agent to be released on Canada thistle in SD in 1993. About 51,000 units of the Canada thistle gall fly have been introduced into SD (Helbig, personnel communication). Most of these in- sects were released in 1994 (40,000 units) with lesser amounts in 1995 (7,500 units) and 1993 (3,000 units). The adult thistle gall fly lays eggs in the stem of the Canada thistle plant. As the larvae develop, a marble to walnut size gall forms in the stem during the summer. The gall becomes a nutrient sink, reducing the plant’s vigor. As Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 23 the vigor decreases, the Canada thistle plant becomes less competitive and less able to resist pathogens and other insect infestations. In addition, plants with galls often produce fewer flowers and seeds. Physiological stress from stem gall formation was greatest in young Canada thistle plants (Forsyth, 1985) and during the growth phase of gall development (Lalonde, 1985). In addition, gall formation on the main shoot results in stunted plants and reduced flowering (Forsyth and Watson, 1985). The adult gall fly has a distinctive black W-shaped markings on its wings. This fly does best in dense stands of Canada thistle, where water is available. It seems to prefer semi-shaded areas to full sun. Areas subject to disturbance (mowing, grazing, chemical treatment) are not conducive to gall fly survival. This species also has difficulty establishing in areas with cold, open winters and hot, dry summers (Rees et al., 1995).

Canada thistle stem weevil (Ceutorhynchus litura)

About 37,000 units of the Canada thistle stem weevil have been released in SD since 1994 according to USDA-APHIS-PPQ records. The Canada thistle stem weevil is a native insect of Europe. Adults feed on stem and leaf tissue and can be found on foliage starting in August and overwinter in soil. The adult weevil has a pronounced white T-shaped marking on the back. Adult feeding is not a major source of damage. Eggs are laid in shoots of plant that are less than 5 cm tall. Developing larvae eat the vascular cambi- um of the stem tissue during spring and early summer which causes mining damage. Once the larvae exit the stem, other organisms (nematodes, pathogens) enter the stem causing secondary damage, which is the damage that causes decline of plant infestations. Stem weevils effectively reduce over- winter survival of Canada thistle, as well as reduce shoot production in the spring (Rees, 1990). Rotting of root tissues reduces shoot production the following spring. Fa- vorable habitats include dry areas where Canada thistle is in high densities and undisturbed by grazing, mowing, or flooding.

Canada thistle bud weevil (Larinus planus)

About 25,000 Canada thistle bud weevils have been released in SD since 1994. Originally, this insect was accidentally introduced into the northwestern states of the U.S. but has shown some promise in Canada thistle control. The larval stage is the most destructive stage of the weevils lifecycle, although high numbers of adults can also cause plant damage on the upper leaves and buds. The adults lay eggs in unopened buds. As larvae develop, they feed on the bud tissues and developing seed, reducing seed production. Unfortunately, most plants regrow from vegetative shoots that are not attacked by this insect. The effectiveness of this insect is questioned because it only attacks terminal buds, leaving late developing buds uninfested. Augmentation with different biocontrol agents has been recommended. 24 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000)

Tortoise beetles (Cassida rubiginosa)

About 10,000 tortoise beetles have been introduced into SD since 1995. Not much work has been done on the Canada thistle tortoise beetle. Even though both adults and larvae feed on plant leaves, the adult is more destruc- tive than earlier insect stages (Ward and Pienkowski, 1978). If this insect is in high enough densities, constant defoliation will reduce plant vigor (Forsyth, 1985; Ang et al., 1994). Unlike chemical control, where injury can be seen within weeks after ap- plication, injury from biological agents may not be observed for months or years after release. To determine if the control agents are reducing Canada thistle vigor, assessment is needed. This study examined the effect of Canada thistle biocontrol agents on Canada thistle growth and root carbohydrate con- tent at three release sites in eastern SD. Biocontrol agents had been released from 1 to 5 years prior to the beginning of the study.

MATERIALS AND METHODS

Experiment sites and insects

This experiment was conducted at three release sites in eastern SD (Table 1). Site 1 was located near Clear Lake, SD. Two locations near the Clear Lake release site were chosen as control sites where biocontrol agents had not been released, and a third location had the Canada thistle stem weevil released. Site 2 was located at Lake Louise State Park near Miller, SD. A control area where

Table 1. Canada thistle biocontrol species introduced to study sites in South Dakota.

SITE SPECIES YEAR RELEASED

Deuel County Clear Lake Canada thistle stem weevil (Ceutorhynchus litura) 1995

Hand County Lake Louise Canada thistle stem weevil (Ceutorhynchus litura) 1995, 1996 Canada thistle bud weevil (Larinus planus) 1995,1996 Thistle stem gall fly (Urophora cardui) 1995 Tortoise beetle (Cassida rubiginosa) 1996

Charles Mix County Lake Andes NWR Canada thistle stem weevil (Ceutorhynchus litura) since 1994

Chytka Canada thistle bud weevil (Larinus planus) since 1994 Thistle stem gall fly (Urophora cardui) Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 25 no insects were released was chosen and a second area was chosen where four bioagents, the Canada thistle stem weevil, thistle stem gall fly, Canada thistle bud weevil, and tortoise beetle had been released. Site 3 was located near Lake Andes, SD. A control location was chosen and 2 release locations were monitored. One release location was near Lake Andes in the Lake Andes Na- tional Wildlife Refugee (LANWR) and the second was on a private landowner’s acreage (Chytka). The biocontrol agents released at these sites were the Cana- da thistle stem weevil, thistle stem gall fly, and bud weevil.

Sample collection

About 30 Canada thistle stem samples were collected from each site three to four times between May and October in 1997 and 1998. When stem sam- ples were collected stem height, leaf condition, stem-mining damage, number of galls, and number of larvae present in stem or gall or both were determined. Stem mining was quantified by splitting the stem in half and measuring the length of the mining tunnel. Five plant roots were selected from each site and analyzed for total nonstructural carbohydrate (TNC) content.

Carbohydrates analysis

Canada thistle root samples were cleaned, air dried, cut into thin slices, and dried at 70ºC. The dry sample was ground with mortar and pestle to pass through a 40-mesh screen. The total nonstructural carbohydrate content (TNC) in 0.1 g of root mate- rial was removed using a modified Weinmann method: the samples were di- gested by boiling with water for 5 min., cooled to room temperature, pH buffer and mylase enzyme solutions were added, and incubated at 40ºC for 24 h. The solution was filtered through a Whatman #5 filter paper, and analyzed using Shaeffer-Somogyl copper-iodometric titration (Smith, 1981).

RESULTS AND DISCUSSION

Clear Lake

Canada thistle plant height. Canada thistle plants at the control site were taller than plants at the release site in May and September 1997 (Table 2). Height differences were not observed in July 1997 (Table 2). However, Cana- da thistle plants at the control site were shorter than plants at the release site in June and July, 1998 (Table 2). Height differences between control and re- lease sites most likely are due to environmental effects. The release site was located on a toeslope, close to Clear Lake, where it was cool and moist com- pared to the control area that was in a summit position. Insect damage may also have contributed to the plant height with injured plants elongating to a greater extent than the noninjured plants in the control area. Canada thistle stem damage. At Clear Lake, stem damage (defined as length of stem that was mined by insect larvae) was first observed in May, 26 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) site site Pr > t Control Release TOTAL NONSTRUCTURAL TOTAL Larvae Stem damage NUMBER OF THISTLE site site site site Pr > t Damaged plants Control Release Control Release PLANTS COLLECTED HEIGHT STEM WEEVIL DAMAGE CARBOHYDRATES Date1997 (no.) (no.) (cm) (cm) (no.) (%) (cm) (%) (total)(no./plant) (%) (%) Table 2.Table damage at Clear Lake in 1997 and 1998. Canada thistle stem weevil and the number Data reported include in the table of damaged plants,percentage of of stem that was damaged and the number the length of stem damage and the percentage per plant, of larvae number in all plants and the average found larvae stem weevil con- and the total nonstructural carbohydrate and release sites. the control from tent of roots bioagent in 1997. any from damage any site did not have The control The control 1998. on the plants in August and one gall fly larvae site had one stem larvae in the were also found larvae bud Two site in August. the control head of one Canada thistle plant from flower MayJuneJuly 10Sept 0 43 301998 30 30May 30 11.9June 31 naJuly 8.5 na 87.9Aug 30 64.8 68.7 0.006Oct 86.7 19 30 53.5 30 na 31 0.45 0.0001 20 30 na na 30 38.8 47 14 28 6.2 8 55.2 0 48.3 na 47 73.9 27 87.9 0.0001 na 0 na na 87.6 0.0001 4.7 4.4 0.56 17 0 7 53.9 7 5 0 16 3 0 na 23 1 0 53 0 14 10 0 3 47 0 8 0 23 20 na 0 82 12 10 0 4>0.20 na 13 na 2.8 4.0 9 0>0.20 0 1 3.3 3.6 0 17 na 0 0 0 0 3.8 0 5.1 3.7 1.7 4.6 0.06 2.3 1.9 0.001 1.8 0.06 na 0.002 7.3 na Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 27

1997. Forty-seven percent of the collected stems from the release site had min- ing damage (Table 2). Stem damage was not observed until June in 1998 and then only 23% of the collected stems had mining damage (Table 2). By Au- gust 1998, 82% of the Canada thistle stems had mining damage whereas in September 1997, no collected stems had damage. Mining damage averaged 4.5 cm of the stem length in 1997 which was 7% of the stem in June but only 5% in July due to stem elongation. In 1998, about 9.5 cm of stem was mined accounting for 10 to 20% of the stem length. These data indicate that Canada thistle stem weevils were active at the release site al- though the larvae were not always observed. Canada thistle stem weevil larvae were found 40% of the stems with an av- erage density of 3 larvae/stem in May 1997. In June 1997, the incidence of lar- vae was 3% with 3 larvae/stem. In 1998, larvae were found only in June in 13% of the stems at a density of 1 larvae/stem. Although fewer larvae were found in 1998 than in 1997, stem mining incidence and mining length were greater in 1998. Observing larvae in the plant in May and June would be consistent with the reported life cycle of the Canada thistle stem weevil. The Canada thistle stem weevil lays its eggs in March and April, with larval feeding occurring in the stem through late June (Rees et al., 1995). The larvae exit the stem and pupate in the soil with adults emerging in August. The adults overwinter in soil and become active again in March of the following year. Canada thistle root carbohydrate content. TNC contents of thistle roots were similar among all samples collected in 1997 (Table 2). In 1998, root TNC content at the control site averaged about 4.2 mg TNC/g of root and av- eraged about 1.9 mg TNC/g of root at the release site, a 60% reduction in TNC in roots at the release site throughout most of the growing season. In Octo- ber, TNC content of the roots in the release site increased by 4-fold. This dramatic increase in TNC root content has been reported for Canada thistle (McAllister and Haderlie 1985) and leafy spurge (Lym and Messersmith 1987). TNC helps the root to overwinter and aids in promoting spring shoot growth. It was unfortunate that Canada thistle in the control site was not col- lected so that a late-season comparison between insect-stressed and non- stressed plants could be made. However, the root TNC content in May 1998 was 1.5 times lower in the release site compared the control site. Biocontrol agents may be adversely affecting overall root health. In the long run, this type of effect should reduce stand vigor, since the main mechanism of Canada this- tle spread and sustainability is due to buds on lateral roots.

Lake Louise

Canada thistle plant height. Canada thistle plants were taller at the re- lease site in June and September 1997 and June and October 1998 when com- pared to plants at the control site during the same months (Table 3). Insect damage may have contributed to differences in plant height with injured stems elongating to a greater extent at the release site than at the control site. 28 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) n site site Pr > t Control Release TOTAL NONSTRUCTURAL TOTAL Larvae Stem damage NUMBER OF THISTLE site site site site Pr > t Damaged plants Control Release Control Release PLANTS COLLECTED HEIGHT STEM WEEVIL DAMAGE CARBOHYDRATES Date1997 (no.) (no.) (cm) (cm) (no.) (%) (cm) (%) (total)(no./plant) (%) (%) Table 3.Table Canada thistle stem damage at Lake Louise in 1997 and 1998. and per- the number Data reported include in the table centage of damaged plants, of of stem that was damaged and the number the length of stem damage and the percentage per plant, of larvae number in all plants and the average found larvae stem weevil con- and the total nonstructural carbohydrate and release sites. the control from tent of roots bioagent in 1997. any damage from any site did not have Plants in the control I 1998, in July, the stem weevil site had damage from plants in the control August, September, and October. was The stem weevil in 4 plants. found with about 4 larvae and August in July found In August, of Cana- in the flowers were found eggs larvae 13 bud da thistle. JuneAugSept 30 301998 31 23May 40June 40 104July 30Aug 133 30 128Oct 81 30 30 131 0.001 54* Stem weevil larvae. 31 141**Galls found on stem and gall fly larvae in galls. 0.54 30 15 16 0.001 30 29 49 29 149 13 94 29 140 62 26 0.005 117 73 29 18 0.0001 65 143 0.0001 10 10 105 0.34 20 11 8 44 0.001 3* 25 67 8 81 3* 83 9** 20 1.3 2** 18 3** 16 67 10 10 2** 62 26 9 8 2.4 14* 23 29* 3* na 6 1* 2.5 3* 22 4.3 >0.5 3* 3.7 1** na 0 na 1** 3.7 na 0 6.5 3.0 2.5 1.7 0.48 4.7 0.002 1.9 7.5 0.39 2.7 0.02 1.1 0.001 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 29

Canada thistle stem damage. Stem damage was observed at the first sam- pling date (June, 1997 and May, 1998) and throughout the rest of the season (Table 3). The percentage of stems with mining damage ranged from 65% to 94% in 1997 and from 62% to 83% in 1998. Only 3 stem weevil larvae were found in 1 stem in June, 1997. In 1998, stem weevil larvae were found in 48% of the stems in May and 60% of the stems in June, averaging 3 larvae per stem. In August, more than 65% of the stems had mining damage, although no stem weevil larvae were present. The length of mined stem varied between years. In 1997, the average mined length was about 13 cm. In 1998, the mined length ranged from about 1 cm (May) to 23 cm (October) which was 5 to 25% of the entire stem length. These data indicate that thistle stem weevils were active in the release site. In 1998, 60% of the sampled stems from the control site had mining dam- age in October and stem weevil larvae were found in 5 stems. These data in- dicate that stem weevils were present at the control site. In our study, we sam- pled control areas prior to sampling release sites and the three locations were sampled on different days. Therefore, the outmigration of the insect may have occurred by natural movement (wind, flight, etc.) rather than our movement between sites. Galls and gall fly larvae were found on stems in August and September 1997, and August 1998 although not in high abundance (Table 3). The ap- pearance of galls and gall fly larvae indicate that the gall fly was active at the Lake Louise release site. Bud damage and bud weevils were observed on one plant at Lake Louise in July 1998. Egg cases were found in buds of 13 more plants indicating that 43% of the sampled plants were infested. The greatest number of egg cases that were found in one bud was five. Bud weevil larvae were found in four plants with the highest density of 2 larvae/bud. The fact that buds were in- fested is encouraging. Destroying Canada thistle seed, while not reducing es- tablished Canada thistle infestations, would reduce the possibility of newly seeded infestations. No tortoise beetles or evidence of damage from tortoise beetles were ob- served. This damage would have been characterized as defoliation of the Canada thistle plant. Canada thistle carbohydrate content. TNC content of the Canada this- tle roots was lower in roots from the release site compared to the content ob- served in the control site throughout 1998, although no differences were ob- served in June 1997. TNC contents in roots from the release site averaged about 2.1 mg TNC/g of root during 1998, whereas TNC in control roots aver- aged about 5 mg/g of root. In October, TNC content of control roots increased about 160% compared to TNC in August, whereas TNC in roots from the re- lease site decreased by 60% during that period. In the long run, this type of TNC reduction should reduce stand vigor. 30 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000)

Lake Andes

Canada thistle plant height. When compared to control plants, plants were taller at LANWR in May and June 1997 and at Chytka in June and Septem- ber 1997 (Table 4). In 1998, Canada thistle plants at Chytka were taller than control plants in May, June, and July whereas Canada thistle plants at LANWR were shorter in May and taller in July than Canada thistle plants from the con- trol area. Canada thistle stem damage. Stem damage was first observed in June at LANWR, and in May at Chytka in 1997 (Table 4a). At LANWR, 28% of the stems collected in June 1997 had damage, whereas in September only 3% of the stems were damaged. At Chytka in 1997, the percentage of stems mined ranged from 15% to 53%, with the lowest percentage in September, and high- est percentage in July. In 1998, some stem damage was observed in May and June samples (3% of plants sampled), but by October 75% of the collected plants had mining damage. The length of stem mined at LANWR ranged from 1 to 21 cm in 1997, and 0.5 to 10.5 cm in 1998, respectively. At Chytka, stem damage ranged from 6 to 15 cm in 1997, and 1 to 22 cm in 1998, respectively. This damage occurred in 4% to 27% of the entire stem length. Stem weevil larvae were only found at Chytka in May 1997. At LANWR in 1998, larvae were found in one stem in both May and June, with a density of 5 and 1 larvae/stem, respectively. At Chytka, stem weevil larvae were found in 4 stems in June and 3 stems in July, with average densities of 2 and 1 lar- vae/stem, respectively. Canada thistle gall damage was observed in both 1997 and 1998 samples from both locations. At LANWR, galls were present on 7 stems with an aver- age larval density of 6 larvae/gall in July 1997, and on 12 stems averaging 5 lar- vae/gall in September 1997. Galls were present on 2 stems in July 1998 al- though no larvae were found, and on 3 stems with about 5 larvae/gall in September 1998. At Chytka, one stem had a gall in July and September 1997, with 4 larvae/gall and 1 larvae/gall, respectively. Galls were found on 7 stems collected in August 1998 with an average density of 3 larvae/gall. Bud damage was observed on six plants at Chytka in July 1998. The av- erage number of larvae found per bud was 16. Stem mining was not observed in stems from the control site in 1997. However in 1998, mining was observed in 4% of stems collected from May through August. Larvae were found in two stems at a density of 1 larvae/stem in August. In October, 54% of the sampled plants from the control site had some mining damage that ran for about 30% of the entire stem length. Galls were also found on control stems in September 1997 (3 plants with 6 lar- vae/gall) and August 1998 (3 plants with 4 larvae/gall). These observations indicate that Canada thistle stem weevil, Canada thistle gall fly, and Canada thistle bud weevil were active in the release sites. There was some movement from release site(s) to the control area. Canada thistle carbohydrate content. TNC contents of the Canada this- tle roots from both release sites were similar compared to TNC in roots from the control (Table 4b). Although stem mining was evident in a number of plants, this damage did not affect the root health. Since most of the TNC in- Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 31 s Larvae damage CHYTKA RELEASE plants Larvae damage site site site site site Pr > t plants NUMBER OF THISTLE LANWR Chytka Control LANWR Chytka Damaged Stem Damaged Stem PLANTS COLLECTED HEIGHT LANWR RELEASE SITE DAMAGE SITE DAMAGE Table 4a.Table Canada thistle stem damage at Lake Andes site in 1998. and percentage the number Data reported include in the table plants,of damaged of stem weevil and the number of stem that was damaged the length of stem damage and the percentage per plant, of larvae number in all plants and the average found larvae content of roots and the total nonstructural carbohydrate and release sites. the control from the gall fly in September of 1997 with three plant site had damage from Plants in the control galls and larvae.having In 1998, and gall fly in August, the stem weevil site had damage from the control plants from 1998 with galls and larvae. plants having two site in October, damage was also observed in the control Stem weevil 1998 with 13 plants damage,having were observed. although no larvae Date1997 (no.)May (no.)June (cm)July (cm) 14Sep. (cm) 32 141998 31 21 34May 30 5.4June 27 (no.) 59July (%) 7 na 30Aug. 121 (cm) 104 30Oct. 7 142 (%) 127 29 101 30 0.001 30 (total)(no./plant) 139*Stem weevil larvae. 0.1 30 139 (no.)**Galls found on stem and gall fly larvae in galls. 28 (%) 12 na 40 0.001 31 9 53 (cm) 0 8 16 (%) 92 28 58 (total)(no./plant) 1 4 0 96 24 113 13 79 50 3 21 120 0.001 134 0 17 0.05 39 138 0.001 1 0 0 1 0.20 38 <1 0 1 0 0 3 0.04 0 0 3 0 7** 0 12** 0.5 0 6** 0 10 5** 6.5 6 0 25 8 11 0 38 16 0 4 10.5 4 1* 53 0 1* 29 27 15 15 5* 2** 1* 8 12 na 3** 2 6 0 na 5** 6 0 2 50 0 5 4* 1** 7 1** 0 7 17 2 4** 3 25 1** 12 1 6 13 75 9 16 4 22 2 4* 7 0 17 5 3* 2* 7** 0 0 1* 3** 0 32 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000)

Table 4b. Total nonstructural carbohydrate control of roots from Lake Andes sites in 1997 and 1998. No differences from the control site were found in ei- ther release site.

1997 1998 Control LAWNR Chytka Control LAWNR p>t Chytka p>t Date (%) (%) (%) (%)

May NA NA NA 3.5 2.8 0.41 2.8 0.41 June 2.6 2.2 2.7 1.9 2.0 0.90 2.0 0.90 July NA NA NA 1.5 2.3 0.29 2.7 0.15 Aug. NA NA NA 3.1 3.8 0.37 1.9 0.15 Oct. NA 2.4 2.3 5.9 6.0 0.95 7.1 0.36 formation came from roots collected in 1998 and there was evidence of stem mining at the control site, there may have been no differences to detect.

SUMMARY

Stem mining by the stem weevil was the most observed damage. Stem mining often was not observed until June but was prevalent in late season sam- pling dates. Stem mining was not observed unless the Canada thistle stem was split down the center, so that tunneling could be seen. Stem weevil larvae were found in the release sites early in the season (May, June), and occasion- ally in the control sites late in the season. Bud weevils also were found in a limited number of plants from Lake Louise and Lake Andes. In most cases, damage was not observed until July and the bud had to be split to find egg cases or larvae feeding on immature seed and/or flower receptacle parts. Galls and gall fly larvae were found on stems late in the season. Defoliation from the tortoise beetle was not noted at Lake Louise even though this insect had been released there. Plant height was not an indicator to determine if biological agents were ac- tive. Peschken and Wilkinson (1981) reported that mining activity did not af- fect thistle height. However, galls on the main stem have been reported to re- duce stem height compared with plants that had galls on the side shoots or plants with uninfested shoots (Peschken and Finnamore, 1982; Forsyth, 1985). Total nonstructural carbohydrate (TNC) content of the root was generally lower in roots from release sites compared to roots from control sites at Clear Lake and Lake Louise in 1998. The test for TNC, although complicated, may be a very good method to determine if the biocontrol agents are affecting the ability of Canada thistle to survive. High root TNC content has been linked to increased winter hardiness of roots. If TNC of the roots of an infested plant is low, then the plant may not survive the harsh SD winters. Since Canada this- tle infestations rely on root survival to perpetuate or increase patch size or both, the reduction of root TNC may be an important mechanism to reduce patch vigor, and ultimately, Canada thistle’s importance in the SD landscape. Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 33

ACKNOWLEDGEMENT

Financial support from the South Dakota Weed and Pest Commission is gratefully acknowledged. South Dakota Exp. Stn. Paper No. 3189.

REFERENCES

Ang, B.N., L.T. Kok, G.I Holtzman, and D.D. Wolf. 1994. Competitive growth of Canada thistle, tall fescue, and crownvetch in the presence of a thistle defoliator, Cassida rubiginosa Muller (Coleoptera: Chrysomelidae). Biol. Control 4:227-284. Forsyth, S.F. 1985. Stress physiology and biological weed control: a case study with Canada thistle (Cirsium arvense (L.) Scop.). Dissertation-ab- stracts-International-B-Sciences-and-Engineering. 45:2763B. Forsyth, S.F. and A.K. Watson. 1985. Stress inflicted by organisms on Canada thistle. Proceedings of the VI International Symposium on Biological Con- trol of Weeds. Agriculture Canada. Ottawa, Canada. 425-431. Lalonde, R.G. 1985. Period of maximum host stress during development of galls of Urophora cardui (Diptera:Tephritidae) on Canada thistle (Cirsium arvense). Proceedings of the VI International Symposium on Biological Control of Weeds. Agriculture Canada. Ottawa, Canada. 439-443. Lym, R.G. and C.G. Messersmith. 1987. Carbohydrates in leafy spurge roots as influenced by environment. Journal of Range Management. 40:139-144. McAllister, F.S. and L.C. Haderlie. 1985. Seasonal variation in Canada thistle (Cirsium arvense) root bud growth and root carbohydrate reserves. Weed Sci. 33:44-49. Nichols, J. The integrated use of biological and chemical control on the nox- ious weed Canada thistle. Rangelands. 15:256-257. Peschken, D.P. and J.L. Derby. 1992. Effect of Urophora cardui (L.) (Diptera: Tephritidae) and Ceutorhynchus litura (F.)(Coleoptera:Curculionidae) on the weed Canada thistle, Cirsium arvense (L.) Scop. Can. Ent. 124:145- 150. Peschken, D.P. and D.P. Finnamore. 1982. Biocontrol of the weed Canada thistle (Cirsium arvense): release and development of the gall fly Uropho- ra cardui (L.) (Diptera: Tephritidae) in Canada. Can. Ent. 114:349-357. Peschken, D.P., and D.P. Wilkinson. 1981. Biocontrol of Canada thistle (Cir- sium arvense): release and effectiveness of Ceutorhynchus litura (Coleoptera:Curculionidae) in Canada. Can. Ent. 113:777-785. Rees, N.E. 1990. Establishment, dispersal, and influence of Ceutorhynchus litura on Canada thistle (Cirsium arvense) in the Gallatin valley of Mon- tana. Weed Sci. 38:198-200. Rees, N.E., P.C. Quimby, Jr., G.L. Piper, E.M. Coombs, C.E. Turner, N.R. Spencer, and L.V. Knutson. 1995. Biological Control of Weeds in the West. Western Soc. of Weed Science, USDA-ARS, Montana Department of Agri- culture, Montana State University. November, 1995. 34 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000)

Smith, D. 1981. Removing and analyzing total nonstructural carbohydrates from plant tissue. University of Wisconsin. Research Rep 2107. p.5. Ward, R.H. and R.L. Pienkowski. 1978. Biology of Cassida rubiginosa, a this- tle-feeding shield beetle. Ann. Entomol. Soc. Am. 71:585-591. Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 35

IDENTIFICATION OF ESTs THAT ARE POSSIBLY RELATED TO THE FHB-RESISTANCE OF WHEAT (TRITICUM AESTIVUM L.) CULTIVAR SUMAI 3

D.H. Xing and Y. Yen Department of Bio/Microbiology and J.C. Rudd Department of Plant Science South Dakota State University Brookings, SD 57007

The epidemics of FHB (Fusarium head blight) can cause a great loss of wheat (Triticum aestivum L.) production. The purpose of this research is to investigate the genetic basis of FHB-resistance at the molecular level and pave the way for marker-assistant breeding of FHB-resistant wheat. Spring wheat cultivars Sumai 3 (FHB-resistant) and Wheaton (FHB-susceptible) were studied with RNA differential display and single-floret inoculation with a FHB isolate was used to initiate FHB. Several expressed sequence tags (ESTs) were re- vealed only in FHB-inoculated Sumai 3. We are characterizing these ESTs.

Keywords

Fusarium head blight, wheat, differential display

INTRODUCTION

Fusarium head blight (FHB, also known as “scab”), mainly caused by fun- gus Fusarium graminearum, is a devastating disease affecting all classes of wheat and other small grains. FHB can occur to wheat from heading to harvest and cause the failure of kernel development, scabby “tombstone” kernels and kernels contaminated with vomitoxin. The epidemic of FHB in recent years have resulted in a great loss in wheat production and the related food indus- tries (McMullen 1997). About $2,606 millon dollars of loss in American agri- culture and food industries between 1991 and 1996 was estimated by Johnson et al (1997). Breeding resistant wheat cultivars is an efficient way to fight FHB. Several sources of resistance have been identified. Different kinds of resistance were revealed (Schroeder et al. 1967, Meidaner 1997). However, the multiple gene nature of wheat resistance to FHB (Bai et al. 1989; Guo 1989; Singh et al. 1995; Ginkel et al. 1996) make the conventional breeding method time- and labor- consuming and low in efficiency. Also, the application of marker assistant breeding and gene transformation were limited due to the lack of basic infor- mation about FHB pathogenesis at the molecular level. Muehlbauer et al. (1999) investigated the expression of several common defense-related genes in 36 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) wheat spikes 48 hours after F. graminearum inoculation. No significant differ- ence was found between resistant and susceptible wheat cultivars, although the expression of those common defense-related genes were induced strongly. Establishment of the infection results from complex interaction between plant and fungus, involving the expression of resistant genes of the plant and virulent genes of the fungus. In both organisms altered gene expression occurs from the onset of the attempted fungal invasion onwards. These genes, which are induced specifically by each other (the host resistant genes by pathogen; the pathogen virulent genes by host), could be identified with differential dis- play of mRNAs between the resistant and susceptible cultivars. The PCR based mRNA differential display technique (DDRT-PCR) devel- oped by Liang and Pardee (1992) is a sensitive and quick method for the anal- ysis of differential gene expression. The principle of DDRT-PCR is to use a set of oligonucleotide primers, one being anchored to the polyadenylate tail of a subset of mRNAs, the other being short and arbitrary in sequence so that it an- neals at different positions relative to the first primer. The mRNA subpopula- tions defined by these primer pairs were amplified after reverse transcriptation and resolved on a DNA sequencing gel. This technique has been successfully used for the gene expression analysis in a plant-fungus interaction in tomato (Benito et al. 1996). The purpose of this research is to identify the specific FHB-resistant genes of wheat and the virulent genes of F. graminearum, to provide molecular markers for breeding resistant cultivar and to enrich our understanding of molecular mechanism of FHB pathogenesis.

MATERIALS AND METHODS

Spring wheat cultivars Sumai 3 (FHB-resistant) and Wheaton (FHB sus- ceptible) were used in this research. Plants were grown in a greenhouse on SDSU’s Brookings campus. Single-floret inoculation was conducted at the be- ginning of anthesis to initialize FHB development. 20 µl of conidiospore sus- pension (100,000 spores/ml) of F. graminearum isolate Fg4 or distilled water (used as control) was placed between the lemma and palea of the first flow- ering floret in the middle of a wheat spike with a pipette. The inoculated plants were Immediately moved into humidity chamber until the floret was harvested ( for the treatments less than 24 hours ) or for 24 hours ( for treatments more than 24 hours ). Caution was taken to prevent inoculum from contaminating other florets. The inoculated and adjacent 4 spikelets were collected in 0, 1, 16, 32 and 64 hours after FHB and water inoculation. Samples are immediately frozen in liquid nitrogen and stored at -80°C until use. High-quality, total RNA was extracted from the samples with Tri Reagent (Molecular Research Center, Inc. Cincinnati, OH, USA). Differential display of mRNA was conducted with RNAimageTM Kit ( GenHunter, Nashville, TN USA). The protocol provided with the kit was followed with necessary modification. Generally, the subsets of mRNAs were defined by the M nucleotide of the dT11M (M is the A, C or G degeneracy) primer anchored to the polyA tail of mRNAs and reversely transcripted. The subsets of cDNAs were then amplified Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 37

with the combination of the corresponding dT11M primer and an arbitrary

13mer primer, which is annealed at a different position relative to the dT11M primer. A total of 50 different primer combinations were tested for displaying differentially expressed mRNAs in the sampled wheat spikelets. The produced ESTs were resolved on 6% denatured acrylmide DNA sequencing gels and vi- sualized with silver staining (Bassam 1991).

RESULTS AND DISCUSSION

The response of Sumai 3 and Wheaton after inoculation

Three independent experiments showed that the response of Sumai 3 and Wheaton to FHB isolate Fg4 is very consistant. When the samples were col- lected 32 hours after inocu- lation, the inoculated single florets of both Wheaton and Sumai3 have got light FHB symptoms ( brownish spots at the glumes); But no FHB symptom was ob- served on the florets next to the inoculated single flo- rets. However, 8 days later after inoculation, the half FHB-treated Wheaton spikes were bleached and dry and no kernel develop- ment was observed. In the case of Sumai 3, the FHB symptoms spread only to the rachis next to the inoc- ulated single florets (Fig. 1) and the kernels developed well. These results indicate that the FHB inducing pro- cedure is very suitable to demonstrate the type II re- sistance of Sumai 3.

Differential Display

Using 50 primer combi- nations, a total of 30,210 Wheaton Sumai 3 ESTs were recognized in the spike samples, with an Figure 1. The responses of Sumai 3 and average of 40.8 (17-60) per Wheaton to F. Graminearum 10 days after primer combination for inoculation. 38 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) each sample. Of these ESTs, 800 are polymorphic among the treatments, an av- erage of 3.2 (0-14) per primer combination. Our data revealed several types of gene expression patterns (Fig. 2). For example, Some genes were expressed only in the spikes of either FHB inoculated Wheaton (Fig. 3a), FHB-inoculated

Figure 2. An example of mRNA differential displays of water-inoculated (1) spikes of Sumai 3 and the FHB-inoculated spikes of Sumai3 (2) and Wheaton (3). Samples are collected in 0, 1, 16, 32, 64 hours after inoculation. Primer combinations used are AP4+H-T11A, AP4+H-T11C and AP4+H-T11G. Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 39

Figure 3. Examples of differentially expressed genes in FHB-inoculated (+) or water-inoculated (-) spikes of a FHB-resistant wheat cultivar Sumai 3 (S) and a FHB-susceptible cultivar Wheaton (W) revealed by mRNA differential display. Samples are collected in 0, 1, 16, 32, 64 hours after inoculation. a: two ESTs expressed only in Wheaton; b: one EST expressed 16 hours after FHB-inocu- lated but is absent from the water-inoculated control; c: three ESTs expressed only in Sumai 3 and one EST showed increased expression in FHB inoculated Sumai 3; and d: one EST showed increased expression in FHB-inoculated Sumai3.

Wheaton and Sumai 3 (Fig. 3a, b), or the water- and FHB-inoculated Sumai 3 (Fig. 3c). Genes that expressed only in FHB-inoculated spikes of Sumai 3 (Fig. 3d) or change their expression level at certain time after FHB-inoculation (Fig. 3a, b, c, d) were also observed. Those ESTs that appeared in both FHB-inocu- lated Sumai3 and Wheaton spikes may be accounted for general resistance. It is also possible that they were resulted from the expression of Fusarium viru- lent genes. Those ESTs that expressed only in the FHB-inoculated Sumai 3 spikes are the most promising and are possible ones that are specifically relat- ed to FHB resistance in Sumai 3. Alternatively, they are Fusarium virulent genes which expressed specifically due to the interaction between Fusarium and Sumai 3. We are conducting northern and southern blots, trying to discriminate these two possibilities with these ESTs as probes. 40 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000)

LITERATURE CITED

Bai, G.-H., Q.-P. Xiao and J.-F. Mei. 1989. Studies on the inheritance of scab (Fusarium graminearum) resistance in six wheat varieties (China). Acta Agri. Shanghai. 5:17-23. Bassam, B.J., G. Caetano-Anolles And P.M. Gresshoff. 1991. Fast and sensitive silver staining of DNA in polyacrylamide gel. Anal. Biochem. 196:80-83. Benito, E. P., T. Prins and J.A.L.V. Kan. 1996. Application of differential display RT-PCR to the analysis of gene expression in a plant-fungus interaction. Plant Molecular Biology. 32:947-957. Ginkel, M.V., W.V.D. Schaar, Z.-P. Yang and S. Rajaram. 1996. Inheritance of re- sistance to scab in two wheat cultivars from Brazil and China. Plant Dis- ease. 80:863-867. Guo, T. 1989. Genetic analysis for resistance to scab in wheat. Acta Agri. Univ. Henansis. 23:40-47. Johnson, D., G. Flaskerud, S. Taylor and V. Satyanarayana. 1997. Measuring the economic losses due to wheat scab. National Fusarium Head blight Forum. St. Paul, MN, Nov. 10-12, 1997, pp. 5-6. Liang, P. And A.B. Pardee. 1992. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science. 257:967-971. McMullen, M., R. Jones and D. Gallenberg. 1997. Scab of wheat and barley: A re-emerging disease of devastating impact. Plant Disease. 81:1340-1348. Miedaner, T. 1997. Breeding wheat and rye for resistance to Fusarium diseases. Plant Breeding. 116:201-220. Muehlbauer, G.J., C. Pritsch, D. Somers, C., B. Buchnell, T. Hohn and C. Vance. 1999. Wheat spike-fusarium graminearum molecular interactions. In: J.A. Wagester, R. Ward, L.P. Hart, S.P. Hazen, J. Lewis and H. Borden (com- piled): The 1999 National Fusarium Head Blight Forum. University Print- ing, East Lansing, MI, USA, pp.28. Schroeder, H.W. And J.J. Christensen. 1963. Factors affecting resistance of wheat to scab caused by Gibberella zeae. Phytophthology. 53:831-838. Singh, R.P., H. Ma and S. Rajaram. 1995. Genetic analysis of resistance to scab in spring wheat cultivar Frontana. Plant Disease. 79:238-240 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 41

AN ICHTHYOSAUR BONE FROM THE JURASSIC OF SOUTH DAKOTA

David C. Parris and Barbara Smith Grandstaff New Jersey State Museum, Trenton, NJ 08625-0530

ABSTRACT

The first Jurassic ichthyosaur to be reported from South Dakota is a right angular, cf. Baptanodon. It is from the Sundance Formation in Rapid City, and found during 1962, but was only recently correctly identified.

Keywords

Ichthyosaur, Jurassic

INTRODUCTION

Although ichthyosaurs are well known from the Jurassic of Wyoming (Marsh, 1879, 1880A,1880B) none have previously been reported from the Jurassic of South Dakota, nor the Black Hills region. While some may exist in collections, the lack of recorded specimens and comparative materials from South Dakota has no doubt hindered their recognition. Some ichthyosaur frag- ments may indeed have been misidentified, as was the one described here. It is to be hoped that this reported occurrence will result in the recognition or discovery of others.

DISCOVERY

In 1962, excavations for a commercial development began on the east side of Jackson Boulevard (2100 block) in Rapid City, Pennington County, South Dakota. Mrs. Elsie Biegler, an enthusiastic amateur collector, was among those who inspected the site. Shelly fossils were numerous, and clearly indicated that the rock unit was the Sundance Formation, notably by the presence of belemnites. Mrs. Biegler also found a bone, which she generously donated to the Museum of Geology at the South Dakota School of Mines and Technology (SDSM). Although recorded and catalogued it remained unidentified until 1967, when one of us (D.C.P.) suggested that it might be a fish spine. This sug- gestion was generally accepted and the specimen long reposed under this name. Re-examined after a chance encounter in the collections during 1998, it proved to be of reptilian affinities instead, and is the only South Dakota spec- imen of a Jurassic ichthyosaur known to us. 42 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000)

SYSTEMATIC PALEONTOLOGY Order Ichthyosauria Blainville 1835 Family Ichthyosauridae Baur 1887 Genus Baptanodon Marsh 1880 cf. Baptanodon sp. (Figure 1)

Referred Material: SDSM 62277, a right angular bone.

Provenience: Sundance Formation (Jurassic) in Rapid City, Pennington County, South Dakota, from an excavation in the Northeast Quarter of the Northeast Quarter of the Southeast Quarter of Section Three, Township One North, Range Seven East (Cattermole, 1969). Glauconitic sandstone adhering to the specimen, as well as its probable position low in the section, indicate that it came from the Stockade Beaver Shale Member of the Sundance Forma- tion. The member has a thickness of up to twenty-five meters in the area and is correlated to the Bathonian and Callovian stages (Gries and Martin, 1985). Remaining exposures of rocks at the site, revisited during 1999, confirmed the provenience of the specimen; identical matrix was collected from the outcrop. Description: The specimen, SDSM 62277, is a mandibular element, the right angular bone. Situated in the ventral edge of the posterior part of the mandible, the angular generally has dorsal grooves for articulation with the surangular and dentary. Presence of these dorsal grooves led to recognition of the bone as reptilian, and its comparison with Jurassic ichthyosaur taxa. The angular bone in ichthyosaurs is elongate and relatively straight com- pared to the angular bones of other reptilian groups and bears a distinctive lat- eral flange on the labial posterior side as well as the previously mentioned dor- sal grooves. The descriptions of McGowan (1973) and Romer (1968) are par- ticularly instructive in this regard. The specimen is a virtually complete angu- lar of actual length 265 mm. Comparison to Yale University specimen YPM 1979, an isolated angular of Ophthalmosaurus (from the Oxford Clay of Eng- land), was essential to identification of the bone. The specimens compare closely even in size and surface texture and in the position and proportions of the posterior/lateral flange. Both specimens bear two dorsal grooves for artic- ulation with the surangular and dentary bones but differ in the proportions of the grooves. They are subequal in SDSM 62277, while in YPM 1979 the me- dial one is considerably larger in size and more extensive in length. The lat- eral groove runs nearly the entire length in SDSM 62277. In YPM 1979 it is well-developed only in the vicinity of the posterior lateral flange, extending on- ly slightly anterior to it and existing only as a slight depression rostrally. The two specimens may be presumed to be from different taxa. The differing groove proportions presumably indicate a surangular and dentary of subequal size (caudoventral thickness) in SDSM 62277, while in YPM 1979 the surangu- lar was of dominant size in this region of the mandible.

IDENTIFICATION

The best-known genus of ichthyosaur from the Sundance Formation is Baptanodon Marsh (1879, 1880A, 1880B), of which several species have been Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 43 described. Although we have no angular bone of Baptanodon for comparison, the expectation that it would be similar to that of Opthalmosaurus (as com- pared here) is reasonable. The two genera have been considered synonyms by some authors, for example Dechaseaux (1955), and McGowan (1991), but discussion of the validity of that synonymy is beyond the scope of this report. We have, of course, noted how the angular of SDSM 62277 differs from that of the specimen of Opthalmosaurus at Yale. We refer the South Dakota specimen to cf. Baptanodon sp. and note that the long expected occurrence of ichthyosaurs in the Jurassic of South Dakota is now documented.

ACKNOWLEDGMENTS

We thank Mrs. Elsie Biegler for her discovery and generous donation of the specimen, and for the additional information she provided at the site so many years later. Mary Ann Turner kindly assisted our comparative studies at Yale University. James E. Martin, William B. Gallagher and Christopher McGowan provided additional biostratigraphic information and reviewed the manuscript. The companionship and assistance of Rapid City Troop 131, Boy Scouts of America (Black Hills Area Council) during the 1999 field inspection was much appreciated.

REFERENCES CITED

Baur, G. 1887. On the morphology and origin of the Icthyopterygia. Ameri- can Naturalist 21;837-840. Blainville, H.M.D. de. 1835. Description de quelques especies de reptiles de la Californie. Nouvelles Annales du Museum díHistoire Naturelle 4:233- 296. Cattermole, J.M. 1969. Geologic map of the Rapid City West Quadrangle, Pen- nington County, South Dakota, United States Geological Survey, Geologic Quadrangle GQ - 828, Scale 1:24,000. Dechaseaux, C. 1955. Ichthyopterygia. IN J. Piveteau (Ed.), Traite de Paleon- tologie, pp 376-408. Paris. Gries, J.P. and Martin, J.E. 1985. Composite Outcrop Section of the Paleozoic and Mesozoic strata in the Black Hills and surrounding areas. IN Rich, F.J. ed. Geology of the Black Hills, South Dakota and Wyoming, American Ge- ological Institute, 2nd edition p. 261-292. Marsh, O.C. 1879 A new order of extinct reptiles (Sauranodonta) from the Jurassic formation of the Rocky Mountains. American Journal of Science (3), xvii, pp. 85-86 Marsh, O.C. 1880A. The limbs of Sauranodon, with notice of a new species. American Journal of Science (3), xix, pp. 169-171. Marsh, O.C. 1880B. Note on Sauranodon. American Journal of Science (3), xix, pp. 160-171. McGowan, C. 1973. The cranial morphology of the Lower Liassic latipinnate ichthyosaurs of England. Bulletin of the British Museum (Natural History), Geology 24:1 - 109. 44 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000)

McGowan, C. 1978. Further evidence for the wide geographical distribution of ichthyosaur taxa (Reptilia, Ichthyosauria). Journal of Paleontology 52:1155-1162. McGowan, C. 1991 Dinosaur, Spitfires, and Sea Dragons. Harvard University Press, Cambridge, Massachusetts, 365 pp. Romer, A.S. 1968. An ichthyosaur skull from the Cretaceous of Wyoming. Con- tributions to Geology, University of Wyoming 7:27-41.

Figure 1. cf. Baptanodon sp. right angular, lateral view. Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 45

A BISON SKULL FROM LYMAN COUNTY, SOUTH DAKOTA

B.S. Grandstaff and D.C. Parris New Jersey State Museum Trenton, NJ 08625-0530

J.E. Martin Museum of Geology South Dakota School of Mines and Technology Rapid City, SD 57701

Keywords

Bison, South Dakota, Missouri River, taphonomy, pathology

ABSTRACT

During August, 1998, a well-preserved bison skull was found in gully fill on the western bank of the Missouri River. The skull was resting upside down near the bottom of a gully incised into the upper part of the Niobrara Forma- tion. The gully was subsequently filled with poorly sorted unconsolidated ma- terial consisting of small to large, poorly-rounded chips of Niobrara chalk in a matrix of tan clay. The gully fill also contains a few chips of the Pierre Shale which crops out at the top of the hill. Sediment in the gully fill surrounding the skull resembles sheetwash deposits. A soil profile with vegetation was de- veloped on the gully fill. The cranium was excavated and jacketed for removal, and later prepared at the New Jersey State Museum. The specimen belongs to a male bison, based on the presence of horn burrs, with a tip-to-tip horn core width (780mm) slightly larger than the published range for Bison bison bison horn cores (510- 778mm for males). Morphology of the skull is similar to Bison antiquus occi- dentalis, but its radiometric age is only 710±40 14C years B.P, making it too re- cent to belong to any fossil species. Horn measurements (length on curve 280mm, straight-line length 250mm) are also slightly larger that those of Bison bison bison. Horn measurements and frontal widths (minimum 285mm, max- imum 362mm) fall within the overlapping ranges published for Bison antiqu- us occidentalis and Bison bison athabascae. Width of the occipital condyles (125mm) and skull length (518mm) fall within the sizes published for B. a. oc- cidentalis and the living subspecies of Bison bison. Skull morphology and di- mensions suggest a closer link with B. b. athabascae than with B. b. bison. This specimen retains some horn sheath, preserved near the tips of the horns. The upper cheek teeth are deeply worn, and the animal probably died of old age. There is no evidence of human activity associated with this skull. 46 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000)

INTRODUCTION

During August of 1998 the South Dakota School of Mines and Technology conducted a field paleontology course in central South Dakota. Wet weather kept the crew out of the usual field areas, but it was possible to do a prelimi- nary survey of outcrops along the Missouri River in Lyman County on land be- longing to the US Army Corps of Engineers. Outcrops of the Niobrara Forma- tion along the river had been cut by gullies in the past, and the gullies subse- quently filled in. One of the authors (B.S.G.) discovered a skull (SDSM 47704) partially exposed in the bottom of one of these sediment-filled gullies. Exam- ination in situ suggested that it might be reasonably complete, and appeared to have preserved horn sheaths. The skull was identified in the field as be- longing to the genus Bison. Excavation was conducted using standard pale- ontologic field methods. The skull and surrounding sediments were encased in a protective plaster jacket and returned to the lab for preparation. SDSM 47704 is the skull of one of the shorter-horned Bison species. Evo- lutionary history of Bison is still poorly understood (Guthrie, 1990a), and a de- tailed consideration of Bison evolution is beyond the scope of this paper. Bi- son evolution in North America can be briefly summarized as follows. During late Wisconsinan to mid Holocene time two indigenous forms of Bison existed in North America. According to McDonald (1981) these were closely related subspecies, B. antiquus antiquus Leidy and B. antiquus occidentalis Lucas Bi- son antiquus antiquus is the older subspecies, ranging in age from at least 45,000 to around 4000 years BP. Bison antiquus occidentalis existed from about 11,000 to 4000 years BP. The two subspecies coexisted for approxi- mately 7000 years, but were adapted to somewhat different environments, with B. a. antiquus being adapted to a savanna or wooded steppe environment and B. a. occidentalis to a more open prairie environment (McDonald, 1981). Mc- Donald believes that Bison antiquus occidentalis evolved from B. a. antiquus approximately 11,000 years ago by genetic drift in isolated populations of B. a. antiquus during a population bottleneck. This population decline was related to climatic and habitat changes following the last glacial advance, to human hunting, or to both (McDonald, 1981; Kurten and Anderson, 1980; Skinner and Kaisen, 1947). However, Guthrie (1970, 1990a) and Wilson (1969) suggested that there were two Bison lineages, a Bison priscus - Bison occidentalis lineage and a separate Bison latifrons - Bison antiquus lineage, in North America; al- though Wilson expressed some doubts about the origin of B. antiquus. McDonald (1981) believes that the two subspecies of Bison antiquus, after an initial period of isolation, came back into contact about 9500 years ago, and may have hybridized with one another. Guthrie (1990a) and Wilson (1969) agree that B. antiquus and B. occidentalis could have hybridized to produce a polyphyletic Bison bison, although Wilson (1974) later considered B. antiquus and B. occidentalis to be conspecific. With or without hybridization, Bison an- tiquus occidentalis eventually evolved into Bison bison, possibly in association with the Altithermal event 6500-4000 BP (Wilson, 1974). It is thus a transitional form between Bison antiquus and Bison bison (McDonald, 1981; Skinner and Kaisen, 1947; Guthrie, 1990a). Bison bison incudes the two living subspecies. Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 47

Bison bison bison Linneaus is a grazer inhabiting open prairie environments, and B. b. athabascae Rhoads inhabits more wooded and upland environments. The two living subspecies originated less than 5000 years ago. Bison latifrons Harlan is known from South Dakota (Green and Martin, 1960; Pinsof, 1985), as is Bison cf. antiquus (Pinsof, 1985). Most other cited remains are of postcranials, and are not identified to species (Pinsof, 1985). Bi- son antiquus occidentalis reportedly has been found in archaeological sites currently under study by the state Archaeological Center (Foshe, personal com- munication). Bison bison bison is extant in South Dakota, and is known from prehistoric sites (McDonald, 1981), as is Bison bison (subspecies not specified) (Semken and Falk, 1987). Two species of European Bison, B. priscus Bojanus and B. alaskensis Rhoads, occurred in North America during the Pleistocene. Their ranges ex- tended as far south as Mexico. Both species died out in North America, prob- ably by about 20,000 years BP (McDonald, 1981). While their ranges overlap with that of B. a. antiquus they did not coexist with B. a. occidentalis or with Bison bison. Bison priscus may extend into the Holocene in Alaska and north- ern Canada (Guthrie, 1990a; Wilson, 1969). Pinsof (1991) states that B. lat- ifrons and B. alaskensis may have survived until very late in the Wisconsinan in Idaho. Names and classification of Bison species and subspecies used in this pa- per follow McDonald (1981). Guthrie (1966, 1990a, 1990b) differs from Mc- Donald in using B.antiquus and B. occidentalis as separate species rather than as subspecies. Some authors (Guthrie, 1970; Wilson, 1974; Kurten and Ander- son, 1980) have placed occidentalis, or even both antiquus and occidentalis, as subspecies of Bison bison (B. b. antiquus and B. b. occidentalis). Bison an- tiquus and B. bison will be used when referring to forms that predate differ- entiation of subspecies for each species, or when no distinction is made be- tween subspecies.

GEOLOGIC SETTING

SDSM 47704 was found buried near the bottom of a gully cut into the Nio- brara Formation. It was located in the SW1/4 of the NE1/4, section 17, T104N R71W in Lyman County, South Dakota. Its GPS position was 43º48’N, 99º21’W. The skull was covered by about 2m of gully fill. Only the rim of the right or- bit and the right premaxilla were exposed in the side of the cliff when the skull was first observed. The gully, as preserved, was some 4m above the current river level, which is elevated because the site is upstream from a dam. Later- al distance from the pre-dam channel is at most about 1km. The gully may have been cut into a terrace formed when river level was higher than it now is. Warren (1952) indicated that terraces are preserved on both sides of the Missouri River in this region. The gully was filled with unconsolidated light tan clay which contained small to large clasts derived from the Niobrara chalk, and a few clasts derived from the Pierre Shale. The clasts were primarily oriented perpendicular to the skull. Their distribution in the jacket was nonrandom. Clasts were sparsely distributed in that portion of the jacket which lay toward 48 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) the head of the gully, and abundant in the region which lay along the gully bottom under the skull, particularly in that part of the jacketed block on the side closest to the gully mouth. The skull was upside down when found. It was oriented with its long ax- is directed S5ºE, with the rostral end of the skull directed toward the south. 1 The palatal surface of the skull plunged 31 ⁄2 degrees to the south. There was an isolated cervical vertebra located about 1m above the skull in the gully fill which might have belonged to the same individual, and a few bone chips in the jacketed sediment on the upgully side of the skull which probably did be- long to the same individual. Reexamination of the gully in 1999 did not pro- duce any additional bones. The gully fill had been penetrated by roots which are presumably mainly of modern age. There were also a few borings in the sediment made by mod- ern beetles. None of these borings penetrated the skull itself.

TAPHONOMY

This skull is exceptionally well preserved. Horn is still present covering the tips of both horn cores and extending much of the length of at least the caudal surface of the left horn core. All bones of the skull are preserved, and all bones are articulated except for the nasals, which are slightly displaced upward. The left horn core is broken near the tip, but does not appear to have been dis- placed very much following the break. None of the bones appear to have been abraded during transport, and none are broken except for the left horn core and minor damage to the auditory bullae. Even delicate structures of the basicranial surface (the paroccipital processes, muscular processes associated with the au- ditory tube, and the auditory bullae) are preserved. This skull clearly has un- dergone only minimal transport. It might have been protected to some degree during transport by remnant soft tissues, but taphonomic evidence suggests the skull was exposed to surface weathering prior to its burial. Longitudinal cracks are present following the bone fibers, and joint sur- faces have undergone mosaic cracking. Slight flaking of the surface layer of bone is evident on the nasals and maxillae, and covers considerably more than 1 cm2 of the bone surface. Thus, the weathering stage applicable to this skull is early stage 2 of Behrensmeyer (1978). The palatal-basicranial surface of skull, which faced upward at the time of discovery, is less weathered than the naso-maxillary surface. The frontals, which were also directed downward, are less weathered than the nasals or maxillae. Behrensmeyer (1978) observed that bone surfaces in contact with the soil may be more modified than upper sur- faces of the same bone, particularly if the bone is in contact with highly alka- line soils where salts such as Na2CO3 or NaCl crystallize on the bone surfaces. This skull was enclosed in an adherent layer of caliche, which did not affect the integrity of the bone surface over the frontals, horn cores, or palatal sur- faces. It is unlikely that formation of the caliche layer produced greater weath- ering confined to only a part of the area it covered. It is more likely that the skull lay exposed with its dorsal side up on the surface and was weathered for a period of time before being transported into the gully, probably by sheet- Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 49 wash following an intense storm. Lack of weathering to the frontals and horn cores may mean that they were protected by soft tissues (e.g. the horn sheaths, flesh, or the “hair cap” covering the forehead) for some time prior to burial. Based on weathering rates that Behrensmeyer (1978) observed in Am- boseli, stage 2 weathering implies that the skull may have lain on the surface for 1 or 2 years before being buried. It is likely that the period of exposure was less than one year, since water in the longitudinal cracks would probably have damaged the skull by frost wedging if it had been exposed over the win- ter (Rothschild and Martin, 1993). Longitudinal cracks in SDSM 47704 remain very narrow. However, Roe (1970) suggested that Bison skulls could last 50 years on the ground surface. The skull was probably buried fairly quickly after being transported into the gully, since the upward facing palatal-basicranial surface remained rela- tively unweathered. Absence of rodent gnawing on the skull may imply a short period of weathering prior to transport and burial, or may simply mean that relatively few rodents lived in the area at the time the Bison died. There was no modification of the skull by carnivores. Rodents, predators, and scavengers can be important agents of bone modification where they are present (Graham, 1993). The absence of other skeletal elements associated with this skull indicates that the skull had completely disarticulated from the atlas prior to being washed into the gully. Toots (1965) and Hill and Behrensmeyer (1984) found that the skull separated from the atlas relatively soon after death. The lower jaw was not found with the skull. Hill and Behrensmeyer found that mandibles also tended to disarticulate relatively quickly, but Toots found that the lower jaw in large herbivores tended to remain articulated with the skull for a rela- tively long period. Difference in observed disarticulation patterns might be en- vironmental, since Toots worked on the northern Great Plains and Hill and Behrensmeyer worked in Africa. Absence of postcranial parts of the skeleton could be explained by the fact that skulls act as lag elements (Behrensmeyer, 1975; Voorhies, 1969). Other parts of the skeleton may have washed away, or even been carried off by scavengers. Alternatively, the skull may have become lodged in the gully because its horns hooked into roots or debris on the gully floor. In either case other bones may have washed further down the gully and been deposited in the Missouri River, or in parts of the gully that have subse- quently been eroded by the river.

DESCRIPTION

SDSM 47704 is the skull of a very elderly male. The teeth are deeply worn, and are at a very advanced level of the S-4 (old age) stage of Skinner and Kaisen (1947). P2 has fallen out on both sides, possibly in vivo. Both P3 and P4 are worn to or below the enamel-dentine junction. M1 is worn down to the roots on both left and right sides. The styles are worn away on both M2s and preserved only as a tiny remnant on the right M3. Fossettes are very reduced on both M2s, and preserved as a reduced remnant on the left M3. They remain relatively large on the right M3. Root canals of P3 to M1 are filled in with sec- 50 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) ondary dentine. There is no sign of decay in these teeth despite their deep wear. The horn cores have large burrs, indicating that this animal was male. For most of their surfaces they have well-developed longitudinal grooves and ridges. The horn cores are well ossified. The orbits are tubular and extreme- ly protrusive (Table 1). The supraorbital foramen is roofed over. The supraoc- cipital suture is totally closed, and the frontal suture is closed except for its ros- tralmost 2.4cm. The lacrimofrontal and lacrimomaxillary sutures are fused at their lateral ends but not at their medial ends, and the nasofrontal sutures are only weakly fused. According to Skinner and Kaisen (1947) these sutures close late in life in Bison. The suture between left and right nasals is unfused. Su- tures between the zygomatic and the lacrimal and maxilla are completely fused. Fusion of the skull bones is characteristic of mature animals (Skinner and Kaisen, 1947; Shackleton et al., 1975). This skull’s appearance agrees best with their plate 10, Figures 3 and 3a. The left horn core of SDSM 47704 presents an abnormality on its dorsal and rostral surfaces adjacent to the horn pedicle. This portion of the left horn core lacks the ridges and grooves seen elsewhere on the same horn core, and on the right horn core. Ridges and grooves running the length of the horn core are a normal aspect of Bison horn cores (Skinner and Kaisen, 1947), and in- deed of bovid horns in general (Dyce and Wensing, 1971; Raghaven and Kachroo, 1964). Longitudinal grooves on the horn core may help resist tor- sional stresses in the horn during male-male combat (Guthrie, 1990a). The horn has an internal blood supply (Bone, 1982) deep to the horn sheath. In bovids branches of the cornual artery run lengthwise along the horn core (Raghaven and Kachroo, 1964). The grooves on the horn core surface are as- sociated in Bos with the rich vascular supply to the corium which generates the horn sheath covering the horn core (Dyce and Wensing, 1971). Absence of the grooves suggests a possible restriction of normal blood supply to that area of the horn. The bone in this region has a felted texture of crisscrossing bone fibers rather than the normal horn core texture of fibers, grooves, and vascular chan- nels oriented parallel to the long axis of the horn. This region is much less porous than the normal texture seen elsewhere in the horn cores of this spec-

Table 1. Protrusion of the orbits in SDSM 47704 as compared to that in other Bison males. Measurements follow McDonald, 1981.

SDSM B. a. B. a. B. b. B. b. B. B. 47704 antiquus occidentalis bison athabascae priscus alaskensis absolute protrusion 38.5 28.3 25.7 28.8 30.3 28.4 31.9 relative protrusion 0.79 0.85 0.85 0.84 0.85 0.84 0.84 absolute protrusion = (max width frontals _ min width frontals)/2 relative protrusion = min. width frontals / max. width frontals Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 51 imen or of other mature Bison skulls. The lack of porosity of this region sug- gests reduction in the number of capillaries passing between the interior and surface of the horn core. Surface appearance of this region is much more sim- ilar to that of the horn pedicle or other bones than the normal appearance of a horn core. Disruption of blood supply to this region could have occurred as the result of a traumatic injury during fighting. If so, the injury appears well healed except for restoration of normal vascular supply to the horn corium. Collateral vessels may have compensated for any reduced blood supply, but horn production could have been inhibited in the affected region of the horn. Direct injury to horn cores can cause asymmetries of horn development (Guthrie, 1966). This injury must have occurred after the animal reached ma- turity, however, since the left and right horn cores are approximately equal in size and only slightly different in orientation (Table 2). Rothschild and Martin (1993) describe several pathologies know to occur in Bison, none of which specifically affect the horn cores. Guthrie (1990a) noted damage to horn core fluting which he attributed to fighting between Bison bulls.

Table 2. Measurements of SDSM 47704 compared with males of various Bison species. Measurements follow McDonald (1981) and Skinner and Kaisen (1947). Bison a. occidentalis SDSM 47704 Bison a. antiquus Bison b. bison Bison b. athabascae Bison alaskensis Bison priscus tip – tip horn core w. 780 765-1067 626-1055 510-778 542-848 751-1064 800-1540 l. core on upper curve 263 203-364 186-392 124-270 165-323 285-416 426-795 l. core on dorsal cord 250 185-330 175-350 120-243 154-277 268-379 294-667 d-v diam. horn base 92.5 81-126 70-114 69-99 81-106 84-116 100-153 min. circ. horn base 263 233-392 237-355 199-324 254-322 293-387 341-493 w. at ext. audit. meatus 265 251-318 238-294 220-270 243-298 248-310 268-340 w. occipital condyles 124.87 132-161 111-151 111-140 118-139 127-165 133-175 d. nuchal line – for. mag 102.85 94-134 89-120 81-115 92-114 91-119 97-134 a-pt diam horn base 98.88 76-129 77-120 67-103 83-109 98-130 122-171 min. width frontals 285 276-352 261-348 237-318 273-313 269-336 281-394 max width frontals 362 338-400 311-394 289-356 326-384 313-415 356-440 M1-M3 length (labial side) l=86.46 105.2-106 90-102 81.8-97.9 ------r=84.75 max width M3 (mesial cusp) l=26.74 29.6-30.0 27.8-29.1 22.3-31.4 ------r=27.28 total length skull 518 (629.0) 511-606 500-583 562-604 -- 602-676 length of frontals 240 260-314 233-287 214-279 240-276 259-286 265-338 < divergence horn r=68º 72-86º 63-83º 58-79º 63-77º 63-79º 64-85º core-skull midline l=66º frontal width / length* 1.19 1.11 1.14 1.10 1.15 1.08 1.11 frontal w. / total length* 0.55 0.50 0.53 0.51 0.51 0.48 0.51

*from Skinner and Kaisen (1947) 52 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000)

TAXONOMIC DIAGNOSIS

Measurements of this skull are compared with the published ranges (Mc- Donald, 1981) for various species and subspecies of Bison in Table 2. There is significant size overlap between different taxa, even in the horn core char- acter suite (Guthrie, 1966). Only male skulls are included in the diagnosis of this individual to decrease the amount of overlap between species. Identification of Bison skulls usually is based primarily on the horn core character suite (McDonald, 1981), although there may be considerable overlap in these characteristics (Guthrie, 1966). Details of the horn core comparisons are given in Table 3. SDSM 47704 retains horn sheath covering the tips of the horn cores, which means that horn core shape at the tip and presence or ab- sence of a dorsal groove at the tip of the horn core cannot be evaluated. These characters would help to distinguish Bison antiquus antiquus and the Euro- pean species of Bison found in North America from Bison antiquus occiden- talis and the two subspecies of Bison bison. Presence of the horn sheath also complicates measurement of horn core length dimensions, which have been estimated from the sheath-covered horn tips. We refer SDSM 47704 to Bison bison cf. athabascae. Measurements, horn core characters, and skull shape characters for this specimen are compared with published data for Bison species from McDonald (1981) and Skinner and Kaisen (1947) in Tables 2 through 4. The tip-to-tip horn length and individu- al horn lengths exceed the published size range for Bison bison bison but does fall within the known size range for B. b. athabascae. The skull is too small to belong to Bison antiquus antiquus, specifically in its total and frontal lengths and in width of the occipital condyles. The horn cores are angled backward more than is the case in B. a. antiquus and horn core curvature does not fit within the known range of variation for B. a. antiquus. Horn cores are depressed below the plane of the frontals, unlike the condition McDonald (1981) described in B. antiquus occidentalis. However, Rasmussen (1974) identified a skull with depressed horn cores as B. a. occidentalis. Horn shape (posterior margin and long axis shapes and spiraling of the longitudinal grooves) and horn rotation are not consistent with the characteristics found in the European Bison species found in North America (B. priscus and B. alasken- sis), and overall size of the specimen is too small for Bison alaskensis. Total skull length is smaller than the range known for B. bison athabascae but length of the frontals does fall within the range published by McDonald (1981). Most measurements of SDSM 47704 lie within the size range for Bison bison athabascae, and also fall within the published size range of Bison. antiquus. occidentalis (McDonald, 1981). Many of these measurements also fall within the range known for Bison bison bison. Some aspects of the Lyman County skull have been difficult to evaluate. The molar teeth of SDSM 47704 are smaller than the range McDonald gave for Bison bison athabascae or Bison antiquus occidentalis (Table 2). McDonald’s sample size for these taxa was very small, however (only 1 to 3 individuals), and comparison to our skull would not be meaningful. Tooth dimensions fall within the published range for Bison bison bison. Interestingly, M3 width is Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 53 identical to that of the one B. b. athabascae listed by McDonald (1981). Skin- ner and Kaisen (1947) indicated that the tooth row shortens with age in Bison, and it is possible that our relatively short tooth row would fit within the size range for Bison bison athabascae if a larger sample of that subspecies were known. The orbits in SDSM 47704 are unusually protrusive (Table 1) in com- parison to McDonald’s data for various Bison taxa. Protrusion of the orbits is known to increase with the age of an individual bison (Skinner and Kaisen, 1947; Wilson, 1974; Shackleton et al., 1975), and our skull belongs to a very el- derly individual. The observed orbital protrusion is presumably correlated with the age of this animal, and is also probably not taxonomically significant.

Table 3. Horn core character complex in SDSM 47704 and other male Bison. Bison a. occidentalis SDSM 47704 Bison a. antiquus Bison b. bison Bison b. athabascae Bison alaskensis Bison priscus base shape: 1 symmetric, 2. asymmetric 1111122 posterior margin: 1 straight, 2 sinuous, 3. concave 1 1 1 or 3 1 or 3 1 or 3 2 2 long axis: 1 straight, 2 arched, 3 sinuous 2 2 2 or 3 2233 growth pattern: 1 spiral, 2 straight 2 2 1 to 2 2 1 to 2 2 2 core rotation: 1 none, 2 forward, 3 backward 3133322 horn curvature: 1 continuous, 2 base straight, 3 recurved 121111,31,3 horn axis rel. frontals: 1 horizontal, 2 downward, 3 upward 2 1 or 2 1 or 3 1 or 2 1 or 2 1 or 2 1 or 2 horn core tip: 1 no twist, 2 post. twist* 1 to 2 222211 index of horn curvature* 132 128-147 121-169 115-182 126-149 107-153 126-152 index of horn compression* 94 83-108 86-104 81-107 84-98 73-103 70-78 index of horn proportion* 106 68-100 85-110 66-104 62-92 100-166 106-134 index of horn length* 98 66-114 88-112 57-92.6 57-90 110-189 109-167

• after Skinner and Kaisen (1947). All others after McDonald (1981) • index of horn curvature = (length on lower curve / straight line length) x 100 • index of horn compression = (d-v diameter / a-pt diameter) x 100 • index of horn proportion = (upper curve length / base circumference) x 100 • index of horn length = (length on upper curve / min. width of frontals) x 100 54 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000)

Certain aspects of the morphology of SDSM 47704 ally it more closely with the Bison antiquus group than the Bison bison group (Table 4). The occiput is slightly concave. There is a fairly well developed external occipital protu- berance, the rugosity for attachment of the funiculus nuchae (Habel, 1977). The occipital condyles protrude slightly from the occipital plane. The back of the skull tends to be flat in Bison bison, but can be slightly to moderately con- cave in Bison antiquus. There is a prominent external occipital protuberance in Bison antiquus, but generally Bison bison skulls lack a prominent protu- berance at the insertion of the funiculus nuchae. The occipital condyles do not protrude from the occipital plane in Bison bison, but do protrude slightly in Bi- son antiquus. Bison bison athabascae is believed to be relatively less derived from the ancestral B. antiquus occidentalis than is Bison b. bison (Skinner and Kaisen, 1947; Guthrie, 1970), and these morphologic similarities to the Bison antiquus group may reflect that closeness.

Table 4. Skull morphology of SDSM 47704 compared with other Bison males (after McDonald, 1981). Bison a. occidentalis SDSM 47704 Bison a. antiquus Bison b. bison Bison b. athabascae Bison alaskensis Bison priscus frontal doming? yes yes sometimes yes yes sometimes sometimes sheath recurved? yes no? yes? yes yes yes yes orbit shape: 1 tubular, 2 faces forward, 3 face to side 1, 2 1, 2 ----- 1, 3 1, 3 1, 2 1, 2 occiput: 1 flat, 2 slightly concave, 3 concave 2 1 to 3 22233 ext. occip. protub.: 1 weak, 2 mod. Prom., 3 prominent 2 3 3 1 to 2 2 2 3 occipital condyles: do not protrude, 2 protrude 2221112 condyles flanged: 1 no, 2 sometimes, 3 frequently 1222223 horns thicken distal to burr? no no no no no yes* yes*

*horn may remain the same thickness distal to burr Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 55

DISCUSSION

Radiocarbon dating proved to be critical to identification of this specimen. A small volume of bone fragments from the skull itself was collected during preparation and sent to Geochron Laboratories for analysis (GX-26570-AMS). Care was taken to avoid contaminating the bone during preparation. Because of the small volume of bone fragments available analysis was conducted using accelerator mass spectrometry. The 13C corrected age determined for SDSM 47704 was 710±40 14C years before the reference year (1950). The specimen is therefore too young geologically to fit within the known temporal ranges of any Bison species other than Bison bison, and SDSM 47704 must belong to one of the two living subspecies. SDSM 47704 is very well preserved. Fusion of the cranial sutures is near- ly complete due to the advanced age of the animal, and this factor undoubt- edly contributed to the completeness of this skull. Late fusion of cranial su- tures means that Bison skulls frequently disarticulate prior to burial (McDon- ald, 1981). Preservation of delicate processes in the basicranium and relative lack of weathering of the bone surfaces both indicate that the skull is tapho- nomically immature, which also contributes to its completeness. The recent geologic age of the specimen also is a factor in its excellent preservation. Based on most dimensions, SDSM 47704 could fall within any of three Bi- son taxa: B. antiquus occidentalis, B. bison bison, or B. bison athabascae. Horn core dimensions would seem to eliminate Bison bison bison from con- sideration. However, horn core dimensions are more variable than those in other parts of the skull (Shackleton et al., 1975; Guthrie, 1966). The large horn cores of this individual might not actually lie outside the normal range of vari- ation in B.b. bison, since this skull is only a few millimeters larger than B. b. bison for each of the horn core measurements. The short total skull length (dimension OP) would likewise seem to eliminate Bison bison athabascae from consideration, but sample sizes for the wood bison are very small (7 in- dividuals for this measurement) and this difference may not be significant. The orbits are extraordinarily protrusive, but relative protrusion of the orbits still dif- fers from McDonald’s published figures by less than 10% of the average, and this protrusion is probably simply a factor of the age of this particular individ- ual. Wear of the teeth is not only extremely deep, but also relatively level. Lev- el wear is characteristic of grazing habits such as those of Bison bison bison rather than browsing habits, and wood Bison tend not to have extremely lev- el tooth surfaces (Guthrie, 1990a). Extremely deep wear in old individuals might produce very level teeth even in a browser, however. The very recent geologic age of the specimen, only about 710 years BP, does serve to eliminate Bison antiquus occidentalis from consideration in iden- tifying SDSM 47704. Morphology of the skull actually appears to be somewhat more helpful in identification than do skull dimensions, and suggests greater similarity to Bison bison athabascae than to Bison bison bison. Identification of this skull as Bison bison athabascae is counter to Mc- Donald’s conclusions that the wood bison is restricted to northern Canada and Alaska. However, this identification might not be unreasonable. Skinner and 56 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000)

Kaisen (1947), Wilson (1974), Lowery (1974), Guthrie (1970) and Hall and Kelsen (1959) all indicate that wood bison were distributed as far south as Col- orado in upland areas. Wilson’s conclusions were based in part on his study of skulls from upland areas of the Bighorn Mountains in Wyoming. In addi- tion, however, this skull is not modern, but is some 700-800 years old. The Pa- cific climatic episode, from 800-400 BP, brought cooler conditions to the north- ern plains (Semken and Falk, 1987). It is possible that the geographic range of wood bison extended further south during this climatic episode than it does now, and SDSM 47704 lived during the Pacific climatic episode. Skinner and Kaisen (1947), in synonomizing Bison bison septemtrionalis Figgins with B. b. bison, note that some of the skulls described were buried and partly fossilized, and might therefore be expected to differ somewhat from the living population. SDSM 47704 is also a buried skull, whose age is close to one quarter of the entire duration of the living subspecies. It might differ from them to some degree just because it is temporally closer to the ancestral Bison antiquus occidentalis, and perhaps morphologically closer as well. Wil- son (1969) suggested that earlier chronomorphs of Bison bison bison would probably have been similar in both size and cranial characteristics to Bison bi- son athabascae. Distinguishing between the living subspecies in the overlap- ping portions of their size ranges, and even distinguishing between Bison bi- son subspecies and Bison antiquus occidentalis, can be very difficult (Skinner and Kaisen, 1947; Wilson, 1974). It is particularly likely to be difficult to iden- tify a skull which may be morphologically different from the living subspecies because of its geologic age. SDSM 47704 preserves an apparent pathology of the left horn core. Mc- Donald (1981) documents some unusual morphologies in Bison populations which he attributes to inbreeding. These morphological variants include ab- normal tooth structure, supernumary teeth, and malformations of the maxillae and nasals. He also indicates that horn core morphology may be altered by genetic drift in isolated populations, for example in “Bison scaphoceras” (=B. antiquus antiquus fide McDonald). Since the observed pathology in this specimen affects only a limited region of one horn core it is more likely that it results from a traumatic injury than from genetic drift. Injuries to the horn or surrounding tissues could occur during combat between Bison males. Horn core damage due to fighting is mentioned by Guthrie (1990a).

CONCLUSIONS

SDSM 47704 probably belongs to Bison bison athabascae. It is more sim- ilar to the wood Bison than to Bison bison bison. Morphologically it is very similar to Bison antiquus occidentalis, and may indicate persistence of the an- cestral morphology into fairly recent times in the northern Great Plains. Taphonomic evidence suggests that the Lyman County skull lay on the sur- face only briefly before its burial. The skull does not have any tooth marks to indicate activity by carnivores, and has not been gnawed on by rodents search- ing for a source of calcium. Duration of its surface exposure is estimated to have been 1-2 years based on weathering of bone in Africa (Behrensmeyer, Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 57

1978), but absence of attack by rodents might imply that the skull was exposed for a period of time closer to the minimum. It is also possible that bone weath- ering is relatively rapid in temperate prairie environments. Surface exposure was not long enough for freeze-thaw cycles to damage the skull (Rothschild and Martin, 1993). Burial probably occurred when the skull was swept into a gully by sheetwash following a heavy rain and incorporated into the poorly sorted, immature sediments accumulating in the bottom of a gully. The gully was later filled in by that or similar sheetwash events. There is no sign of human activity in connection with this skull. There are no cut marks on the bone surface. No flakes or points were found either dur- ing field excavation or preparation of the jacketed skull in the lab. Based on tooth wear it is likely that the animal died as a result of old age. The great orbital protrusion in this particular individual helps point up the importance of considering the effects of age on skeletal morphology both when attempting to define a species and when attempting to draw conclusions about morphologic variability within a species. It also presents a caution about the care which must be employed when selecting taxonomically significant traits for analysis, as some aspects of morphology will change allometrically with age even after an animal reaches maturity—even in mammals. More important, SDSM 47704 demonstrates the importance of getting ra- diometric dates on subfossil materials whenever possible. Without the guid- ance provided by radiometric dating we would have continued to believe that this was a skull of Bison antiquus occidentalis, the taxon with which its di- mensions and morphology are in best agreement. It should also serve as a cau- tionary note about attempting to carry identifications of incomplete remains too far. In this instance a beautifully preserved complete Bison skull has proven very difficult to identify even to species, yet fragmentary material may in some cases have been identified to subspecies. This skull suggests that identifica- tions of partial Bison skulls can seldom be made with complete confidence.

ACKNOWLEDGEMENTS

The authors wish to thank the US Army Corps of Engineers for their on- going support of work by field paleontology courses at the South Dakota School of Mines and Technology. We also thank the Corps for their assistance in transportation and storage of this specimen. Thanks also go to the Friends of the New Jersey State Museum for supporting the costs of radiocarbon dat- ing for this skull. The authors would also like to thank Dr. Paul Orsini of the University of Pennsylvania School of Veterinary Medicine for allowing access to modern Bison bison skulls in his collection. Thanks to Dina Brandt for her invaluable assistance with the smoothing out of a logistical problem in our fieldwork. We also thank Craig DeTample of the South Dakota Discovery Cen- ter and Aquarium for assisting with the dig, and particularly all the members of the 1998 field paleontology class who helped excavate the skull. GPS data were provided by Mr. Ken Langer. 58 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000)

LITERATURE CITED

Behrensmeyer, A. K. 1975. The Taphonomy and Paleoecology of Plio-Pleis- tocene Vertebrate Assemblages East of Lake Rudolph, Kenya. Bulletin of the Museum of Comparative Zoology 146(10):473-578. Behrensmeyer, A. K. 1978. Taphonomic and ecological information from bone weathering. Paleobiology 4:150-162 Bone, J. F. 1982. Animal Anatomy and Physiology. Reston Publ. Co., Reston VA. 521pp. Dyce, K. M. and C. J. G. Wensing. 1971. Essentials of Bovine Anatomy. Lea and Febiger, Philadelphia. 211pp. Graham, R. W. 1993. Processes of time averaging in the terrestrial vertebrate record. in: Kidwell, S. M. and A. K. Behrensmeyer (eds.) Taphonomic Ap- proaches to Time Resolution in Fossil Assemblages. Short Courses in Pa- leontology #6. Published by the Paleontological Society. pp. 102-124. Green, M. and H. Martin. 1960. Bison latifrons in South Dakota. J. Paleo. 34(3):548-550. Guthrie, R. D. 1966. Bison horn cores—character choice and systematics. J. Paleo 40(3):738-740 Guthrie, R. D. 1970. Bison evolution and zoogeography in North America dur- ing the Pleistocene. Quarterly Rev. Biol. 45(1):1-15 Guthrie, R. D. 1990a. Frozen Fauna of the Mammoth Steppe, the story of Blue Babe. U. Chicago Press. 323pp. Guthrie, R. D. 1990b. Late Pleistocene faunal revolution—a new perspective on the extinction debate. in: Agenbroad, L. D., J. I Mead, and L. W. Nel- son (eds.) Megafauna and Man, discovery of America’s heartland. Mam- moth Site of Hot Springs, SD, Inc. Scientific Papers v.1, pp 42-53 Habel, R. E. 1977. Guide to the dissection of the domestic ruminants, 3rd edi- tion. Published by R. E. Habel, Ithaca, NY. 165pp. Hall, E. R. and K. R. Kelson. 1959. The Mammals of North America, vol. 2. Ronald Press Co. pp1024-1026. Hill, A. and A. K. Behrensmeyer. 1984. Disarticulation patterns of some mod- ern East African mammals. Paleobiology 10(3):366-376. Kurten, B. and E. Anderson. 1980. Pleistocene Mammals of North America. NY: Columbia University Press. 442pp. Lowery, G. H. Jr. 1974. The Mammals of Louisiana and its Adjacent Waters. Louisiana Wildlife and Fisheries Commission. Louisiana State University Press. 565pp. McDonald, J. N. 1981. North American Bison, their classification and evolu- tion. University of California Press. 316pp Pinsof, J. 1985. The Pleistocene vertebrate localities of South Dakota. in: Mar- tin, J. E. (ed.) Fossiliferous Cenozoic deposits of western South Dakota and northwestern Nebraska. Dakottera v. 2 pt. 2, pp. 233-264. Pinsof, J. 1991. A cranium of Bison alaskensis (Mammalia:Artiodactyla: Bovi- dae) and comments on fossil Bison diversity in the American Falls area, southeastern Idaho. JVP 11(4):509-514. Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 59

Raghaven, D. and P. Kachroo (eds.) 1964. Anatomy of the Ox, with compar- ative notes on the horse, dog, and fowl. Indian Council of Agricultural Re- search, New Delhi. 760pp. Rasmusen, D. L. 1974. Bison occidentalis from northeastern Montana. North- west Geology vol.3:59-61 Roe, F. G. 1970. The North American Buffalo, A Critical Study of the Species in its Wild State. 2nd edition. University of Toronto Press. 991pp., 1 map Rothschild, B.M. and L. D. Martin. 1993. Paleopathology, Disease in the Fos- sil Record. CRC Press, Boca Raton. 386pp. Semken, H. A. Jr. and C. R. Falk. 1987. Late Pleistocene/Holocene Mammalian Faunas and Environmental Changes on the Northern Plains of the United States. in: Graham, R. W., H. A. Semken Jr., and R. A. Graham (eds.): Late Quaternary Mammalian Biogeography and Environments of the Great Plains and Prairies. Illinois State Museum Science Paper vol. 22:176-313. Shackleton, D. M., L. V. Hills, and D. A. Hutton. 1975. Aspects of variation in cranial characteristics of plains bison (Bison bison bison Linnaeus) from Elk Island National Park, Alberta. Jour. Mammalogy 56(4):871-887 Skinner, M. F. and O. C. Kaisen. 1947. The fossil Bison of Alaska and pelim- inary revision of the genus. Bull. Amer. Mus. Nat. Hist. vol. 89 article 3, pp125-256, plates 8-26 Toots, H. 1965. Sequence of disarticulation in mammalian skeletons. Univ. Wyoming Contributions to Geology 4(1):37-39. Voorhies, M. R. 1969. Taphonomy and population dynamics of an early Pliocene vertebrate fauna, Knox County, Nebraska. University of Wyoming Contributions to Geology Special Paper 1. 69 pp. Warren, C. R. 1952. Probable Illinoian age of part of the Missouri River, South Dakota. GSA Bull. 63(11):1143-1156. Wilson, M. 1969. Problems in the speciation of American fossil Bison. Chac- mool 1969. University of Calgary Archaeological Association. pp. 178-199. Wilson, M. 1974. History of the Bison in Wyoming, with particular reference to early Holocene forms. in: Wilson, M. (ed.) Applied geology and ar- chaeology: the Holocene history of Wyoming. Geol. Survey Wyo., Rept. Invest. #10. pp91-99. 60 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000)

Figure 1. Diagram of Lyman County Bison outcrop.

Figure 2. Orientations of pebbles in the Bison skull jacket. Orientations were taken in the lab, and do not reflect true compass orientations of the pebbles. Most clasts strike at high angles to the long axis of the skull. Plot produced using the Stereo program produced by RockWare, Inc. Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 61

Figure 3. Dorsal view of the Lyman County Bison skull, SDSM 47704. Tips of the horn cores are still covered by sheathing material. Longitudinal grooves can be seen on the right horn core.

Figure 4. Detail of the left horn core base in SDSM 47704. The midline of the skull is toward the left, and the horn tip toward the right. Note the absence of longitudinal grooves and the irregular transverse fabric of the bone in this re- gion of the horn core.

Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 63

USING THE OCEAN OPTICS R-2000 RAMAN SPECTROMETER IN THE UNDERGRADUATE LABORATORY

Deanna L. Donohoue, Gary W. Earl and Arlen Viste Department of Chemistry Augustana College Sioux Falls, SD 57197

ABSTRACT

The purpose of this work was to explore the productive use of a new Ra- man spectrometer in the undergraduate chemistry laboratory. The instrument is the Ocean Oprics R-2000, (Ocean Optics, 2000). Infrared and Raman spectra were compared with each other and with normal mode calculations in Gaus- sian 98 and HyperChem, for the molecules CCl4, CS2, C2Cl4, C6H6, and C5H5N.

Raman spectra were also observed for N2 and O2, for which there is no corre- sponding IR spectrum.

INTRODUCTION

Vibrational spectroscopy includes both infrared (IR) and Raman spec- troscopy. Although Raman and IR spectra both explore the normal modes of vibration of a molecule, their intensity patterns may be quite different and dis- tinctive. IR activity requires a changing dipole moment, while Raman activity requires a changing polarizability. For molecules of reasonably high symme- try, the irreducible representation for a particular normal mode may corre- spond to IR activity, Raman activity, both, or neither. For example if the molecule contains a center of inversion, the rule of mutual exclusion applies, and a normal mode cannot be active in both the Raman and the IR, although occasionally it might be inactive in both. (Herzberg, 1945, Cotton, 1990, Nakamoto, 1997). A discussion of Raman and IR spectra of four small molecules, together with quantum chemistry calculations on them, was pub- lished while the present work was in progress. (McClain, 2000).

METHODS

The Raman R-2000 was installed on a 486 PC under Windows 95. Beyond initial installation and testing, the R-2000 has been used in several chemistry courses during 1999-2000. The first two were an Honors section of Introduc- tion to Chemistry, and Physical Chemistry. In the Honors general chem course the R-2000 was used by freshman honors students as part of their working on the identification of an organic unknown. They were informed that the un- known might possibly contain the elements C, H, O, Cl. With the help of in- structor and lab assistants, each of the lab groups ran five types of spectra: Ra- 64 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) man, FTIR, GC/MS, NMR, and UV-visible. With these and other simple obser- vations (such as water solubility), they proceeded to identify the unknown. Each lab group was successful in their identification. The Raman instrument is similarly being used in Organic Chemistry, as well as in Physical Chemistry, Quantum Chemistry, and Independent Study/Research.

RESULTS AND DISCUSSION

Raman and infrared spectra often have quite different patterns of intensi- ties. This is particularly true with molecules of high symmetry. A classic exam- ple is CCl4 (Shoemaker, 1996). This tetrahedral molecule, point group Td, has ν -1 nine normal modes of vibration, of which 1 (A1) at 460 cm is a Raman active ν -1 ν symmetric stretch, 2 (E) at 214 cm is a Raman active ClCCl bend, 3 (T2) at -1 ν 793 cm is an asymmetric stretch active in both IR and Raman, and 4 (T2) at 314 cm-1 is a bend active in both both IR and Raman (Nakamoto, 1997). The Raman spectrum shown was taken with the Ocean Optics R-2000 instrument, with spectral range ca 200-2850 cm-1, and the FTIR spectrum was recorded with a Nicolet Avatar 360 FT-IR Spectrometer, with spectral range 400-4000 cm-1.

Figure 1. CCl4 Raman spectrum.

Figure 2. CCl4 FTIR spectrum.

ν All four vibrations are clearly shown in the Raman spectrum, while only 3 ν ν appears in this FTIR spectrum. The 1 and 2 vibrations are symmetry forbid- ν den in IR, and 4 is outside the range of our FTIR instrument. Frequencies and intensities were calculated for the normal modes of CCl4 with Gaussian 98 for Windows. At the Hartee-Fock 6-31G(d) level, the frequency scaling factor is 0.8929 (Foresman, 1996). The Raman activities calculated for frequencies 1-4 -1 -1 -1 were 19.0 for 449 cm (A1), 3.4 for 218 cm (E) , 10.5 for 805 cm (T2), and for -1 5.1 for 311 cm (T2), The Gaussian calculations are in good agreement with the observed spectra. Note that the most intense Raman band is the breathing mode (symmetric stretch), which is forbidden in the IR spectrum. Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 65

Raman spectra were observed for liquid N2 and liquid O2, as shown in Fig- ure 3 and Figure 4.

Figure 3. Liquid N2 Raman spectrum. Figure 4. Liquid O2 Raman spectrum.

-1 -1 The observed frequencies are 2329 cm for liquid N2 and 1538 cm for liq- ϖ ϖ ϖ uid O2. From the gas phase data, and using 0 = e - 2 exe, literature values ϖ -1 -1 of 0 are 2330 cm for N2 and 1556 cm for O2, in satisfactory agreement with the liquid phase data observed here. (Huber and Herzberg, 1979, Shoemaker,

1996) After a few minutes, the liquid O2 apparently absorbs a significant amount of N2, as indicated by the growth of the Raman band of N2.

The linear molecule CS2 has 3N-5 = 4 normal modes of vibration. Like CO2, ν Σ + CS2 belongs to the D∞h point group and has a symmetric stretch 1 ( g ), a two- ν Π ν Σ + fold degenerate bend 2 ( u), and an asymmetric stretch 3 ( u ). The first is ν ν Raman active, and 2 and 3 are IR active. (Nakamoto, 1997, Herzberg, 1945) ν -1 ν -1 Figure 5 and Figure 6 show 1 at 649 cm and 3 at 1506 cm , in reasonable ν -1 ν -1 ν agreement with CS2 gas, for which 1 = 658 cm , 2 = 397 cm , and 3 = 1533 -1 ν -1 cm (Nakamoto, 1997, p 166). Although 2 falls just below the 400 cm limit of our FTIR, and is limited also by the cutoff of the NaCl windows, the Raman ν ν spectrum shows the classic Fermi resonance of 1 with the overtone band 2 2. This is the origin of the Raman band observed at 796 cm-1. In terms of sym- Π Π Σ + Σ - ∆ Σ + metry analysis, u x u = g + g + g. Of these, g becomes involved in Fer- ν Σ + mi resonance with the 1 ( g ) fundamental, and thereby gains intensity. (Nakamoto, 1997, p 62, Herzberg, 1945, p 277).

Figure 6. CS2 IR spectrum.

Figure 5. CS2 Raman spectrum. 66 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000)

Current practice in organic chemistry courses in the USA includes intro- duction and use of infrared spectroscopy, NMR spectroscopy, and mass spec- troscopy on a routine basis. Raman spectroscopy is currently much less com- mon. However Raman spectra can often provide information on vibrational modes which may not be evident in the infrared spectrum. C=C vibrations serve as a case in point. Lambert provides a fine discussion of vibrational spec- troscopy of organic compounds, including both IR and Raman spectra, and Nakamoto is an excellent resource on the inorganic side. (Lambert, 1998,

Nakamoto, 1997) The Raman and IR spectra observed for C2Cl4 are shown in Figure 7 and Figure 8.

Figure 8. C2Cl4 IR spectrum. Figure 7. C2Cl4 Raman spectrum.

Since the point group of C2Cl4 is D2h, which includes a center of inversion, the rule of mutual exclusion applies, and a given normal mode cannot be ac- tive in both the Raman and the IR. The observed spectra are in accord with this. Table 1 summarizes the spectral observations and normal mode calcula- tions in Gaussian 98W, using 6-31G(d). The normal modes are numbered as in Herzberg (Herzberg, 1945, p 107, 329), but with rotation axes and irre- ducible representations consistent with international recommendations (Salt- house, 1972, p 77). Note particularly the C=C stretch that is clearly present in the Raman spectrum but absent in the IR spectrum. Normal mode 1 is a sym- -1 metric stretch (breathing mode), Ag symmetry, observed at 1576 cm . The Gaussian 98W results for the Raman bands are reasonably consistent with the observed spectrum, except that the 1576 cm-1 C=C band appears somewhat less intense on our instrument than the Gaussian 98W prediction.

Benzene, C6H6, belongs to point group D6h. Since there is a center of in- version, the rule of mutual exclusion applies. With N=12, 3N-6 = 30 normal modes. These are given by Herzberg. (Herzberg, 1945, p 118, 364) The Raman and IR spectra are shown in Figure 9 and Figure 10. The four most prominent Raman bands within our spectral range are at -1 ν 605, 990, 1178, and 1594 cm . These are assigned by Herzberg as 18 (E2g) at ν ν ν -1 605.6, 2 (A1g) at 991.6, 17 (E2g) at 1178.0, and 16 (E2g) at 1584.8 cm , with rel- ative integrated intensities 2.1, 10.0, 2.2, and 1.9. Two additional bands in- Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 67

Table 1. Spectral observations and normal mode calculations in Gaussian 98W.

Normal Raman G98W/cm-1, mode obs/cm-1 obs/cm-1 Raman activity Irr Rep Herzberg/cm-1

1 1576 1629, 103.1 Ag 1571 s

2 452 435, 16.9 Ag 447 vs

3 255 235, 5.0 Ag 237 s

4Au silent

5 not observed 997, 1.3 B3g 1000 vw

6 354 338, 5.5 B3g 347 m

7 <400 B3u 332 ?

8 515 574, 7.0 B2g 512 m

9 909 B2u 913 vs

10 ? B2u ?

11 777 B1u 782 vs

12 <400 B1u 387 ?

Figure 10. Benzene IR spectrum.

Figure 9. Benzene Raman spectrum.

ν -1 cluded by Herzberg are 11 (E1g) at 848.9 cm , intensity 0.9, and combination band 2+18 (E2g) at 1606.4 cm-1, intensity 1.6. (Herzberg, 1945, p 364) In Gaussian 98W, 6-31G(d) with frequency scaling factor 0.8929 (Foresman, 1996), the frequencies of the four prominent Raman bands are calculated as 594 cm-1 -1 -1 (E2g, activity 3.9), 968 cm (A1g, activity 54.6), 1156 cm (E2g, activity 7.2), and -1 1605 cm (E2g, activity 12.8). The other Herzberg fundamental is calculated as -1 859 cm (E1g, activity 3.3). An estimate of the Herzberg combination band ν ν -1 2+ 18 (E2g), from our observed Raman spectrum, is 605+990 = 1595 cm , an- other instance of Fermi resonance. The four prominent normal modes are sketched in Figures 11-14, based on the Gaussian 98W calculations, with visu- alization through HyperChem. (HyperChem, 1999) The breathing mode (sym- ν -1 metric stretch) of the benzene ring, 2 (A1g) at 990 cm , is particularly note- worthy, and is the strongest band in the Raman spectrum. 68 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000)

Figure 14. Figure 13. Figure 12. ν -1 ν -1 16 (E2g) 1594 cm ν -1 17 (E2g) 1178 cm 2 (A1g) 990 cm

Figure 11. ν -1 18 (E2g) 605 cm

Pyridine, C5H5N, is isoelectronic with benzene (C6H6), but one CH pair has become N in the ring. The point group is C2v. Since there is no longer a center of inversion, the rule of mutual exclusion does not apply. Pyridine has 27 nor- mal modes of vibration instead of the 30 of benzene. However the vibrational spectra of pyridine are recognizably similar to those of benzene, as shown in Figure 15 and Figure 16.

Figure 16. Pyridine IR spectrum.

Figure 15. Pyridine Raman spectrum.

The C2v point group is a subgroup of D6h. With conventional choices of ax- es (Salthouse, 1972), irreducible representation E2g of D6h becomes A1 + B2 in

C2v, and A1g of D6h becomes A1 in C2v. The most intense Raman bands of pyri- -1 -1 ν dine occur at 991 and 1027 cm , in the vicinity of the 990 cm 2 (A1g) breath- ing mode of benzene. In Gaussian 98W, 6-31G(d) with frequency scaling fac- tor 0.8929 (Foresman, 1996), the frequencies of these two most prominent Ra- -1 -1 man bands are calculated as 976 cm (A1, activity 23.6) and 1003 cm (A1, ac- tivity 23.1), with visualization in HyperChem shown in Figure 17 and Fig- ure 18. Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 69

Figure 17. Figure 18.

Pyridine A1 mode Pyridine A1 991 cm-1 (calc 996) mode 1027 cm-1 (calc 1003)

CONCLUSION

Raman spectroscopy with the Ocean Optics R-2000 instrument is useful and practical in the undergraduate chemistry laboratory. (Ocean Optics, 2000). Observing both Raman and FTIR spectra provides significantly more thorough and complete vibrational information than the FTIR spectrum alone. Quantum chemistry calculations with Gaussian 98W and HyperChem software facilitate visualization of normal modes of vibration, and add support to spectral inter- pretation. Introduction of Raman spectra into the organic chemistry laboratory is currently in progress. Lambert and Nakamoto provide very good discussions of Raman and IR spectra for organic and inorganic compounds. (Lambert, 1998, Nakamoto, 1997).

LITERATURE CITED

Cotton, F. Albert. 1990. Chemical Applications of Group Theory. John Wiley, New York, NY. Foresman, James B., and Æleen Frisch. 1996. Exploring Chemistry with Elec- tronic Structure Methods. 2nd ed. Gaussian, Pittsburgh, PA. Herzberg, Gerhard. 1945. Molecular Spectra and Molecular Structure. II. In- frared and Raman Spectra of Polyatomic Molecules. Van Nostrand, Prince- ton, NJ. Huber, K. P., and G. Herzberg. 1979. Molecular Spectra and Molecular Struc- ture. IV. Constants of Diatomic Molecules. Van Nostrand, Princeton, NJ. HyperChem 5.11 Pro for Windows. 1999. Hypercube, Gainesville, FL. Lambert, Joseph B., Herbert F. Shurvell, David A. Lightner, and R. Graham Cooks. 1998. Organic Structural Spectroscopy. Prentice-Hall, Upper Saddle River, NJ. McClain, Brian L., Sara M. Clark, Ryan L. Gabriel, and Dor Ben-Amotz. May 2000. Educational applications of IR and Raman spectroscopy: A compari- son of experiment and theory. J. Chem. Ed. 77:654-660. Nakamoto, Kazuo. 1997. Infrared and Raman Spectra of Inorganic and Coor- dination Compounds, Part A and Part B. 5th ed. Wiley-Interscience, New York, NY. 70 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000)

Salthouse, J. A., and M. J. Ware. 1972. Point Group Character Tables and Re- lated Data. Campridge University Press, Cambridge, UK. Shoemaker, David P., Carl W. Garland, and Joseph W. Nibler. 1996. Experiments in Physical Chemistry. 6th ed. McGraw-Hill, New York, NY, Expt. 36. Ocean Optics. 2000. Raman Systems R-2000 Raman Spectrometer. http://www.oceanoptics.com/ProductSheets/R2000.asp

ACKNOWLEDGMENTS

The Ocean Optics R-2000 was acquired by the Augustana College Depart- ment of Chemistry, with partial support from Ocean Optics through Grant Award # OOI99-407, and with support also from the Roland Wright Chemistry Endowment of Augustana College. We gratefully acknowledge spectral and computational work on pyridine shared by fellow Quantum Chemistry student John Gilbertson, as well as con- genial interaction with other student colleagues in Physical Chemistry and Quantum Chemistry at Augustana College. Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 71

13C NMR SPECTRA AND QUANTUM CHEMISTRY CALCULATIONS FOR BENZONITRILE AND 4-AMINOBENZONITRILE

Steven R. Moeckly, Gary W. Earl and Arlen Viste Department of Chemistry Augustana College Sioux Falls, SD 57197

ABSTRACT

The purpose of this work was to measure the 13C NMR spectra of Ben- zonitrile and 4-Aminobenzonitrile, and to relate the 13C chemical shifts with re- sults of molecular orbital calculations using Gaussian 98W and HyperChem, and to compare with assignments by Wehrli and coworkers. (Wehrli, 1969).

Keywords

Carbon-13, nmr, ab initio, chemical shift.

INTRODUCTION

In 1998, Anasazi Instruments upgraded the Augustana College NMR in- strument (originally a Varian EM-360A) to an Anasazi EFT-60 NMR spectrome- ter, providing extensive multinuclear nmr capability. In this paper, carbon-13 NMR spectra observed with this instrument are compared with literature results and with quantum chemistry calculations, both ab initio and semiempirical.

METHODS

Proton-decoupled 13C NMR spectra of benzonitrile and p-aminobenzonitrile were acquired using the Augustana 60 MHz FT-NMR, an Anasazi EFT-60 NMR spectrometer. Samples were run as solutions in acetone. The carbonyl carbon of acetone was used as reference. The calculations of the NMR shielding ten- sor in Gaussian 98W were carried out as recommended by Foresman and Frisch. (Foresman and Frisch, 1996) This entailed geometry optimization at the B3LYP/6-31G(d) level, followed by a RHF/6-311+G(2d,p) NMR calculation.

RESULTS AND DISCUSSION

δ σ δ σ The chemical shift is related to the isotropic shielding by sample = ref - σ δ σ σ sample. Wehrli reported - versus CS2 reference, thus sample - ref, increasing in the 72 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) upfield direction. For simplicity in comparing with Wehrli and with Gaussian σ σ 98W calculations of isotropic shielding, we have similarly tabulated ( sample - ref) δ = - sample. Results are presented in Table 1.

Table 1. Results.

σ σ ( sample- ref), ppm isotropic shielding σ σ σ ( sample- ref), ppm vs acetone ref tensor , ppm Charge from AM1

Sample vs CS2 ref, Wehrli this work Gaussian 98W HyperChem

Benzonitrile C-1 82.9 93.6 72.3 -0.016 C-2, C-6 62.7 72.8 50.0 -0.090 C-3, C-5 65.6 76.4 58.9 -0.135 C-4 62.7 72.8 51.7 -0.107 CN 76.4 87.3 58.5 -0.096

4-Aminobenzonitrile C-1 101.7 109.5 89.8 -0.088 C-2, C-6 65.2 73.2 43.8 -0.036 C-3, C-5 85.4 93.1 80.2 -0.228 C-4 46.1 53.7 30.9 -0.146 CN 78.3 86.4 67.3 -0.083

CONCLUSION

Trends in the 13C isotropic shielding (-δ values) were reasonably consistent between the literature (Wehrli, 1969), our measured 13C NMR spectra, and

Gaussian 98W quantum chemistry calculations. Different solvents (CS2 and ace- tone) versus the gas phase calculation in Gaussian 98W lead to differences in absolute values. The semiempirical AM1 calculations in HyperChem were also for the gas phase, and are less successful in correlating the chemical shifts of the five types of carbons. Wehrli found a correlation of chemical shifts at the 1 position with pi electron density at C-1. This more limited correlation is also consistent with our AM1 charges.

LITERATURE CITED

Wehrli, F. W., J. W. de Haan, A. I. M. Keulemans, and O. Exner. 1969. 9. Lin- eare Korrelation der 13C-Kernresonanzfrequenzen mit den LCAO-MO-π- Electrondichten in substituierten Benzonitrilen. Helv. chim. Acta, 52:103- 107. Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 73

Foresman, James B., and Æleen Frisch. 1996. Exploring Chemistry with Elec- tronic Structure Methods. 2nd ed. Gaussion, Pittsburgh, PA.

ACKNOWLEDGMENTS

The NMR was upgraded through a 1996 research grant of Prof. Gary Earl from NSF/EPA, CHE-9613052, “Preparation of More-Biofriendly Quaternary Ammonium Compounds and their Decomposition to Useful Reagents,” with matching support from Augustana College for the NMR.

Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 75

SYNTHESIS AND ANALYSIS OF QUATERNARY COMPOUNDS CONTAINING PHOSPHATE AND BORATE ANIONS

David Stone and Gary Earl Augustana College Sioux Falls, SD

ABSTRACT

The purpose of my research was the successful synthesis and analysis of anion exchange from the dihydrogenated tallow dimethyl ammonium methyl- carbonate quaternary compound to the borate quat, phosphate quat, and phos- phate bis-quat. The preparation of phosphate quat utilized the acid dissociation constant of phosphoric acid (pKa1=2.16, pKa2=7.21 pKa3=12.32) to drive an anion ex- change with the methyl carbonate quat (pH=10.3) by the following reaction:

+- +- H3PO4 + Quat OCOOCH3 Quat H2PO4 + CH3OH + CO2

By titrating equimolar amounts of the methylcar- bonate quat into phospho- ric acid, dissolved in iso- propanol, the phosphate quat was synthesized. The pH was monitored and at the termination of titration found to be ~4.85. Con- tinuing the addition of an- other molar equivalent of Figure 1. Phosphate quat compared to sodium the methylcarbonate quat dihydrogen phosphate (right) using 31PNMR. allowed for the formation of the bis-quat phosphate (pH~9.77). Analysis of the phos- phate quats was performed utilizing 31P FT-NMR and FT-IR. Both the phosphate quat and phosphate bis- quat where compared to a phosphoric acid (85%) ref- erence and sodium dihy- drogen phosphate or di- Figure 2. Phosphate bis quat compared to sodium monohydrogen disodium monohydrogen phosphate (right). 76 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) phosphate, respectably. The NMR spectra of each quat, mono and bis-quat, were compared to their sodium phosphate counterpart to verify that the sepa- ration from the phosphoric acid reference was caused by the change in anionic properties around the phosphorus atom. Comparison of the above spectra suggests that the anion exchange to the phosphate anion had occurred. FT-IR spectra were also run to confirm that the anion exchange of the methyl carbonate anion to the phosphate anion had occurred. The comparison of the phosphate bis-quat and methylcarbonate quat showed some distinct changes in the spectra that suggested a definite change to the phosphate anion. First, there was significant reduction in the C=O (~1650 cm-1) region. Also, there were significant in- creases in the fingerprint region P=O stretching Figure 3. Phosphate bis quat compared to -1 (1470 cm ), P-O stretching methylcarbonate quat. (1100-930 cm-1), and O-P- O bending (~500 cm-1). (Nakamoto, p266) The spectra of the phosphate quat and phosphate bis-quat correlate very well except for one distinct area, ~500 cm-1 , in which the bis-quat emits one peak and the mono quat emits two; the difference being caused by the hin- drance of a second cation group on the bis-quat. The preparation of the borate-quat took slightly more effort because of the predominant species occur- ring at (~ 11.64), which eliminates the possibility of direct conversion from the methyl carbonate quat to the borate quat. To get around this problem the hydroxide quat was pro- Figure 4. Comparison of phosphate bis-quat duced using the reaction and mono-quat using FT-IR. below:

+- +- Quat OCOOCH3 Quat OH + CO2 + CH3OH Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 77

The reaction was carried out with the methylcarbon- ate quat dissolved in t-butyl alcohol and mixed with >6M NaOH for approxi- mately one-half hour. All this was done in the ab- sence of heat to prevent Hoffman Elimination back to a tertiary amine. Figure 5. Borate quat compared to sodium After this preparation borate (right) using 11BNMR. of the hydroxide quat, the production of the borate quat B(OH)4 was carried out by the direct addition of boric acid crystals. 11B NMR was run with 1M boric acid as a reference. The same pattern as noted for the phosphate quats except the borate quat moves down- field from the reference be- cause of the boron atom in Figure 6. Borate quat compared to methylcar- the quat sharing more elec- bonate quat. trons than the boron atom in boric acid. When comparing the changes of the boric quat and the methylcarbonate quat using FT-IR there were significant changes that suggest the conversion to borate anion. A significant reduction occurred at ~1650 cm-1 signifying the loss of the methylcarbonate an- ion. A more intricate pattern for the OH peaks at ~3000 cm-1 suggests that the -OH anion is no longer present. Other peaks of significance occur at ~1200-900 cm-1, which is attributed to B-O stretching by Nakamoto.

CONCLUSION

There is adequate evidence that the methylcarbonate quat was converted to the phosphate and borate quats. More research possibilities exist synthe- sizing molybdate, dichromate, and nitrite quaternary compounds and explor- ing the different properties of the specific anions.

REFERENCES

Nakamoto, Kazuo; IR and Raman Spectra of Inorganic and Coordination Compounds, 5th ed.; Wiley, New York

Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 79

THE SYNTHESIS OF METHYL CARBONATE QUATS AND THEIR ANION CONVERSIONS

Drew J. Paulson and Gary W. Earl Department of Chemistry Augustana College Sioux Falls, SD 57197

ABSTRACT

The purpose of this research was to become more familiar with the reac- tion conditions and properties of methyl carbonate quats. A series of replicate reactions were run inside a 300mL and a 1L pressure reactor using dihydro- genatedtallow methyl amine as the model amine for the research project. Dimethyl carbonate, an alternative alkylating agent, was used to produce the dialkyl dimethyl quaternary ammonium methyl carbonate.

+ - (C18H37)2NCH3 + CH3OCOOCH3 (C18H37)2N (CH3)2 OCOOCH3

This reaction was run under an oxygen-free atmosphere. FT-NMR and HPLC instruments were used to monitor the rate of the reaction by analysis of peak area ratios. 80 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000)

The reaction ratio of dimethyl carbonate to amine was 10:1, respectively. Re- search was done to see if the dimethyl carbonate / methanol mixture recov- ered from a quaternization reaction could be recycled in future reactions. Other experimentation was done to synthesize new ester amines using N-methyldiethanolamine and lauric acid in the reaction:

C11H23COOH + (HOCH2CH2)2NCH3 (C11H23COOCH2CH2)2NCH3

This reaction was also run under an oxygen-free at- mosphere in a pressure re- actor. The ester product was characterized by FT-IR and FT-NMR, as shown re- spectively.

The reaction ratio of lauric acid to the amine was 2.3:1, respectively. The es- ter amine was quaternized using dimethyl carbonate as the alkylating agent in the reaction:

+ - (C11H23COOCH2CH2)2NCH3 + CH3OCOOCH3 (C11H23COOCH2CH2)2N (CH3)2 OCOOCH3

As usual, this reaction was also run under an oxygen-free atmosphere, with the reaction ratio of dimethyl carbonate to amine being 10:1, respectively. FT-IR and HPLC instruments were again used to characterized the ester quat product and to determine the reaction half-life. Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 81

Continued research investigated dialkyl dimethyl quaternary anion exchange using nitric acid and sulfuric acid in the following reactions:

+ - + - (C18H37)2N (CH3)2 OCOOCH3 + HNO3 (C18H37)2N (CH3)2 NO3 + CO2 + CH3OH

+ - + -2 (C18H37)2N (CH3)2 OCOOCH3 + H2SO4 [(C18H37)2N (CH3)2]2 SO4 + 2CO2 + 2CH3OH

Physical properties of the methyl carbonate, nitric, and bis-sulfate dialkyl dimethyl quaternary ammonium compounds were also examined. 82 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000)

CONCLUSION

It was determined that the half-life of the dihydrogenatedtallow methyl amine quaternization reaction was 1 hour, and this reaction was complete af- ter 7 hours at a temperature range between 135º–140º C. Also, the dimethyl carbonate / methanol mixture recovered from this reaction was successfully reused in five subsequent quaternization reactions. Results from the synthesis of the ester amine yield that the reaction time is about 4 hours at a tempera- ture between 160º–165º C. The half-life of the ester quaternization using dimethyl carbonate was 4.5 hours at a temperature range between 135º–140º C and 20 hours at a temperature range between 90º–95º C. It was also discov- ered that different anions exhibit slightly different physical properties.

ACKNOWLEDGMENTS

Special recognition was given to the NSF-EPA research grant # CHE- 9613052, the 1999 Summer Science Institute, fellow colleagues J. Bjergaard, J. Gilbertson, S. Moeckly, L. Stanley, D. Stone, D. Wineinger, and Augustana Col- lege for making this research opportunity possible. Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 83

MOLECULAR MODELING OF SELECTED ANTIBIOTICS AND ANTIBIOTIC RESISTANT ENZYMES

Kari Lunder, Gary W. Earl and Arlen Viste Department of Chemistry Augustana College Sioux Falls, SD 57197

ABSTRACT

The purpose of this research was to gain insight on the properties of se- lected antibiotics and the growing problem of antibiotic resistance. The peni- cillin family is of particular importance in this study. An interesting structural aspect of the penicillin family is the four-membered ring attached to a five- membered ring that contains a nitrogen and a sulfur. The active site of 1TEM, downloaded from Brookhaven Protein Data Bank, was found and studied along with a computer-aided design inhibitor that was bound in the active site.

INTRODUCTION

Antibiotics are among the most widely used pharmaceutical in the world. People are increasingly using antibiotics needlessly, and it has developed a problem of antibiotic resistance among the general public. (Henry, 2000) The β-lactamase 1TEM is studied in detail. 1TEM is a class A β-lactamase. β-lac- tamases are responsible for resistance to penicillins, cephalosporins, and other related compounds. In recent years many new β-lactam molecules have been developed which have retained antibiotic activity even in the presence of β-lactamases in bacteria. A common practice is to use a cephalosporin in con- junction with penicillin. β-lactamases can be inhibited by the cephalosporin and still maintain the structure of the penicillin and eventually kill the bacteria. (Miyashita, 1995) In recent years certain bacteria have become resist to even this sort of treatment and a different method must be asserted. One such de- sign is 6α-(hydroxymethyl)penicillanic acid as a potent inhibitor for the 1TEM β-Lactamase inhibitor. (Myashita, 1995, Maveyraud, 1996, Kelly, 1998, Mobash- ery, 1999) This molecule is believed to displace the hydrolytic water molecule with its hydroxymethyl functional group. This interaction prolongs the acyl- enzyme intermediate which makes inhibition possible. This compound acy- lates the active site at serine which it is bound to, but it can still resist deacy- lation by being incorporated because of its functionality replace the hydrolytic water molecule, WAT 712. (Maveyraud, 1996) The discoveries of known pathogens that have recently become resistant to most antibiotics, and even pathogens that have become resistant to all antibiotics, has been quite alarm- ing. Presented in this paper are some ways that have been developed to help fight this growing problem. Implementing molecular modeling into the study 84 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) of antibiotics can greatly reduce time and money spent on developing new an- tibiotics. (Leach, 1996) The study of the frontier orbitals, electrostatic poten- tial maps, and charge density can provide clues on the properties of antibiotics. The study of the antibiotic’s properties and their reactivity will provide insight on the problem of antibiotic resistance.

METHODS

Molecular Modeling tools such as HyperChem, Protein Explorer, and the Brookhaven Data Bank were implemented for the study of the antibiotics and the antibiotic resistant enzymes. (HyperChem, 1999, Caffery, 1998, Protein Ex- plorer, 2000, Protein Data Bank, 2000). HyperChem was used to study the physical properties of the antibiotics and the antibiotic resistant enzymes. A useful tool for the study of antibiotic resistant enzymes is the Brookhaven Pro- tein Data Bank. These enzymes were downloaded from the protein data bank and imported into HyperChem for further study. Antibiotics were built in Hy- perChem and their properties were studied. A script was written to find the active site of the penicillin resistant enzyme 1TEM that was downloaded from the Brookhaven Protein Data Bank and inserted in to HyperChem using an ENT file. Raswin is another tool for visualization of proteins. (Rasmol, 2000)

RESULTS AND DISCUSSION

HyperChem, and the Protein Explorer were useful tools for the molecular modeling of the penicillin family and the antibiotic resistant enzyme 1TEM, shown in Figure 1 (Protein Data Bank, 2000). In Figure 2, 1TEM is visualized using Raswin, (Rasmol, 2000) Structural analysis of these enzymes are bene- ficial to find antibacterial agents that are resistant to their inhibitory effects. A structural analysis was also performed on the penicillin family, an example of which is shown in Figure 3 (Delgado, 1998). The frontier orbitals of methicillin were calculated using the AM1 semi-em- pirical method and they are shown in Figure 4 and Figure 5. The frontier orbitals of penicillin G were also calculated using AM1 with the results shown in in Figures 6–8. It was of interest that the frontier orbitals particularly involved the distinctive structural features of the square four-mem- bered ring and the sulfur.

Figure 2. 1TEM in Raswin. Figure 3. Methicillin. Figure 1. 1TEM. Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 85

Figure 4. Methicillin Figure 5. Methicillin HOMO. LUMO.

Figure 6. Penicillin G. Figure 7. Penicillin G Figure 8. Penicillin G HOMO. LUMO.

The active site of 1TEM was found using a selected script file implement- ed in HyperChem. After the active site had been selected, the active site and the inhibitor bound together were selected using the same method. Research by J.A. Kelley et al. located the active site with Serine 70, Lysine 73, Serine 130, Aspartate132, Glutamate 166, Aspartate 170, Lysine 234, Serine 235, and Argi- nine 244. (Kelley, 1998) This is represented in Figure 9.

Figure 10. 6α- Figure 9. Active site of (hydroxymethyl) 1TEM. penicillinate

The active site was found bound to 6α-(hydroxymethyl)penicillanate rep- resented in Figure 10. This is the inhibitor that was designed to inhibit the β-lactamase enzyme 1TEM. A view of the active site with the 6α-(hydroxymethyl)penicillanate bound in the active site as seen in HyperChem in Figure 11. Physical properties are very important for the study of the activity of cer- tain molecules. The physical properties of the penicillin family were studied in detail. Also, the physical properties of 6α-(hydroxymethyl)penicillanate bound in the active site was studied in detail to provide clues to how this in- 86 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) hibitor works. Electrostatic potential maps are shown for methicillin in Figure 12, 6α-(hydroxymethyl)penicillanate in Figure 13, and 6α-(hydrox- ymethyl)penicillanate in the presence of the active site in Figure 14.

Figure 11. Active site with in- hibitor bound. Figure 12. 3D isosurface elctrostatic mapping of methicillin.

Figure 13. Electrostatic potential map of 6α- (hydroxymethyl) Figure 14. 6α-(hydroxymethyl) penicillinate. penicillinate electrostatic po- tential map in presence of ac- tive site.

LITERATURE CITED

Caffery, M. L., P. A. Dobosch, and D. M. Richardson. 1998. Laboratory Exercis- es Using Delgado, Jaime N., and William A. Remers, Ed. 1998. Wilson and Gisvold’s Textbook of Organic Medicinal and Pharmaceutical Chemistry. 10th ed., Lippincott-Raven Publishers, Philadelphia. Henry, Celia. March 6, 2000. Antibiotic Resistance. Chemistry and Engineer- ing News. 78(10):41-58. HyperChem 5.11 Pro for Windows. 1999. Hypercube, Gainesville, FL. Kelly, J. A., A. P. Kuzin, P. Charlier, and E. Fonze. 1998. X-ray studies of en- zymes that interact with penicillins. CMLS. Cell. Mol. Life Sci. 54:353-358. Leach, Andrew R. 1996. Molecular Modelling: Principles and Applications. Ad- dison Wesley Longview Limited, Essex, England. Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 87

Maveyraud, Laurent, Irina Massova, Catherine Birck, Kazuyuki Miyashita, Jean- Pierre Samama, and Shahriar Mobashery. 1996. Crystal structure of 6α-(hy- droxymethyl)penicillinate ciomplexed to the TEM-1 β-lactamase from Es- cherichia coli: Evidence on the mechanism of action of a novel inhibitor designed by a computer-aided process. J. Am. Chem. Soc., 118:7435-7438. Mobashery, Shariar; Irina Massova , Kazuki Miyashita, and Pascale Taibi. 1995. Design, Synthesis, and evaluation of a potent mechanism-based inhibitor for the TEM B-lactamase with implications for the enzyme mechanism. J. Am. Chem. Soc. 117: 11055-11059. Mobashery, Shariar*; Jalal Haddad, and Sergei Vakulenko. 1999. An antibiotic cloaked by its own resistance enzyme. J. Am. Chem. Soc. 121:11922-11923. Mobashery, Shariar*; Wenlin Lee, Zhi-Hong Li, and Sergei Vakulenko. 2000. A light-inactivated antibiotic. J. Med. Chem. 43:128-132. Protein Data Bank. 2000. Research Collaboratory for Structural Bioinformatics. http://www.rcsb.org/pdb/ Protein Explorer. 2000. http://www.umass.edu/microbio/chime/explorer/in- dex.htm Rasmol. 2000. http://www.bernstein-plus-sons.com/software/rasmol/

ACKNOWLEDGMENT

We thank the Augustana College Department of Chemistry for software and hardware support which is essential for these molecular modeling activities.

Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 89

AMINO ACID CONCENTRATION OF SOYBEAN WITH PROTEIN VARIATION IN DIFFERENT ENVIRONMENTS

R.A. Scott and K.D. Kephart Plant Science Department South Dakota State University Brookings, SD 57007

ABSTRACT

Soybean [Glycine max (L.) Merr.] is a valuable source of protein both in an- imal and human diets. The economic value of soybean as a protein source is diminished, especially in animal diets, by the lack of sufficient levels of sulfur- containing amino acids (methionine and cystine). Animal diets often must be fortified with synthetic methionine to meet minimum standards, and can be- come expensive. There has been increasing interest in raising amino acid con- centrations in soybean, but additional research is needed to relate protein quantity to quality. The objective of this study was to determine the effects of protein changes on methionine, cystine, and threonine concentrations when soybean cultivars are grown in different environments. ‘Lambert’, ‘Parker’, and ‘Sturdy’ soybean were grown in 14 environments over three years. Seeds were analyzed for protein, methionine (met), cystine (cys), and threonine (thr) con- centrations. Significant environmental effects were found for protein, met, and cys concentrations (P=0.01). Significant differences (P=0.05) were found among cultivars for met and cys, but variations were small (13.3-13.8 and 13.4-14.3 g kg-1 for met and cys, respectively). Amino acid concentrations ranged from 12.5 to 15.2 g kg-1 met, 12.2 to 16.4 g kg-1 cys, and 34.1 to 41.1 g kg-1 thr among the three cultivars and 14 environments. Significant differences (P=0.05) among environments were found for protein of Lambert (360 - 439 g kg-1), and Park- er (367 - 421 g kg-1). Decreases in met, cys, and thr concentrations as protein concentration increased across 14 environments were small (0.019-0.037 g kg-1), but consistent. Fluctuations of protein concentrations across different en- vironments did not significantly lower protein quality.

INTRODUCTION

Soybean is an important source of protein in animal diets, but lacks suffi- cient levels of certain essential amino acids. In terms of nutritional importance, methionine, cystine and threonine are the most limiting amino acids in soybean (Boulter, 1980). Methionine should be increased from 16 g kg-1 to 30 g kg-1 to meet the Food and Agriculture Organization standard of egg protein (Burton 1982). Soybean breeders have successfully been increasing protein concentration in soybean (Brim and Burton, 1979; Burton, 1991; Hartwig and Kilen, 1991; Lef- 90 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) fel, 1992; Voldeng and Saidon, 1991), but increase in protein have not signifi- cantly changed the amino acid concentration (Burton et al., 1982; Zarkadas et al., 1993). On the other hand, thr concentration decreased significantly when protein was increased 10.1% by backcrossing, but met and cys did not change significantly (Zarkadas et al., 1994). Efforts to increase amino acid concentra- tion have been hampered by lack of genetic variability and cost of amino acid assays (Wilson, 1987). Seretti et al. (1994), however, found significant variation in amino acid concentrations of high-protein lines developed by Leffel (1992). Krober (1956) found differences in methionine concentration of soybean grown in different environments, but did not relate these changes to protein variation. There has been reports addressing what happens to amino acid concen- tration when protein concentration is increased by breeding (Burton et al., 1982; Seretti et al., 1994; Zarkadas et al., 1993, 1994), but there are no pub- lished reports of the fate of amino acid concentration when protein concen- tration changes with changes in environment. With the recent increase in in- terest in improving protein quality, it will be beneficial to know what effects protein changes due to environmental variation will have on amino acid con- centration. The objective of this study was to determine the fate of met, cys, and thr concentrations when protein concentration of soybean cultivars change due to changes in environment.

MATERIALS AND METHODS

Three early-maturing soybean cultivars (maturity group 0-II) were grown in 14 environments across South Dakota in 1992 and 1994 in a randomized complete block design with three replications. Each cultivar represented a dif- ferent maturity group. The environments consisted of five different soil series. Years were ignored in the analysis, and tests grown at the same geographic lo- cation in different years were different environments. Irrigated and non-irri- gated tests at the same geographic location also were different environments. Since each cultivar was grown only in locations where the maturity group was adapted, all three cultivars were not always grown at the same environments. The geographic locations were Brookings (44ºN; Brandt silty clay loam— fine-silty, mixed, Udic Haploboroll), Watertown (45ºN; Brookings silty clay loam—fine-silty, mixed, pachic, Udic Haploboroll), Pierre ( 44ºN; Betts clay loam—fine-loamy, mixed (calcareous), mesic, Typic Ustorthents), Beresford (43ºN; Egan silty clay loam—fine-silty, mixed, mesic, Udic Haplustoll), and Highmore (44ºN; Stickney silt loam—fine-montmorillonitic, mesic, Glossic Na- trustoll). Based on soil tests, nutrient supply levels were judged adequate, and no fertilizer was added during any growing season. Each plot consisted of four rows, 6 m long (trimmed to 5.2 m at maturity) with 76-cm row width. Mature-plant populations were approximately 370, 000 plants ha-1. The center two rows were harvested for protein and amino acid evaluations. Protein and amino acid assays were determined on samples from each replicate separately. Protein concentrations were determined on a mois- ture-free basis using near infrared reflectance spectroscopy (Model 5000, Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 91

NIRSystems, Silver Spring, Maryland). Amino acid analysis was done according to procedures described by AOAC (1990). The procedures involved standard HPLC post column derivitization with ninhydrin and quantified colorimetrical- ly. Calculations were made by comparison to a calibration set using cysteic acid monohydrate (Sigma C-7630) and methionine sulfonate (Sigma M-0751) as standards. Protein concentrations were expressed as proportions of total dry matter and amino acid concentrations were expressed as proportions of total protein concentrations. Data were analyzed using the General Linear Models procedure of the Sta- tistical Analysis Systems (SAS Institute, 1975). In the analysis of variance, culti- vars were fixed and all other effects random. Regression analysis was used to examine relationships between protein and amino acids.

RESULTS AND DISCUSSION

Highly significant (P=0.01) environmental effects on the cultivars were found for protein, met, and cys concentrations. Although protein concentra- tions were inconsistent (P=0.01), met, cys, and thr concentrations of the culti- vars were consistent across environments (P=0.05). Significant cultivar differ- ences were found for met and cys concentrations (Table 1). Met and cys con- centrations of Lambert were greater than Sturdy, and Lambert also had signif- icantly greater cys concentration than Parker. Protein concentrations of Lambert was 4 g kg-1 greater than Parker, which was 2 g kg-1 greater than Sturdy, but the three cultivars were not significantly different. Significant negative rela- tionships were found between protein concentrations and both met and cys concentrations (r = -0.51 and r = -0.53, respectively). This indicated that the met and cys differences among the cultivars probably were not due to the small protein differences. Serretti et al. (1994) did not find consistent met and cys dif- ferences between normal and high protein cultivars, confirming that protein differences may not necessarily reflect differences in met, cys, and thr. Significant protein, met, and cys differences were found among environ- ments for both Lambert and Parker (Table 2). Parker also showed significant

Table 1. Mean protein and amino acid comparisons of three soybean cultivars grown in 14 SD environments.

PROTEIN AND AMINO ACID CONCENTRATIONS Methionine Cystine Threonine Protein Cultivar† (g kg-1 dry matter)

------g kg-1 protein------Lambert 13.8 a 14.3 a 36.6 a 398 a Parker 13.4 ab 13.6 b 36.6 a 394 a Sturdy 13.3 b 13.4 b 36.2 b 392 a

†Cultivars with the same letter are not significantly different at 0.05 probability level. 92 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000)

Table 2. Protein and amino acid concentrations of soybean cultivars grown in 14 SD environments (df = 24).

PROTEIN AND AMINO ACID CONCENTRATIONS† Lambert Parker Sturdy Environment Pro Met Cys Thr Pro Met Cys Thr Pro Met Cys Thr

------g kg-1 protein------A 439 12.9 13.0 34.9 421 12.6 12.8 34.1 433 12.5 12.9 35.4 B 421 13.1 12.9 34.3 413 13.6 13.0 36.7 427 13.2 13.0 35.9 C 417 13.0 13.5 35.7 410 14.0 13.0 36.7 412 13.3 12.4 34.5 D 415 13.4 14.0 37.1 399 12.5 12.7 35.2 399 13.3 13.8 36.8 E 413 13.1 14.4 34.2 399 13.3 13.8 35.6 396 12.7 13.7 40.0 F 403 13.9 15.2 35.8 395 12.7 12.2 34.3 392 12.9 12.6 35.3 G 397 14.0 14.7 38.0 390 13.0 12.8 35.2 391 13.4 13.3 36.5 H 390 13.4 13.8 41.1 390 13.7 14.5 38.3 387 13.4 13.3 36.0 I 386 13.9 13.8 35.9 388 12.8 12.4 35.8 385 13.6 13.4 36.2 J 384 14.0 13.9 36.3 387 13.9 14.3 38.4 385 12.7 13.9 36.1 K 384 14.8 14.9 38.4 386 13.7 12.8 36.1 377 13.3 13.1 36.7 L 377 14.8 16.0 37.9 375 14.0 14.5 36.3 363 14.0 15.3 38.6 M 364 15.2 16.4 37.6 372 14.0 14.7 37.6 359 14.4 15.2 38.0 N 360 14.0 15.1 35.9 367 14.3 14.9 36.7 354 14.4 14.8 37.0

LSD (5%) 17.8 1.2 1.1 5.1 17 1.2 1.7 2.2 20 1.6 1.2 3.3 CV (%) 2.6 4.8 4.5 8.0 2.4 5.2 7.2 3.5 2.3 7.0 5.2 5.2

†Pro = protein; Met = methionine; Cys = cystine; Thr = threonine. Protein concentration expressed as g kg-1 of to- tal dry matter; environments ranked according to protein concentration for each cultivar.

differences among environments for thr. Significant protein and cys differences were found for Sturdy, which showed no differences for met and thr. These data indicated that protein and amino acid concentrations of individual culti- vars varied with variation in environment. Krober (1956) studied met concen- tration in group III and IV soybean cultivars, and found differences among en- vironments only for group III. Different cultivars, however, may react differ- ently to changes in environment. The three cultivars in this study involved three different maturity groups. Therefore, it is possible that the differences found among these cultivars could be due to maturity group differences. Met concentration differences between two recurrent selection populations ob- served by Burton et al. (1982) apparently were due to environmental differ- ences. Regression analyses (Fig. 1) showed that as protein concentrations in- creased across the 14 environments there was a small, but consistent decrease in concentrations of all three amino acids in all three cultivars. The significant environmental effects on protein and amino acid concentrations were enough Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 93

Figure 1. Changes in amino acids of soybean with protein variations. 94 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) to cause significant differences, but not significantly reduce amino acid con- centrations. More than 50% of the variation in met and cys concentrations was accounted for by relationships of these amino acids with protein concentrations in Lambert and Sturdy (Fig. 1). For Parker, 29-38% of the variation was ac- counted for. For threonine concentration, only 17 to 24% of the total variation was accounted for among all cultivars. The ranges in decrease among cultivars for each amino acid were small. These data were consistent with Zarkadas et al. (1994), who found consistent, but non-significant decreases in met and cys concentrations when a large increase in protein concentration was achieved within the same cultivar by backcrossing. Zarkadas et al. (1994), however, found significant decreases in thr concentrations in the same study. Zarkadas et al. (1983) found non-significant decreases in met and cys and increase in thr when protein concentrations were increased significantly by breeding. In- creasing protein concentrations by recurrent selection (Burton et al., 1982) did not significantly change met concentrations either. Krober and Cartter (1966) observed met decreases with increasing protein for protein concentrations be- low 370 g kg-1, and small decreases for protein concentrations above 370 g kg-1. This study demonstrated that protein changes resulting from environmen- tal variation did not significantly decrease met, cys, and thr concentrations of the protein. Therefore, integrity of the most limiting amino acids in these soy- bean cultivars were maintained across a wide range of environments. Al- though changes in environment did affect amino acid concentrations, these changes were not caused by changes in protein concentrations.

REFERENCES

Association of Official Analytical Chemists. (1990). Official methods of analysis of the Association of Official Analytical Chemists. 15th ed. AOAC Inc., Ar- lington, VA. Boulter, D. 1980. Ontogeny and development of biochemical and nutritional at- tributes in legume seeds. p. 127-134. In R.J. Summerfield and A.H. Bunting (ed.) Advances in legume science. Proc. International Legume Conference. Royal Botany Garden Publishing Kew, UK. Brim, C.A. and J.W. Burton. 1979. Recurrent selection in soybeans. II. Selection for increased percent protein in seeds. Crop Sci. 19:494-499. Burton, J.W. 1991. Development of high-yielding high-protein soybean germplasm. p. 109-117. In R.F. Wilson (ed.) Designing value-added soy- beans for markets of the future. American Oil Chemists’ Soc., Champaign, IL. Burton, J.W., A.E. Purcell, and W.M. Walter, Jr. 1982. Methionine concentration in soybean protein from populations selected for increased percent pro- tein. Crop Sci. 22:430-432. Hartwig, E.E. and T.C. Kilen. 1991. Yield and composition of soybean seed from parents with different protein, similar yield. Crop Sci. 31:290-292. Krober, O.A. 1956. Methionine content of soybeans as influenced by location and season. J. Agricultural and Food Chemistry 4:254-257. Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 95

Krober, O.A. and J.L. Cartter. 1966. Relation of methionine content to protein levels in soybeans. Cereal Chem. 43:320-325. Leffel, R.C.1992. Registration of high protein soybean germplasm lines BARC- 6, BARC-7, BARC-8, AND BARC-9. Crop Sci. 32:502. Seretti, C., W.T. Schapaugh, Jr., and R.C. Leffel. 1994. Amino acid profile of high seed protein soybean. Crop Sci. 34:207-209. Sionit, N., and P.J. Kramer. 1977. Effects of water stress during different stages of growth of soybean. Agronomy J. 69:274-278. Voldeng, H.D., and G. Saidon. 1991. Registration of a high protein soybean germplasm line OT89 16. Crop Sci. 31:1100. Wilson, R.F. 1987. Seed metabolism. In J.R. Wilcox (ed) Soybeans: Improve- ment, production, and uses. Agronomy 16:643-686. Zarkadas, G., Z. Yu, H.D. Voldeng, and A. Minero-Amador. 1993. Assessment of the protein quality of a new high-protein soybean cultivar by amino acid analysis. J. Agric. Food Chem. 41:616-623. Zarkadas, G., Z. Yu, H.D. Voldeng, H.J. Hope, A. Minero-Amador, and J.A. Rochemont. 1994. Comparison of protein-bound and free amino acid con- tents of two northern adapted soybean cultivars. J. Agric. Food Chem. 42:21-33.

Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 97

MISADVENTURES IN SOYBEAN TRANSFORMATION: PARTICLE BOMBARDMENT VS. AGROBACTERIUM AND COTYLEDONARY NODES

S.A. Hansen and C.D. Carter Plant Science Department South Dakota State University Brookings SD 57007

INTRODUCTION

Transformation and regeneration efforts have met with limited success in soybean. Embryogenic regeneration systems have been successful in only a few lines. Organogenic regeneration systems are applicable to more genotypes but transformation rates have remained low. The two methods most often used to deliver transgenes in soybean are par- ticle bombardment and Agrobacterium tumefaciens. In particle bombardment, gold or tungsten particles coated with plasmid DNA containing the transgenes are shot into plant cells. Insertion into the genome occurs largely at random lo- cations, and may result in the insertion of multiple copies of the transgenes in- to the host plant genome. Agrobacterium methods use the Ti-vector system to insert the transgenes. Insertion into the plant genome is by recombination in- volving the Ti right- and left-border sequences and usually results in incorpo- ration of one copy of the transgene into the host plant genome. To improve the chances of recovery of transgenic plants, a selectable marker gene, e.g., kanamycin or a herbicide resistance gene, may be included in the transgene package. A reporter gene, e.g., beta-glucuronidase (GUS) or green fluorescent protein (GFP), is also often used to allow visualization of transgenic cells or plants. GUS produces a blue color in the presence of a glucuronide substrate. The cotyledonary node system, an organogenic regeneration system, has been widely used for transformation and regeneration of soybean, primarily with gene delivery via Agrobacterium tumefaciens. Recent improvements, in- cluding use of the selectable marker gene bar, have been made by Zhang et al. (1999), resulting in higher transformation rates. The bar gene provides re- sistance to the herbicide glufosinate and thus allows selection for transgenic cells on media containing glufosinate. We have been attempting to obtain transgenic soybean plants from cotyle- donary nodes using both particle bombardment and Agrobacterium transfor- mation methods. We have performed transformation experiments on thousands of explants, and have at times produced putative transgenic plants by both methods, but none of these have yet been confirmed to be transgenic by Southern analysis of the genomic DNA of the putative transgenics or their progeny. In this report, we present results of an experiment to identify potential causes of the low transformation rates that we had obtained using particle 98 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) bombardment with a cotyledonary node regeneration system, and compare these results with transformation rates obtained using Agrobacterium.

MATERIALS AND METHODS

Particle Bombardment Experiments

Soybean (Glycine max L.) cv. Jack seeds were sterilized in 20% bleach so- lution, rinsed in sterile water and germinated on Murashige and Skoog (1962) (MS) medium containing 30 g/l sucrose and 6 g/l agar, at pH 5.8. After 3 days, the hypocotyl, primary shoot and distal half of the cotyledon were removed and discarded. The remainder of each explant was placed on media contain- ing 6 g/l agar and 0.5M sorbitol. The following day, explants were bombarded with gold particles coated with pBI121, using a Particle Inflow Gun (Finer et al., 1992). Explants were transferred to shoot initiation (SI) medium consisting of MS media components plus Gamborg B5 vitamins (Gamborg et al. 1968), 0.5 g/l casein enzymatic hydrolysate, 0.6 g/l MES, 30 g/l sucrose, 8 g/l agar, 5 mg/l thidiazuron, 0.04 mg/l NAA. The medium was autoclaved for 20 min at 120C and 15 psi and filter-sterilized glutamine was added to a final concentration of 2 g/l. SI medium was poured into 100 x 15 mm sterile polystyrene Petri plates. Plates were sealed with Parafilm and placed in an incubator at 24C with ap- proximately 50 µE•m-2•s-1 cool white fluorescent light for 14 hr daily. After 1 week, explants were placed on fresh SI medium containing TDZ at 0.05 mg/l, and explants were transferred to fresh media each week. Experimental treatments were: 1. bombardment with gold particles coated with pBI121 (Clontech), containing a kanamycin resistance gene and a GUS gene linked to a CaMV35S promoter; 2. bombardment with gold particles alone, and 3. no bombardment. There were 30 explants per treatment. Trans- formation rate, as reflected by GUS expression, was analyzed from 3 days to 4 weeks after explant preparation. GUS expression was analyzed by incubating the explants in 1 mM 5-bromo-4-chloro-3-indolylglucuronide (X-Gluc) (Jeffer- son, 1987) overnight; chlorophyll was removed by incubation in 70% ethanol. For histological analyses, explants were fixed 4-5 hours in FAA, dehydrat- ed and paraffin embedded, sectioned on a rotary microtome at 10 um, and stained in safranin-fast green.

Agrobacterium Experiments

Agrobacterium tumefaciens strain LBA4404 and the binary vector pBI121 were obtained from Clontech. Agrobacterium tumefaciens strain EHA101 was obtained from S. Gelvin (Purdue U.), and the plasmid pPTN140 was obtained from T. Clemente (U. Nebraska-Lincoln). The binary vectors were transferred into the appropriate Agrobacterium strains by triparental mating (Maldonado- Mendoza et al., 1996). Agrobacterium cultures were prepared for transforma- tion experiments as per Zhang et al. (1999). Soybean seeds were treated according to the methods of Zhang et al. (1999). Briefly, soybean seeds were germinated for 5 days, the hypocotyl and Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 99 primary shoot were removed, and the cotyledonary node was wounded using a scalpel blade. Explants were co-cultured with Agrobacterium for 3 days and placed on shoot initiation medium containing 500 mg/l ticarcillin, 100 mg/l ce- fotaxime and glufosinate at 10 mg/l. After 2 weeks, explants were transferred to shoot elongation media (Zhang et al., 1999). GUS expression was analyzed as described above.

RESULTS

Of the 92 explants observed in the particle bombardment experiment, on- ly 14 were found to have the cotyledonary node region. Shoots regenerated only when the cotyle- donary node was present. When shoots were present, they appeared to arise from the axillary meristem (Fig- ure 1) within the cotyle- donary node, or, in one case where the primary shoot was not completely removed, from the rem- nants of the primary shoot. No GUS expression was observed on explants that were not bombarded or that were bombarded with gold particles alone, i.e., without DNA, 3 days after bombard- ment (Table 1). However, GUS expression was posi- tive on 85% of explants bombarded with particles coated with pBI121, and there were an average of 30 Figure 1. Axillary shoot arising from the blue spots (GUS-positive re- cotyledonary node of a soybean explant.

Table 1. GUS expression 3 days after particle bombardment. Data are aver- ages of 30 explants per treatment.

GUS-positive Blue spots Percent of blue spots Treatment explants (%) per explant within the cot-node

No particles 0 0 0 Gold particles alone 0 0 0 Gold particles with pBI121 85% 30 6% 100 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) gions) on these explants. Approximately 6% of the blue spots were within the cotyledonary node region (Table 1). Two weeks after bombardment, only 4% of explants bombarded with pBI121 were GUS positive, there were few blue spots per explant, and none of the spots were within the cotyledonary node region (Table 2.)

Table 2. GUS expression 2 weeks after particle bombardment. Data are averages of 30 explants per treatment.

Percent of GUS- Blue spots Percent of blue spots Treatment positive explants per explant within the cot-node

No particles 0 0 0 Gold particles alone 0 0 0 Gold particles with pBI121 4% 2.5 0

Agrobacterium tumefaciens LBA4404/pBI121 carries both the GUS gene and nptII, a kanamycin resistance gene, linked to a CaMV35S promoter. Trans- formation rates, as indicated by GUS expression at 4 weeks, were approxi- mately 5% (Table 3). However, kanamycin resistance is notoriously ‘leaky,’ re- sulting in inefficient selection, as indicated by the survival of up to 40% of the regenerated plantlets on selective media (Table 3).

Table 3. Average GUS expression and survival on selective media 4 weeks af- ter treatment with Agrobacterium tumefaciens EHA101 plus pPTN140. Data are averages of >10,000 explants.

Agrobacterium Percent of GUS- Percent of blue spots Survival on /plasmid positive explants within the cot-node selective media

LBA4404/pBI121 5% 5% 40% EHA101/pPTN140 5%–20% 5% <1%

Agrobacterium tumefaciens EHA101/pPTN140 has produced transforma- tion rates ranging from 5% to 20%, with approximately 5% of the GUS-positive spots in the cotyledonary region. Survival on selective media, i.e., media con- taining glufosinate, was typically 1% or less. Several shoots that are complete- ly or largely GUS-positive have also been obtained (Figure 2). Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 101

DISCUSSION

Soybean shoots regenerated only from the cotyledonary node. During ex- plant preparation, the cotyledonary node is frequently, although inadvertently, removed along with the hypocotyl and primary shoot. Explants lacking cotyledonary nodes did not regenerate shoots. There- fore, care must be used to ensure that the meristemat- ic regions in the cotyle- donary node are retained during preparation of the explants. Transient GUS expres- sion, i.e., expression of the reporter gene within a few days of bombardment, was used to determine whether particles were able to enter Figure 2. GUS-positive soybean shoot ob- the plant cells and what tained following transformation by Agrobac- proportion of the particles terium tumefaciens. The GUS positive re- reached the regenerating gions of this chimeral shoot stain dark blue area, i.e., the cotyledonary (black in the photograph). node region. Transient GUS expression rates of 85% in- dicated that the transgenes often entered the cell. However, after 2 weeks GUS expression rates had diminished to 4%, suggesting that the transgenes are sel- dom integrated into the plant genome. Relatively few of the particles entered or were expressed in the cotyle- donary node region. Analysis of GUS expression 2 weeks after particle bom- bardment indicated that none of the transgenes were still present in the cotyle- donary node. These results suggest that cotyledonary nodes are difficult to tar- get, or that particles do not readily enter or become integrated into the chro- mosomes of cells in the cotyledonary node region, i.e., the region from which shoots regenerate. In contrast to the low transformation rates with particle bombardment, transformation by Agrobacterium resulted in positive GUS expression even 4 weeks after treatment. GUS expression was observed in the cotyledonary node region and GUS-positive shoots were produced. Glufosinate was much more effective than kanamycin for selecting transgenic plants. In summary, the cotyledonary node appears to be relatively inaccessible to particle bombardment unless targeting can be substantially improved. By con- trast, transformation of wounded cotyledonary nodes via Agrobacterium tume- faciens, especially when used in conjunction with a robust selectable marker, e.g., glufosinate, resulted in GUS expression within the regenerating region and in regenerated shoots. 102 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000)

LITERATURE CITED

Finer JJ, Vain P, Jones MW, McMullen MD. 1992. Development of the particle inflow gun for DNA delivery to plant cells. Plant Cell Reports 11: 323-328. Jefferson RA. 1987. Assaying chimeric genes in plants: The GUS gene fusion system. Plant Molecular Biology Reporter 5: 387-405. Maldonado-Mendoza IE, Lopez-Meyer M, Nessler CL. 1996. Transformation of tobacco and carrot using Agrobacterium tumefaciens and expression of the B-glucuronidase (GUS) reporter gene. In: Trigiano RN and Gray DJ, Eds., Plant Tissue Culture Concept and Laboratory Exercises. CRC Press, NY, pp 261-274. Murashige, T. & F. Skoog. 1962. A revised medium for rapid growth and bioas- says with tobacco tissue cultures. Physiol. Plant. 15: 473-479. Zhang Z, Xing A, Staswick P, Clemente TE. 1999. The use of glufosinate as a selective agent in Agrobacterium-mediated transformation of soybean. Plant Cell, Tissue and Organ Culture 56:37-46. Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 103

SUMMER HABITAT ASSOCIATIONS OF RARE FISHES IN SOUTH DAKOTA TRIBUTARIES TO THE MINNESOTA RIVER

Douglas J. Dieterman Department of Wildlife and Fisheries Sciences Minnesota Department of Natural Resources Glenwood, MN 56334

Charles R. Berry South Dakota Cooperative Fish and Wildlife Research Unit U.S. Geological Survey, Biological Resources Division South Dakota State University Brookings, SD 57007

ABSTRACT

Four fish species found in western tributaries to the Minnesota River are listed as rare in South Dakota. We found only two northern redbelly dace (Phoxinus eos), but sample size of other species was sufficient to examine re- lationships between fish abundance and habitat with correlation and regression analyses. Relative abundance of hornyhead chub (Nocomis biguttatus) and blackside darter (Percina maculata) was related to mean stream width (+) and water temperature (-). Relative abundance of rosyface shiner (Notropis rubel- lus) was related to the amount of riffle habitat (-). These fish-habitat associa- tions identify habitat conditions that might be important for managing habitat for these species.

INTRODUCTION

A small part of the upper Mississippi River basin is in South Dakota as wa- tersheds of the Whetstone, Yellowbank, and Lac Qui Parle rivers, which are tributaries to the Minnesota River. The fish communities in these rivers vary from 13 to 22 species for a total of 27 species (Dieterman and Berry 1994). Dominant species are cyprinids such as fathead minnow (Pimephales prome- las), blacknose dace (Rhinichthys atratulus), creek chub (Semotilus atromac- ulatus), and common shiner (Luxilus cornutus). Four species are present that are considered rare in South Dakota but are more common in Minnesota (Wilcox et al. 1978, Underhill 1989, Kavanaugh 1993): northern redbelly dace (Phoxinus eos), hornyhead chub (Nocomis biguttatus), blackside darter (Percina maculata), and rosyface shiner (Notropis rubellus). These species are examples of fish that are common elsewhere in the Mississippi River basin but occur as relict populations in streams of the se- mi-arid prairie on the western edge of their range (Cross and Moss 1987). Our objective was to determine habitat associations of these rare species. 104 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000)

STUDY SITE

The Minnesota River is the largest tributary of the Mississippi River in Min- nesota, crossing the state from its headwaters on the Coteau des Prairies in South Dakota to its confluence with the Mississippi river (Fasching 1983, Ka- vanaugh 1993). About 2% of the catchment is composed of the sub-basins of the Whetstone, Yellowbank, and Lac Qui Parle rivers in South Dakota. These rivers originate as swift creeks on the highlands of the Coteau in South Dako- ta, meander across low plains, and then plunge through ravines into the Min- nesota River (Waters 1977). The streams flow through the Prairie Coteau Escarpment ecoregion, which is a distinctive ecosystem rising 100-300 m from the Minnesota River Valley to the brow of the Coteau. The streams are distinctive for prairie streams because they are relatively natural, high-gradient, cool, and forested. In South Dakota we sampled five tributaries to the Lac Qui Parle River in Deuel County (Monighan Creek, Cobb or Florida Creek, Lost Creek, Crow-Timber Creek, Crow Creek), and the North and South Forks of the Yellowbank and Whetstone rivers in Grant County.

METHODS

One reach was selected at the top, middle, and base of the Coteau on each stream. An exception was the North Fork Whetstone River, which was sam- pled only at the base. Each reach was about 100 m long and encompassed pool, riffle, and run habitats. Habitat data were collected within each reach us- ing line-transect methods similar to those proposed by Simonson et al. (1994). Transects were set across the middle of each pool, riffle, and run, and at the transition points between. Depth, substrate, and cover type were recorded at quarter points along each transect. Substrates were classified as boulder, rubble/cobble, gravel, sand, silt, clay, (Gordon et al. 1992) or as muck (fine ma- terials with substantial organic matter), or detritus (plant fragments). Percent substrate and cover type were calculated as the number of points where a sub- strate or cover type was found divided by the total number of points multiplied by 100. We measured dissolved oxygen (mg/L), temperature (ºC), pH, con- ductivity (uS/cm), turbidity (JTUs), and water velocity (m/s) once in the center of each pool, riffle, and run. The length of each pool, riffle, and run was di- vided by the total reach length to determine the percentage of each. Fishes were collected by electrofishing between block nets (for details see Dieterman and Berry 1994). Relative abundance of each species in each reach was expressed as the number collected per hour of electrofishing. Relation- ships between fish relative abundance and habitat characteristics were ex- plored using correlation and regression techniques. Habitat variables (N = 29) were assessed for correlations with fish relative abundance. Significant correlations (P ≤ 0.10) were further assessed for corre- lations among themselves (Hubert and Rahel 1989). For inter-correlated vari- ables, we chose the variable most highly correlated with fish data. We exam- ined remaining variables for normality and log-transformed them if the as- Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 105 sumption was not met. Relationships were further explored with regression models. All significant models (P ≤ 0.10) were evaluated by examining raw da- ta plots, plots of residuals, tolerance values, and studentized residual frequen- cies (Hatcher and Stepanski 1994).

RESULTS AND DISCUSSION

The general habitat data for these small (width = 3.7-9.9 m) streams re- flected the unique ecosystem setting. Average midsummer temperature was 22 C, which is typical of streams classed as intermediate between cold and warm water streams (Moyle and Cech 1982:397). Temperatures were similar at the top (mean=21.5, SD=1.2), middle (mean=21.7, SD=1.9), and base (mean=21.6, SD=3.7) of the Coteau. Water temperature usually increases from upstream to downstream unless moderated by shading or groundwater inputs (Allan 1995), which probably influenced our study sites. Water was clear, well oxygenated, and alkaline (Table 1). The steep gradi- ent caused relatively swift velocity and the development of pools, riffles and runs (Allen 1995). Most upstream reaches were narrower and had higher per- centages of gravel, rubble, and boulder substrates than did downstream reach- es. In-stream habitat for fish consisted of a complex of boulders, undercut banks, submerged vegetation, and woody debris. Most rare species were collected together, possibly indicating similar habi- tat requirements. For example, hornyhead chubs were present at all rosyface shiner sites and at > 50% of blackside darter sites. We collected two individu- als of the Northern redbelly dace, so habitat associations could not be deter- mined. Ernst (1972) did not find Northern redbelly dace in the adjacent Yel- low Medicine River. However, the species was found at numerous sites in Min- nesota in the 1990s (Kavanaugh 1993). Catch rate for the hornyhead chub was 13.5 fish/hr, but catch rate for the others was <1 fish/hr (Table 2). We found hornyhead chub at 9 of 22 reach- es (Table 2) in five streams; North Fork Yellowbank River, South Fork Yellow- bank River, Monighan Creek, Cobb Creek, and North Fork Whetstone River. Relative abundance was negatively correlated with water temperature and pos- itively correlated with mean stream width (Figure 1). However, these two vari- ables were significantly correlated with each other (P=0.032, r= -0.48, N=20). We eliminated water temperature from further analysis because it had a lower correlation with hornyhead chub relative abundance than did mean stream width. There was a significant positive relationship between mean stream width and relative abundance of hornyhead chub (Table 3). Stream width av- eraged 7.3 m (± 1 SD = 1.2) in reaches where hornyhead chubs were collect- ed, which is larger than the 6.0-m mean width for all reaches (Table 1). Horny- head chubs were was most common in reaches at the base of the Coteau where stream widths tended to be widest. In Wisconsin streams (Becker 1983) the frequency of occurrence of hornyhead chubs in five stream-width cate- gories was 1-3 m (17%), 3-6 m (24%), 6-12 m (18%), 12-24 m (26%), and >24.0 m (16%). In our study, hornyhead chubs were not present where widths were < 5.6 m. Hornyhead chubs and blackside darters used riffle habitat more than expected (Becker 1983, Cross and Collins 1995, Pflieger 1997). 106 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000)

Table 1. Physical habitat and water quality characteristics measured at stream reaches (N) in eight Minnesota River tributaries in northeast South Dakota, during the summer of 1993.

VARIABLE N REACH AVERAGE (±1 SD) RANGE

Reach characteristics Mean width (m) 22 6.0 (1.6) 3.7-9.9 Mean depth (cm) 22 42.7 (12.0) 19-63 Maximum depth (cm) 20 100.7 (20.6) 60-150 Riffle velocit y(m/s) 20 0.62 (0.22) 0.23-1.13 Pool velocity (m/s) 20 0.26 (0.17) 0.03-0.55 Run velocity (m/s) 20 0.37 (0.22) 0.04-0.76

Water quality Dissolved oxygen (mg/L) 22 8.8 (1.0) 7.2-12.2 Turbidity (JTU's) 20 57.2 (58.1) 7.0-245.0 Water temperature (ºC) 20 22.1 (2.3) 16.0-26.0 pH 21 9.6 (0.3) 9.0-10.2 Conductivity (uS/cm) 21 891.2 (264.3) 495.0-1461.0

Habitat type (%) Pools 22 42.4 (21.0) 0-87 Riffles 22 25.7 (14.7) 0-49 Runs 22 32.0 (22.8) 0-84

Substrate (%) Boulder 22 7.1 (9.8) 0-31 Rubble 22 12.7 (13.3) 0-52 Gravel 22 34.2 (19.5) 0-79 Sand 22 28.5 (14.4) 0-59 Silt 22 14.6 (13.7) 0-58 Clay 22 2.6 (4.5) 0-18 Detritus 22 0.1 (0.4) 0-2 Muck 22 0.2 (0.9) 0-4

Cover type (%)1 No cover 13 45.5 (21.2) 10-83 Woody debris (< 7.5 cm diameter) 13 9.2 (5.6) 2-25 Log (> 7.5 cm diameter) 13 1.2 (2.4) 0-7 Undercut bank 13 6.5 (7.3) 0-21 Rubble/Boulder 13 28.7 (19.7) 4-56 Instream vegetation 13 4.2 (4.1) 0-15 Overhanging vegetation 13 4.8 (6.2) 0-22

1Cover type measurements added after study was underway. Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 107

Table 2. South Dakota Natural Heritage Program designation, percent of reach- es where found, and mean(± 1 SD) electrofishing catch-per-effort (#/hr) of four rare fishes collected in Minnesota River tributaries in South Dakota, during 1993.

Species Designation Reach % Catch rate

Hornyhead chub rare 41 13.45 (28.09) Blackside darter rare 32 0.86 (1.55) Rosyface shiner rare 14 0.70 (2.15) Northern redbelly dace state threatened 9 0.19 (0.67)

Table 3. Linear regression models for the relation between relative abundance of three rare fish species and habitat in tributary streams of the Minnesota River in South Dakota.

REGRESSION EQUATION N R2 P

Hornyhead chub -56.0 + 11.5 (mean stream width) 22 0.45 0.0006

Blackside darter 9.8 - 0.41 (water temperature) 20 0.43 0.0016

Rosyface shiner 2.14 - 0.05 (percent riffle) 21 0.15 0.0790

Figure 1. Relationship between catch per unit effort of rare fish and selected physical and chemical conditions in Minnesota River tribu- taries in South Dakota. 108 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000)

Blackside darters were collected at 7 of 22 reaches (Table 2) in five streams; North Fork Whetstone River, North and South Forks Yellowbank River, Lost Creek, and Cobb Creek. Relative abundance of blackside darter was negatively correlated with water temperature and positively correlated with mean stream width (Figure 1). Water temperature was retained in lieu of mean stream width because it was more highly correlated with relative abundance. The result was a significant negative relationship between water temperature and blackside darter relative abundance (Table 3). Mean water temperature was 20.3ºC (SD=2.6) at sites with blackside darters and 22.9ºC (SD=1.7) at sites without them. Reaches at the top and middle of the Coteau had ground water inputs and blackside darters. Rosyface shiners were collected in three streams; North Fork Whetstone River, North and South Fork Yellowbank River (Table 2). Both correlation and regression analyses indicated a negative relationship between rosyface shiner relative abundance and percent riffle habitat (Figure 1; Table 3). A negative relationship with percent riffle habitat may indicate an association with pools and runs, which are habitat types important for this species elsewhere (Beck- er 1983, Etnier and Starnes 1993, Cross and Collins 1995). Our average per- cent riffle habitat was 26% in a 100-m stream reach (Table 1), and all reaches where this species was collected had less than 25% riffle habitat. Long stretch- es of pools and runs appear important to this species in South Dakota also. Most reaches with rare species were located at the base of the Coteau, which agrees with our finding of a relationship between hornyhead chub rel- ative abundance and mean stream width, and the negative relationship be- tween rosyface shiner abundance and length of riffles. Stream reaches at the base of the Coteau have more water permanency than those on top (e.g. Lost, Crow, and Crow-Timber creeks) that are sometimes intermittent (McCoy and Hales 1974). Hornyhead chubs and rosyface shiners are associated with per- manent flows (Cross and Collins 1995, Pflieger 1997), which might explain their absence from these creeks. Intermittency in the summer can stress aquatic bio- ta through increased water temperatures and reduced dissolved oxygen levels (Matthews 1998). Hornyhead chubs and rosyface shiners are intolerant of hy- poxia and hyperthermia (Smale and Rabeni 1995). The habitat of the Prairie Coteau escarpment is unique (Milewski and Willis 1989, Dieterman and Berry 1994, Higgins et al. 1997). The permanent, cool-wa- ter flows in medium-sized streams are not usually found in other prairie streams today, but may have been more common in the past (Rabeni 1996). Focusing management (e.g., Isenhart et al. 1995) on these factors may help conserve fish communities of streams flowing off the Coteau.

ACKNOWLEDGMENTS

We thank Dr. C. Scalet for fish identification, T. TerMeer, T. Albee, S. Eide, C. Fisher, and S. Gurtin for field assistance, and K. Pope, C. Milewski, and D. Willis for comments on a draft. The Cooperative Research Unit is jointly sup- ported by the South Dakota Department of Game, Fish, and Parks, the U. S. Geological Survey, Biological Resources Division, South Dakota State Universi- ty, and the Wildlife Management Institute. Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 109

LITERATURE CITED

Allan, J. D. 1995. Stream ecology: structure and function of running waters. Chapman and Hall, New York, New York. Becker, G. C. 1983. Fishes of Wisconsin. University of Wisconsin Press, Madi- son. Cross, F. B., and J. T. Collins. 1995. Fishes in Kansas. 2nd ed. University Press of Kansas, Lawrence. Cross, F. B., and R. E. Moss. 1987. Historic changes in fish communities and aquatic habitats in plains streams of Kansas. Pages 155-165 in W. J. Matthews and D. C. Heins, editors. Community and Evolutionary Ecology of North American Stream Fishes. University of Oklahoma Press, Norman. Dieterman, D. J., and C. R. Berry. 1994. Fishes in seven streams of the Min- nesota River Drainage in northeastern South Dakota. South Dakota Acade- my of Science 73:23-30. Ernst, Carl, H. 1972. Fishes of the Yellow Medicine River in Southwestern Min- nesota. The Minnesota Academy of Sceince 38:14-16. Etnier, D. A., and W. C. Starnes. 1993. The fishes of Tennessee. University of Tennessee Press, Knoxville. Fasching, Paul. 1983. Drainage basin characteristics of the Minnesota River. Journal of the Minnesota Academy of Science 49:10-14. Gordon, N. D., T. A. McMahon, and B. L. Finlayson. 1992. Stream hydrology. John Wiley and Sons Ltd, West Sussex, Hatcher, L., and E. J. Stepanski. 1994. A step-by-step approach to using the SAS System for univariate and multivariate statistics. SAS Institute Inc., Cary, North Carolina. Higgins, K., R. Johnson, M. Dorhout, and W. Meeks. 1997. Occurrence of small nongame mammals in South Dakota's eastern border counties. South Dakota Academy of Science 76:65-74. Hubert, W. A., and F. J. Rahel. 1989. Relations of physical habitat to abun- dance of four nongame fishes in high-plains streams: A test of habitat suit- ability index models. North American Journal of Fisheries Management 9:332-340. Isenhart, T. M., R. C. Schultz, and J. P. Colletti. 1995. Design, function, and management of multi-species riparian buffer strip systems. Pp 4-5 in C. M. Milewski, C. R. Berry, and S. Wyman, eds. Watershed management work- shop for the James, Vermillion, and Big Sioux rivers. South Dakota Agri- cultural Experiment Station B 720. Brookings: South Dakota State Univer- sity. Kavanaugh, C. 1993. Minnesota River Basin assessment project. Pages 5- 22 in L. Hesse, C. Stalnaker, N. Benson, and J. Zuboy, editors. Restoration Planning for the Rivers of the Mississippi River Ecosystem. Biological Re- port 19, National Biological Survey, Washington, D. C. Matthews, W. J. 1998. Patterns in freshwater fish ecology. Chapman and Hall, New York, New York. McCoy, R. W., and D. C. Hales. 1974. A survey of eight streams in eastern South Dakota: physical and chemical characteristics, vascular plants, in- sects and fishes. South Dakota Academy of Science 53:202-219. 110 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000)

Milewski, C.L. and D.W. Willis. 1989. Reproduction, recruitment, and survival of brown and rainbow trout in a prairie Coteau stream. Prairie Naturalist 21:147-156. Moyle, P., and J. Cech. 1982. Fishes: An introduction to Ichthyology. Prentice- Hall Inc. Englewood Cliffs, New Jersey. Pflieger, W. L. 1997. The fishes of Missouri, revised edition. Missouri De- partment of Conservation, Jefferson City. Rabeni, C. F. 1996. Prairie legacies-fish and aquatic resources. Pages 111-124 in F. Samson and F. Knopf, editors. Prairie Conservation. Island Press, Washington, D. C. Simonson, T. D., J. Lyons, and P. D. Kanehl. 1994. Guidelines for evaluating fish habitat in Wisconsin streams. General Technical Report NC-164. For- est Service, North Central Forest Experiment Station, U. S. Department of Agriculture, St. Paul, Minnesota. Smale, M. A., and C. F. Rabeni. 1995. Hypoxia and hyperthermia tolerances of headwater stream fishes. Transactions of the American Fisheries Soci- ety 124:698-710. Underhill, J.C. 1989. The distribution of Minnesota fishes and late Pleistocene glaciation. Journal of the Minnesota Academy of Science 55:32-37. Waters, T.F. 1977. The Streams and Rivers of Minnesota. University of Min- nesota Press, Minneapolis. Wilcox, D.B., B. Lorenz, and V. Kranz. 1978. Fish species, some uncommon, collected from the lower Minnesota River. Journal of the Minnesota Acade- my of Science 44:7-12. Abstracts of Senior Research Papers

presented at The 85th Annual Meeting

of the South Dakota Academy of Science

Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 113

USE OF SHELTERBELTS AS BREEDING HABITAT BY BIRDS IN EASTERN SOUTH DAKOTA

Dale L. Droge and Jeffrey S. Palmer College of Natural Sciences Dakota State University Madison, SD 57042-1799

ABSTRACT

We censused birds present in shelterbelts during the breeding season to determine if shelterbelts are critical resources for maintaining or stabilizing non-game bird populations in South Dakota. We conducted our survey in Lake County, South Dakota where shelterbelts represent the most significant per- centage of the woodland habitat and may be critical to the reproductive suc- cess of many birds in this area. We censused 10 shelterbelts in 1997 and 8 shel- terbelts in 1998. In order to investigate the effect of habitat area on species composition, we censused both large (more than 500 m in length) and small (less than 300 m long) shelterbelts. Census points were established no closer than 50 m to the end of a shelterbelt. Each point was at least 150 m from oth- er points in the same shelterbelt. We used the point count method to survey the bird community of the study site. The observer stood at the census point and recorded all bird species seen or heard inside the shelterbelt within 50 m of the census point during a five minute period. During the course of the study we recorded the presence of 36 different species. The species richness of individual shelterbelts ranged from 23 species to 9 species. This difference between sites suggests that the age and tree species composition of the shelterbelt may be important influences on breed- ing bird diversity. There were 15 species of birds that were recorded at 10% or more of the census points in at least one of the two years. House Wren and Common Grackle were the most frequently observed species, followed by Mourning Dove and Song Sparrow. American Robins were three times more frequent in 1998 compared to 1997. The length (area) of the shelterbelt did not have a strong effect on the number of species observed. We conclude that shelterbelts provide critical nesting habitat for a signifi- cant number of resident and migratory birds. A decrease in the number or quality of shelterbelts will negatively affect songbird populations in eastern South Dakota.

Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 115

INVESTIGATING LEATHERBACK TURTLE (DERMOCHELYS CORIACEA) EGG LAYING AND EGG SURVIVAL

Valerie E. Wilson and Donna Hazelwood Dakota State University Madison, SD 57042

ABSTRACT

Archie Carr first described the plight of the decreasing leatherback turtle (Dermochelys coriacea) population in the 1960s, and since then scientists and volunteers have saved thousands of the species from extinction. Agencies in- volved are the Department of Planning and Natural Resources, division of U.S. Fish and Wildlife Service, and Earthwatch. As a member Earthwatch Expedi- tion’s “Saving the Leatherback Turtle,” Team V, June 1-11, 1999, I studied the nesting behavior activities of the female leatherback turtle (Dermochelys cori- acea) at Sandy Point National Wildlife Refuge, on St. Croix, U.S. Virgin Islands. Three Co-Principal Investigators and my team monitored successive nightly pa- trols. I measured 22 females for carapace length and width, distance from in situ nest to vegetation and from nest to high-tide water mark. Other identifi- cation procedures included scanning PIT (Passive Integrated Transponder) tag numbers, recording arrival and departure times, and diagnostics (i.e. distin- guishing marks). Eleven nesting females were recorded laying eggs within the erosion-zone territory, thus we reburied the eggs in a neutral site. The mean for eggs laid per female leatherback per year were 78.4 yolked and 32.2 yolk- less (sterile) per clutch. My studies have contributed to ongoing leatherback turtle research at Earthwatch Expedition.

Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 117

WATER QUALITY AND PHYTOPLANKTON DYNAMICS OF THE OTTERTAIL POWER PLANT COOLING POND, MILBANK, SD

Amy L. Gronke and Nels H. Troelstrup, Jr. Department of Biology and Microbiology South Dakota State University Brookings, SD 57007

ABSTRACT

Ottertail Power Company maintains a 350-acre cooling pond to provide water to the boilers in their plant. Over the past several years, plant managers have witnessed blooms of algae, fish kills and calcium carbonate build-up on structures in the plant. The objectives of this project were to (1) develop base- line data describing current physical, chemical and biological conditions with- in the pond (2) develop a long-term monitoring plan for the pond and (3) draft recommendations to facilitate improvement of conditions within the pond. Wa- ter temperature, dissolved oxygen, conductivity, total hardness, total nitrogen, total phosphorus, water transparency, chlorophyll a and total and relative abundance of phytoplankton were measured monthly from five locations and three depths during 1998 and 1999. Ratio of total nitrogen (range 2.55 to 5.31 mg/L) to total phosphorus (range = 0.32 to 4.40 mg/L) averaged 3:1. Water tem- peratures varied by site and date (range 3 to 45ºC). Dissolved oxygen ranged from 0.2 to 15.0 mg/L. Total phytoplankton counts ranged from 11,776 to 66,423 cells/ml. Chrysophyta, Chlorophyta and Euglenophyta were found in greater abundance during the winter months (range = 0 to 43,101 cells/ml) while Cyanobacteria dominated during the summer months (range = 0 to 28,709 cells/ml) at all sites. Calcium carbonate precipitation appears to be a function of high primary production and high calcium concentrations. Low ni- trogen to phosphorus ratios appears to favor summer blooms of Cyanobacte- ria. Summer fish kills appear to coincide with high summer water tempera- tures and low oxygen levels. Water temperatures during a fish kill in July 1998 approached 40ºC while oxygen levels from mid-depth to the bottom were less than 4.0 mg/L. Results of this effort provide a baseline against which future changes can be measured in the pond. Monitoring protocols have been es- tablished to facilitate future monitoring of the pond and corrective manage- ment prescriptions are under development.

Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 119

CHARACTERIZATION OF A RURAL STREAM TO DEFINE CRITICAL REACHES INFLUENCED BY NON-POINT SOURCE POLLUTION

A.M. Larson and N.H. Troelstrup, Jr. Department of Biology and Microbiology South Dakota State University Brookings, SD 57007

ABSTRACT

The objective of this research was to define the current ecological integri- ty of Bachelor Creek in Moody County, South Dakota and define critical reach- es influenced by non-point source pollution. The Bachelor Creek watershed drains an area of 37,852 acres, of which 1,563 acres are considered highly erodible land. Land-use in the watershed area is primarily agricultural with ap- proximately 83% cropland, 5% grassland, and 7% farms and shelterbelts. Samples and measurements were taken to characterize conductivity, dis- solved oxygen, pH, alkalinity, total dissolved and suspended solids, ammonia, nitrate, sulfate, iron, manganese, sodium, and total dissolved phosphorus of 15 sites on Bachelor Creek once per month from April through September in 1998 and 1999. Bottom substrate and embeddedness, stream flow, channel alter- ations, pool/riffle ratio, bank stability, bank vegetative stability, and streamside cover were evaluated using USEPA’s Rapid Bioassessment Protocol (RBP) habi- tat scoring methods at Bachelor and Brookfield sites. Macroinvertebrate sam- ples were collected at three randomly chosen riffle locations from each site on Bachelor and Brookfield Creeks. RBP habitat scores for Bachelor reach 5 (upstream) ranged from 31-53% relative to Brookfield reference sites. Bachelor reach 5 consistently failed to support aquatic life uses. Unstable bottom substrate accounted for a majority of the low habitat scores of the degraded Bachelor Creek sites. Bottom sub- strate composition at reach 5 was 98-100% sand, silt, or clay, whereas the bot- tom substrate composition at the reference stream was 8-17% sand, silt, or clay. Ammonia levels were often high at reach 5, ranging from 0.06-2.87 mg/L. Sites on reach 5 were considered moderately impaired due to low EPT index (0-1) and taxa richness (4-12) values and high coefficient of community loss (2.25- 7.50) in comparison to the reference stream. Results suggest that stream in- vertebrate communities are highly correlated with impaired habitat conditions and elevated ammonia concentrations in the upper channelized reaches of Bachelor Creek.

Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 121

DISTRIBUTION, BREEDING PATTERNS, AND DEVELOPMENTAL PHYSIOLOGY OF SOME SOUTH DAKOTA TERRESTRIAL ISOPODS

J. Likness and L.G. Johnson Biology Department Augustana College Sioux Falls, SD 57197

ABSTRACT

This research focused on several current hypotheses regarding terrestrial isopod distributions, and an effort to relate distribution information to breed- ing phenology and developmental physiology. Jonathan Wright has made sev- eral proposals regarding terrestrial isopod distribution patterns in South Dako- ta and across the Great Plains (Wright, 1997a, 1997b). He hypothesizes that terrestrial isopods in South Dakota are strictly synanthropic, that is they are lim- ited to sites around present or past human habitations that provide the kind of niches (especially loose rubble heaps) which they need to survive temperature extremes and periods of low humidity. He also hypothesizes that because of the increased likelihood of introduction and greater availability of suitable habitats, more species of terrestrial isopods will be found in larger communi- ties than in smaller ones. We have made observations relevant to Wright’s synanthropy hypothesis. We found Trachelipus rathkei in a rural wood lot near Britton, and in Sica Hol- low State Park. Wright also reported T. rathkei along Spearfish Creek in the Black Hills (Wright, 1997b). We propose to seek more examples of remote populations of terrestrial isopods by examining several additional rural sites and State Parks. With regard to Wright’s small versus large community hy- pothesis, we have found Armadillidium vulgare in Rapid City (J. P. Likness). That species had previously been found only in Sioux Falls (by L. G. Johnson, see Wright 1997b). Since Rapid City is our second largest city, but is relative- ly remote from larger population centers, it would be interesting to see whether the several additional species found in Sioux Falls and Aberdeen are present there as well. We also plan to choose several smaller communities in Eastern South Dakota for further testing of Wright’s small versus large community hy- pothesis. Earlier studies of breeding phenology, indicate that breeding in Porcellio spinicornis occurs in waves separated by inter-breeding periods when embryos are not present (McQueen, 1976). However, we found that apparently contin- uous breeding of Trachelipus rathkei went on throughout the summer. Con- tinuous breeding could be either a special adaptation to habitats found in South Dakota or simply a difference between the species examined in the two studies. 122 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000)

Developmental rates of terrestrial isopods clearly are temperature depen- dent (e.g. Helden and Hassell, 1998), but there has been little research com- paring temperature tolerances of developing embryos with those of adults. Knowing these tolerances for terrestrial isopods could help to define their habi- tat limitations in South Dakota. Wright (1997b) has found that adult terrestrial isopods’ temperature tolerance ranges shift seasonally such that winter-accli- matized animals can tolerate slightly lower temperatures than can summer-ac- climatized ones with the opposite being true of higher temperatures. Are there such seasonal shifts in tolerance characteristic of embryos as well, or are there genetically fixed tolerance ranges for embryos that are limiting in determining the extent of each species’ breeding season? In experiments on temperature tolerance on Trachelipus rathkei embryos in mid-summer, 1999, we found that development proceeded normally at temperatures between 6º C and 30º C. We have yet to test higher temperature effects on summer acclimatized embryos and tolerance ranges of embryos collected during early spring breeding when the weather is much cooler. We propose to do such experiments during the coming season.

REFERENCES

Helden, A. J. and M Hassell (1998) Phenotypic plasticity in growth and devel- opment rates of Armadillidium vulgare (Isopoda: Oniscidea). Israel J. Zo- ol. 44:379-394. McQueen, D. J. (1976) Porcellio spinicornis Say (Isopoda) demography. II. A comparison between field and laboratory data. Can. J. Zool. 54: 825-842. Wright, J. C. (1997a) Pillbug fact sheet. HTTP://Lupus. Northern.edu:90/Nat- source/invert1/pillbu1.htm Wright, J. C. (1997b) Winter survival and overwintering behavior in South Dakota Oniscidea (Crustacea, Isopoda) In press: Proc. S. D. Acad. Sci. Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 123

INVERTEBRATE COMMUNITY CHARACTERISTICS IN RELATION TO ECOREGION, SHORELINE MANAGEMENT AND HABITAT IN EASTERN SOUTH DAKOTA LAKES

C.W. Braskamp and N.H. Troelstrup Jr. Department of Biology and Microbiology South Dakota State University Brookings, SD 57007

ABSTRACT

The objective of this effort was to define relationships between measures of invertebrate community structure, littoral zone habitats and shoreline charac- teristics. Littoral invertebrates, vegetative biomass, substrate texture, water tem- perature, conductance, dissolved oxygen, pH, shoreline slope, shoreline as- pect, canopy cover and shoreline land-use were sampled from 14 lakes within the Northern Glaciated Plains (NGP, n=8) and Northwestern Glaciated Plains (NWGP, n=6) ecoregions in eastern South Dakota. Five random sites were sam- pled from each basin during the growing seasons once in 1996 and twice in 1997-1999. Dissolved oxygen, cobble/boulder substrate and canopy-cover were higher in the NGP while water temperature, macrophyte biomass and shoreline vegetation densities were higher in NWGP. Swimmers and Corixidae were greater in NGP sites, while gliders, collector-filterers, shredders, and Gas- tropoda were more abundant in NWGP sites. Residential areas (41%) dominat- ed lake shorelines adjacent to NGP sites while game production areas (29%) were prevalent adjacent to NWGP sites. Water temperature, macrophyte biomass and fine sediments were positively correlated with invertebrate met- rics, while dissolved oxygen and shoreline vegetation densities were negative- ly correlated. Oligochaeta and engulfers were positively correlated with coarse substrate, while burrowers, collector-filterers, clingers, shredders, scrapers and Gastropoda were negatively correlated. Macroinvertebrate data were also found to vary between shoreline land-uses. Gliders, scrapers and Gastropoda were more abundant adjacent to game production areas compared to open grassland areas. Game production areas also harbored a greater abundance of gliders in adjacent littoral areas when compared to littoral areas adjacent to pastured areas. Oligochaeta and engulfers were found in greater abundance adjacent to residential areas compared to open grassland areas. Results of this effort lead toward development of invertebrate community signatures of lake shoreline condition. These signatures may be used to facilitate lake manage- ment efforts within an ecoregion context.

Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 125

IMPACTS OF LAND USE ON AQUATIC INVERTEBRATE ABUNDANCES AND THEIR CORRELATION TO NUTRIENT CONCENTRATIONS IN PRAIRIE POTHOLE WETLANDS

Brandon Jahnke and Diane Rickerl Plant Science Department South Dakota State University Brookings SD 57007

ABSTRACT

Many of the wetlands in the Prairie Pothole Region (PPR) of Eastern South Dakota occur on privately owned land and are subject to disturbances from dif- ferent land uses. The objectives of this study were to evaluate the effects of land use on invertebrate abundances in Prairie Pothole wetlands and to deter- mine the correlation between orthophosphate or nitrate-N and aquatic inverte- brate numbers. Eleven seasonal wetlands located on three different land management ar- eas (conventional and organic farming systems and a native prairie setting) in Eastern South Dakota were sampled from May through August during the sum- mer of 1998 and 1999. Wetland water samples were obtained using a one-liter glass jar covered with 2 ply medical gauze to strain out coarse particulate mat- ter. Water samples were only taken if wetland water depth was greater than 20 cm. Wetland water orthophosphate and nitrate-N concentrations were de- termined using a Hach DR2000 spectrophotometer. Wetland invertebrates were sampled using a benthic corer and sorted using a magnesium sulfate floatation method. Five taxa of invertebrates (Daphnidae, Helodidae, Sciomyzidae, Chironomidae Rhynchobdellae) exhibited significant differences due to conventional, organic and native prairie land use areas during the 1998 season. Three taxa of inver- tebrates (Tubificidae, Gyrinidae, Muscidae) exhibited significant differences due to the conventional, organic and native prairie land use areas during the 1999 season. Orthophosphate concentrations ranged from 0.01ppm to 1.71ppm in 1998 and 0.01ppm to 1.49ppm in 1999. Nitrate-N concentrations ranged from 0.0ppm to 1.8ppm in 1998 and 0.1ppm to 1.5ppm in 1999. Twelve taxa of invertebrates exhibited significant correlations with nutrients in the water column. The highest r2 values corresponding to orhtophosphate were (Gomphidae r2=0.78, Gyrinidae r2=0.75, Helodidae r2=0.59 and Daphnidae r2=0.47). Results indicated that land use did have a significant impact on wetland water nutrient concentrations and mean abundances for certain taxa of inver- tebrates. Results also indicated that significant correlations existed between wetland water nutrients and certain invertebrate taxa. Since most invertebrates 126 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) cannot directly absorb nutrients from the water column nutrient transfer must occur by other means. Significant positive correlations were probably a result of food web dynamics where nutrients are linked to primary producers and pri- mary consumers. Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 127

DETECTING INDICATORS OF POOR ABSORPTION OF COLOSTRAL ANTIBODY IN SOUTH DAKOTA BEEF CALVES

Amanda K. Weaver, William B. Epperson, Timothy A. Wittig, Richard J. Pruitt, Donald M. Marshall, and Alan R. Bender South Dakota State University Brookings, SD 57007

ABSTRACT

The purpose of this study was to identify factors that impact absorption of colostral antibody through passive transfer in South Dakota beef calves by measuring total protein levels, which closely correlate to colostral antibody ab- sorption. Calf data and blood samples were collected from 1235 calves at four South Dakota State University production sites from 1996 through 1999. In- formation was collected for the following variables: birthdate, days into the calving season before birth, time of birth, calf sex, calving ease, birthweight, twin or single birth, dam age, needing assistance nursing, precipitation in wa- ter on birthdate, minimum temperature on birthdate, illness, and death. Re- fractometry was used to measure total protein levels of the blood samples. T- tests showed calves with low total protein levels had an increased risk of ill- ness and death (p<0.001). After univariate analysis, a step-wise regression model was developed. The variables ranch, year, calf sex, calving ease, birth- weight, days into the calving season before birth, precipitation in water on birthdate, minimum temperature on birthdate, weather interaction variable, dam age, and dam age-squared were available for inclusion (n=1128). Twin births were excluded because they were outliers. The significance level of en- try was 0.2 and the significance level of exclusion was 0.1. The step-wise re- gression revealed that precipitation in water on birthdate, ranch, year, dam age, dam age-squared, and minimum temperature on birthdate significantly influ- enced total protein levels (p<0.02). The R2 value for the step-wise regression was 0.11, indicating measured factors did impact total protein, though other variables should also be considered in further studies.

Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 129

LOSSES ASSOCIATED WITH GASTROINTESTINAL NEMATODES IN YEARLING, STOCKER, GRASS CALVES

Keith Mertz, Bill Epperson, and Mike Hildreth Depts. of Veterinary Science and Biology/Microbiology South Dakota State University Brookings, SD 57007

ABSTRACT

In the competitive business of the beef cattle industry, it is becoming in- creasingly critical for producers to maximize profits where possible and like- wise to cut losses from their operations. Producers need to evaluate several factors, among which include genetics, nutritional requirements, environmen- tal factors, disease prevention, and parasite control. Gastrointestinal nema- todes are often an overlooked factor when producers consider making im- provements to their operation. Problems associated with gastrointestinal ne- matodes are less obvious because visible signs that animals are infected by the parasites don’t occur until clinical levels of infection are attained. At clinical levels, noticeable changes occur in cattle, such as loss of weight, bottle jaw, and a rough hair coat. At sub-clinical levels, the effects of the parasites can possibly lead to lowered weight gains, decreased rates of production, and re- duced immunity to various other diseases. Although sub-clinical nematodiasis is not visual, producers need to be aware of the losses due to the parasite. The objective of this project was to measure losses due to gastrointestinal nema- todes in yearling, stocker, grass calves. Six herds were utilized for this study over a three-year period (1997-1999). The herd dynamics for each trial of the study consisted of pasture groups of steers, or spayed heifers. The stocking densities for each pasture group varied as well as the overall pasture condi- tions. The experimental model for each trial was to randomly treat 10% of the members from each herd with 1 SR Ivomec bolus before release onto pasture for summer grazing. Another 10% of the herds were also randomly selected as paired controls. Both groups were weighed at the beginning and end of the study and average daily gains calculated. After controlling for confounders such as trial site, start weight, and sex, it was possible to combine all the data and perform a regression analysis on the control and treated cattle. The treat- ed animals outperformed the control animals in ADG by 0.1 lbs. (p=0.004). EPG were also calculated at the end of the study to observe the different amount of infections between the treated and control animals.

Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 131

EFFECTS OF MELANOCORTINS (MSH) AND AGOUTI PROTEIN ON MELANOGENESIS IN B16 MELANOMA CELLS

Partha Ramasastry and Nels H. Granholm Department of Biology and Microbiology South Dakota State University Brookings, SD 57007

ABSTRACT

Two fundamentally different kinds of receptor binding molecules attach to melanocortin receptors (MC1R) of pigment cells to regulate the synthesis of ei- ther black or yellow melanin (eu- or phaeomelanogenesis, respectively). Melanocyte Stimulating Hormone (MSH) acts as an agonist to stimulate while Agouti Protein (AP) acts antagonistically to inhibit cell signaling through the MC1R of pigment cells. The balance of these two peptides dictates whether black or yellow melanin is synthesized. Monoacetyl-alpha- Melanocyte Stimu- lating Hormone (monoac-aMSH), the melanogenically active MSH, is an acety- lated form of the nonacetylated POMC-derived immature molecule - desacetyl- alpha-MSH (desac-aMSH). The lethal yellow mouse (Ay/a) exhibits yellow pig- ment synthesis and yellow hair. Excess AP and/or abnormal levels of desac- aMSH may be causal in the synthesis of yellow pigment. We tested the hypothesis that desac-aMSH would induce a weak melanogenic response in B16 melanoma cells when compared to monoac- αMSH; if so, such a weakened response could result in yellow pigment syn- thesis. We also tested the hypothesis that AP acts antagonistically to monoac- αMSH; if so we would expect to see a graded decline in melanogenesis as AP is increased. B16 melanoma cells were cultured, treated with the three signal- ing peptides (two species of MSH and AP), harvested, and scored for parame- ters of cell signaling (synthesis of cyclic AMP) and melanogenesis—Tyrosine Hydroxylase (tritiated tyr assay) and Dopa Oxidase (Winder MBTH assay) ac- tivities of tyrosinase and total melanin (colorimetric assay) synthesis. Since de- sac-αMSH was comparable to monoac-αMSH in its ability to stimulate signal- ing and melanogenesis in all four assays, these data do not support the notion that an imbalance in the relative concentrations of the two species of αMSH is causal in the production of yellow coats in Ay/a mice. Furthermore, studies on the melanogenic effects of increasing concentrations of AP showed a dose-re- sponse effect, i.e., as the concentration of AP increased, melanogenesis de- creased in a stepwise fashion. Melanogenesis in B16 melanoma cells is up- regulated by the agonists desac- and monoac-αMSH and downregulated by AP. Somehow, AP signals B16 cells by: 1) modulation of MC1R, 2) interaction with another receptor like an AP receptor, 3) promoting another kind of signaling mechanism, or 4) various combinations of 1-3 resulting ultimately in the down- regulation of melanogenesis. Study supported by SDSU AES-SD86H, Eagles Ehrmann Fund and NIH (AR42757).

Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 133

MEASUREMENT OF ESTRONE AND PROGESTERONE IN MOUSE SERUM USING ENZYME LINKED IMMUNOASSAYS

Maureen Diggins and Julia Spiry Department of Biology Augustana College Sioux Falls, SD

Nels H. Granholm Department of Biology and Microbiology South Dakota State University Brookings, SD 57007

ABSTRACT

The lethal yellow mouse exhibits a mutation at the agouti locus on the sec- ond chromosome. In the homozygous condition (Ay/Ay), the mutation is lethal. However, in the heterozygous condition (Ay/a), the individual lives and exhibits a collection of characteristics known as the lethal yellow syndrome (LYS). The LYS includes a yellow coat color (due to the inability to make eu- melanin in the hair), decreased immunocompetence, increasing obesity with age, and reduced fertility. Our hypothesis is that the decreased fertility (decreased follicle stimulating hormone, decreased rates of ovulation, and decreased mating success) is due to a hormonal imbalance related to the obesity. Specifically, increased body fat results in increased serum levels of estrone. Estrone is a form of estrogen produced by fat cell alteration of an adrenal androgen. The estrone may ex- ert a negative feedback on the hypothalamic/pituitary complex, resulting in de- creased production of follicle stimulating hormone and decreased ovulation. Measurement of estrone in mouse serum was accomplished by adapting a protocol for an enzyme linked immunoassay developed for use with human serum. In addition, a protocol for measurement of progesterone in human serum by enzyme linked immunoassay was adapted for use with mouse serum. Elevated serum progesterone levels indicate pregnancy in the mouse and can be used in the assessment of fertility.

Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 135

COMPARISON OF NATIVE AND INTRODUCED PLANTS IN SELECTED FIELDS AND ROADSIDES IN TALL GRASS PRAIRIE IN OTTERTAIL COUNTY, MN

Donna Hazelwood Dakota State University Madison, SD 57042

ABSTRACT

Of the most economically important weeds in the prairie ecosystem, most are introduced plant species. Successful plant invaders share several charac- teristics including, but not limited to, rapid growth, flowering, and seed set un- der a wide range of temperature conditions and daylength, long range seed dispersal abilities, seed and or crop morphology mimicry, and a high popula- tion growth rate. In the northern great plains, the Native Americans were not agrarian, and tall grass prairie native plant species were adapted to soil that was essentially undisturbed. Settlers replaced the native plant species with crops and introduced agricultural practices. In addition, settlers imported, sometimes intentionally and often unintentionally, species that were well adapted to survival on land disturbed by agriculture. Several of these species became successful invaders to the detriment of native plants. The incidence and frequency of native and introduced plant species was recorded summer 1998 and 1999 for plants growing in sandy soils of the tall grass prairie region of Ottertail County, MN. Transects were set up in three farm fields that were abandoned about 30 years ago, and additional observations were made in a Christmas tree plantation, and along gravel roads and one county paved road- side. Contrasting color stakes were used to identify the location of grasses and forbs both years. The most common species in all locations with the excep- tion of the Christmas tree plantation, was the introduced species, smooth brome, Bromus inermis. In contrast, the most abundant plant in the Christmas tree plantation in June 1998 and 1999 was hoary alyssum Berteroa incanata, with smooth brome a close second. In the abandoned farm fields and along road sides, grasses outnumbered forbs with greater species diversity observed in fields that were further from the road and on roadsides that had not been subjected to reseeding or to construction efforts during this study. At each lo- cation, introduced species outnumbered native species. Native species detect- ed in 1998 were also detected in 1999 and additional species were evident in 1999. Spring 1999 experience greater rainfall than spring 1998. In conclusion, native species were outnumbered by invading plant species but in this study of the native species present, they appeared to survive year-to-year at the same location.

Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 137

SEED BIOLOGY OF HONEY LOCUST IN THE NORTHERN GREAT PLAINS

Soja Sekharan, Arvid Boe, Paul Johnson and Shannon Puckett Department of Plant Science South Dakota State University Brookings, SD 57007

ABSTRACT

Honey locust (Gleditsia triacanthos L.) grows naturally in diverse environ- ments from southern Texas to southeastern South Dakota and is commonly planted as an ornamental shade tree throughout and beyond its natural range. The foliage and green pods provides feed for cattle and deer, while many birds and mammals eat the ripe seeds. During winter 1999, we collected pods from several trees in South Dakota and Iowa. Our objective was to determine the relationship between seed po- sition in the pod and seed weight, seed positional seed predation by a bruchid beetle (Amblycerus robiniae) (Fabricius), and variation in seed predation across environments. For each tree, we removed seed from at least 10 randomly selected pods. Seed weight and predation were recorded according to the seed position in the pod. Individual seed weights ranged from 30 to 280 mg. Average seed weights of proximal, central and distal sections of the pod were 179.8, 181.8 and 178.2 mg, respectively. Seed predation did not appear to be related to seed position. The bruchid beetle appears to be bivoltine. About 75% of the beetles over-win- tered in the pods as larvae, pupae or adults. The seed weight-seed position data indicated that honey locust produced well- developed seeds in all seed positions. These data are interesting because hon- ey locust pods contain up to 30 seeds. Seed predation varied markedly across locations in the region with the highest infestations in southeastern South Dakota and Iowa.

Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 139

EVALUATING SITE-SPECIFIC POST EMERGENCE WEED CONTROL STRATEGIES

C.M. Cole, S.A. Clay, D.E. Clay, K.J. Dalsted, C.L. Reese and C.G. Carlson South Dakota State University Brookings, SD 57007

ABSTRACT

Site-specific weed management attempts to minimize the amount of herbi- cide applied by treating only localized areas of the field where weed species have been determined to exist at an economically detrimental level. Individu- al treatments can then be made to target species with specific herbicides. The presumed advantage, from a producer’s perspective, is that yield losses or tech- nology fees associated with additional scouting and subsequent precision ap- plications do not outweigh savings gained by the reduction of herbicide inputs. From an environmental perspective, lower inputs should reduce impacts of herbicides on soil, air, and water resources. This experiment was designed to compare the effectiveness of a traditional, uniform field application (chosen by the producer) with three site-specific post emergence weed management strategies: (i.) a combination of early and late post emergence applications, (ii.) early post emergence applications, and (iii.) late post emergence applications. Two study fields located at the Southeast Research Farm in Beresford, SD, were planted to corn, DK595TC at a seeding rate of 31,700/A on April 30 and May 1, 1999. Both fields were in a ridge-till system. Weed counts were taken every 50 feet (6 counts/plot) on the centerline of each plot using a 0.1 square-meter frame. Each site-specific treatment plot was divided into two equal subplots and scouted individually. Recommendations made were based by qualitatively estimating whether weed densities required a herbicide application. No partic- ular weed-density economic threshold was used in the decision making pro- cess. If assessed weed densities were greater than a pre-set value a herbicide recommendation was made to target only the weed specie(s) present. Site-spe- cific treatments did not receive the same herbicide application. Plots were har- vested October 1-October 8, 1999 with a commercial combine equipped with a yield monitor with DGPS signal correction. Yields were calculated using the GPS coordinates for each plot and a Geographic Information System. A cash market price of $2/ bushel and a site-specific technology fee of $5/ acre were used in determining profitability of weed management. The fee associated with the early plus late post treatment was doubled to account for two appli- cations during the season. It is also assumed that the producer has not incurred any additional capital expenditures such as Variable Rate Sprayers or GPS hard- ware and service. Yields in the conventional treatment (applied early post) were similar to the precision treatments that received only an early post appli- cation. Five plots did not receive any herbicide application during the growing season, and had significantly lower yields and returns than the other treat- 140 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) ments. The treatments that received only a late post treatment generally had lower yields and returns compared to the other treatments. Herbicide inputs for the 1999 growing season could have been cut in half without sacrificing yield when comparing the conventional uniform application to the early plus late post treatment. If technology premiums for precision herbicide applica- tions could be reduced or eliminated, more positive returns and environmen- tal impacts would favor a precision weed management technique. Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 141

USING SPECTRAL REFLECTANCE AND REMOTE SENSING TO COMPARE CROPS AND WEEDS

C. Poppen, S.A. Clay, D. Clay, K. Dalsted, C.G. Carlson, and M.M. Ellsbury South Dakota State University Brookings, SD 57007

ABSTRACT

Remote sensing is a tool that provides field-wide information. Multispec- tral broadband imaging, using band widths of 50 nm or wider, has been used to classify nutrient, weed, disease, and insect problems. The use of hyper- spectral data, using narrow bands with 2-10 nm in width, may allow for better discrimination between classes, especially if band widths are chosen where maximum differences occur in the spectrum. The objective of this study was to determine if crop and weed species have different spectral signatures. Each treatment was replicated three times and reflectance from greenhouse grown plants was measured at four dates. Reflectance range was from 300 to 1000 nm. The research showed that there were maximum differences between soybean and velvetleaf spectral signatures three weeks after planting. Differences oc- curred between 600 and 700 nm, (visible spectrum), and between 750 and 950 nm [near infrared spectrum (NIR)]. Later growth stages did not have many dif- ferences. Three weeks after planting, the maximum reflectance from soybean was about 55% between 550 and 600 nm and between 750 and 900 nm. The maximum reflectance from velvetleaf was 30% between 550 and 600 nm and 20% between 750 and 900 nm. The reflectance patterns changed with time so that by six weeks after planting, spectra were similar. While velvetleaf spectra remained similar to the 3 weeks data in the visible range, soybean reflectance in the 550 to 600 nm range decreased from 55 to 30%. Velvetleaf reflectance increased from 20 to 40% in the NIR range. Examining differences in every 10 nm segment from 600 to 680 nm indicated differences between specific ranges of wavelengths at specific dates. Between 660 and 670 nm the differences oc- curred up to 35 day after planting. A similar pattern occurred between 670 and 680 nm. The methods used for these two plant comparisons will be used for the rest of the experiment to compare other weeds and crops at several growth stages. This research indicated that there are differences between crop and weed spectral signatures at certain times of growth. The differences may be used to distinguish crop and weeds early in the season by using remote dig- ital information.

Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 143

DETERMINING CONDITIONS FOR GROWTH AND MAINTAINANCE OF USTILAGO MAYDIS UNDER LABORATORY CONDITIONS

Kazmann J. Foster and Donna Hazelwood Dakota State University Madison, SD 57042

ABSTRACT

Ustilago maydis, causal agent of common corn smut, is a warm tempera- ture wound pathogen that infects corn at all growth stages but is most harm- ful to young vigorously growing corn (Zea mays) sedlings. Growth require- ments include temperatures that range from 17-20 C to 30-25 C and better rates of growth hav ebeen reported at higher temperatures. In South Dakota, an in- crease in incidence and severity of infection in field corn was reported for fields in Lake County 1999 and may be correlated with hot dry weather. The long range prediction is for a hot dry summer 2000 and therefore U. maydis may lead to economic problems for growers in SD. Field corn and sweet corn are susceptible to infection. Because sweet corn is more susceptible than field corn, a commercially available cultivar ‘Early Sunglow’ was chosen for this study. The purpose of this study was to examine growth requirements of a field collected isolate of U. maydis, and to establish conditions for infecting greenhouse grown sweet corn seedlings. An isolate of U. maydis was collected from Lake County SD fall 1999 and a pure culture was selected and maintained on potato dextrose agar.. Growth of U. maydis was examined at five different temperature regimes ranging from 15 C to 34 C. The best growth occurred in a 34 C incubator. Inoculation of wounded ‘Early Sunglow’ corn seedlings involved and application of a liquid suspension of U. maydis incubated in potato dextrose broth . Corn seedlings were inoculated at two, three, and four leaf stages. My study selected a field collected isolate of U. maydis, established growth requirements under labora- tory conditions, determined a method for inoculating sweet corn, and will serve as the foundation for field studies of the epidemiology of corn smut sum- mer 2000.

Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 145

EVALUATING YIELD DIFFERENCES OF PHYTOPTHORA RESISTANT AND NON-RESISTANT SOYBEAN VARIETIES BY SOIL ELECTRO-CONDUCTIVITY ZONES

C.L. Reese, D.E. Clay and C.G. Carlson Plant Science Department South Dakota State University Brookings, SD 57007

ABSTRACT

Phytophthora megaspermavar. sojae is a fungus that causes seedling and root rots of soybeans. The fungus is indigenous to all soils. The occurrence and intensity of the disease are strongly related to environment and soil/land- scape position. The disease is more prevalent on poorly drained soils, partic- ularly when the soils are water saturated for several days prior to soybean emergence. The seed industry has recognized that some soybeans are more tolerant of Phytophthora and have developed Phytophthora tolerant cultivars. The hypothesis of this experiment was that Phytophthora tolerant (PRR+) vari- eties would yield higher in poorly drained field areas whereas Phytophthora non-tolerant (PRR-) varieties would be more productive on well-drained areas. Soil EC was evaluated as tool to identify soybean variety management zones. Research was conducted at 2 fields in Eastern South Dakota, named the Auro- ra field and the Beresford field. The PRR+ and PRR- soybean varieties were planted in spring 1998. Yield data was collected from both fields in fall 1998 with a differential global positioning system and yield monitor system. Soil electrical conductivity data was collected in fall 1999. Average yield data for each variety by soil EC management zone area was collected using SSToolbox (SST Development Group). The data was imported into a spreadsheet and the difference between variety (DIFF = (PRR-) – (PRR+)) by EC zone was calculat- ed. A regression analysis between yield difference and soil EC was complet- ed. At the Aurora field, a relationship between EC and soybean yield showed that the PRR+ soybean had higher yields when soil EC > 15 mS/m and the PRR- soybean had higher yields when soil EC < 16 mS/m. At Beresford, the PRR+ yielded better than the PRR+ variety when soil EC < 45 mS/m. These results show that planting Phytophthora tolerant varieties in low, wet field areas with low soil EC have the potential to increase yield.

Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 147

THE INFLUENCE OF HABITAT ON SPECIES RICHNESS AND ABUNDANCE OF WETLAND BIRDS AT ORDWAY PRAIRIE, SOUTH DAKOTA

Mary C. Miller and Lester D. Flake South Dakota State University Brookings, SD 57007

ABSTRACT

The influence of glacial wetland habitat on wetland bird abundance and species richness at Samuel H. Ordway Prairie, in McPherson Co., South Dako- ta was investigated during 1998 and 1999. Four bird surveys were conducted from mid-May through mid-August. Habitat was quantified by collecting data on five general wetland characteristics including wetland classification, emer- gent cover, submergent vegetation, shoreline, and upland vegetation. Stepwise multiple and logistic regressions were used to investigate wetland characteris- tics associated with abundance, species richness, and presence of wetland bird species. Surface water area explained 45% (P = 0.0001) of the variation in to- tal abundance. Species richness was associated with surface water area and negatively associated with the percent of the wetland covered by emergent vegetation. These two variables combined explained 38% (P = 0.0001) of the variation in species richness. Surface water area was the main variable associ- ated with the presence of the three most abundant waterfowl species, Blue- wing teal (Anas discors), Gadwall (Anas strepera), and Mallard (Anas platyrhyncos). The amount of shoreline area containing vegetation greater than 15 cm in height was negatively related to the presence of migratory shore- birds. The increased amount and height of shoreline vegetation in 1999 with the subsequent reduction in bare mud area seems to be related to a reduction in migratory shorebirds present during the 1999 surveys. In 1998, the Virginia Rail (Rallus limicola) was not detected in any surveys. Results show this may be related to the reduced height and abundance of emergent vegetation avail- able in 1998. This baseline information illustrates how wetland birds at Ord- way Prairie might be influenced by wetland characteristics, management, and environmental conditions.

Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 149

CHROMOSOME ARM DOSAGE EFFECTS ON THE EXPRESSION OF THE HIGH MOLECULAR WEIGHT GLUTENIN PROTEINS OF WHEAT

Jill D. Kruse, Jesse D. Munkvold, Erin E. Lee, Elizabeth A. Hamann, Ellen J. Hamann, and Michael K. Wanous Department of Biology Augustana College Sioux Falls, SD 57197

ABSTRACT

Baking quality of bread wheat, Triticum aestivum L. is determined by gluten, a mixture of more than 50 proteins. The important qualities of gluten are contributed largely by the high molecular weight (HMW) glutenins, en- coded on the long arm of the group 1 chromosomes 1AL, 1BL, and 1DL. The goal of this study was to identify and locate to specific chromosome arms, genes that modify the expression of the structural genes for HMW glutenins. Using cytogenetic stocks, a dosage series for as many as possible of the 42 chromosome arms of wheat was assembled, generally varying from zero to three doses in the endosperm. For each dosage series, expression of specific HMW glutenin bands was measured on SDS-PAGE gels, using an imaging den- sitometer. An increase in the intensity of a specific band indicates a dosage ef- fect for that chromosome arm, on the respective structural gene protein sub- unit. A dosage effect for a chromosome arm which does not contain the struc- tural gene indicates the presence of a modifier locus. Modifiers for the seed storage proteins may be found that regulate expression either in the positive (enhancer) or negative (repressor) direction. Significant effects on protein band D1 were found for the following chromosome arms: 1AL, 1BL, 1DS, 1DL, 2BS, 2DL, 3BS, 4AS, 5AS, 5BS, 5DS, 6AL, 6BS, 7AL, 7BS, 7BL. Significant ef- fects on protein band B2 were found for the following chromosome arms: 1BL, 1BS, 1DL, 2AL, 2BS, 2DL, 5AS, 5BS, 6AL, 6DS, 6DL, 7AL, 7BS, 7DL. Sig- nificant effects on protein band B10 were found on the following chromosome arms: 1AS, 1AL, 1BL, 1DS, 1DL, 2BS, 2DL, 3DL, 4AS, 5AS, 5BS, 5DS, 6AL, 7AL, 7BS. Significant effects on protein band D5 were found on the following chro- mosome arms: 1AL, 1BL, 1DS, 1DL, 2AL, 2BS, 2DS, 2DL, 4AS, 5AS, 5BS, 6AL, 6BS, 6DS, 6DL, 7AS, 7AL, 7BS, 7BL, 7DL. This research should lead to the iden- tification of genes modifying wheat quality, which may ultimately be used in development of improved cultivars. Support for this research was provided by the South Dakota Wheat Commission, the USDA NRICGP, and the NSF.

Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 151

CARBON ISOTOPE DISCRIMINATION AS A MARKER FOR HEAT-TOLERANCE IN WHEAT

Kara J. Fuehrer, Steven R. Moeckly, Michael E. Chapman, and Michael K. Wanous Department of Biology Augustana College Sioux Falls, SD 57197

Patrick F. Byrne Department of Soil & Crop Sciences Colorado State University Ft. Collins, CO 80523

ABSTRACT

Carbon isotope discrimination is correlated with water use efficiency

(WUE) in C-3 plants. The basis for this relationship is that CO2 uptake and wa- ter vapor loss occur through the same channel: the stomates. Atmospheric 12 13 CO2 contains approximately 99% C and 1% C. When water is not limiting, stomates remain open and the concentration of CO2 in leaf tissue remains high. 13 CO2 containing C is discriminated against during carbon fixation due to its slightly higher molecular weight than the more abundant 12C. When water is limiting, stomates close to prevent water loss, and internal CO2 concentration 13 decreases. Under these conditions discrimination against CO2 is diminished. Thus, the proportion of 13C in plant tissues represents a record of WUE inte- grated over the life of the plant. δ13C is a measure of 13C discrimination. Heat- tolerant and heat-susceptible wheat cultivars were grown under heat-stress conditions in the field. Tissue samples were analyzed using an isotope ratio mass spectrometer. A Fisher’s protected LSD test showed that the three heat- tolerant cultivars, Kauz, Glennson 82, and Seri 82 grouped together and had significantly lower δ13C (lower WUE) than the heat-susceptible cultivar MTR-

WA116. 34 F2 families of a cross between Kauz and MTRWA116, along with the parents themselves, were also examined. Kauz had the lowest δ13C and there δ13 were only two F2 families with C higher than MTRWA116, with the remain- ing F2 families falling between the two parents. This observation was consis- tent with the hypothesis that genes controlling δ13C were segregating in the population. We also found that δ13C was negatively correlated with yield (r=- 0.37) and kernel number (r=-0.69). Under heat-stress conditions in the field, WUE as indicated by δ13C was negatively correlated with yield. This probably indicates that under stress conditions, heat-tolerant plants are able to continue transpiration, and thus lose more water than heat-susceptible plants, due to physiological mechanisms such as deeper rooting, epicuticular wax load, or leaf architecture. Thus, δ13C may be a valuable marker for selection of field heat-tolerance in wheat.

Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 153

A MATHEMATICAL MODEL OF TWO COMPETING SPECIES IN A SIMPLE CHEMOSTAT WHEN THE GROWTH FUNCTION OF EACH SPECIES IS OF CONTINUED FRACTION FORM

Melissa A. Wittgenstein and A.S. Elkhader Department of Mathematics Northern State University Aberdeen, SD 57401

ABSTRACT

A chemostat is laboratory device used in ecological studies of microorgan- isms. A simple mathematical model for two microorganisms competing for the same limiting resource in a chemostat is considered. The model consists of three ordinary differential equations of nonlinear type. The system has four pa- rameters: The washout rate, the input concentration, the maximum growth rate and the half saturation rate. It had been shown that coexistence is not possi- ble when these parameters are constant and when the growth functions are of Michaelis-Menten form or other monotone forms. In this work, we studied a modified chemostat model when the growth and consumption functions of each species are allowed to be of continued fraction form. We used the math- ematical software PHASER to study the chemostat model under these modifi- cations. The study has shown that coexistence is not possible under the new modifications.

Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 155

EDB CONTAMINANT PLUME AT NEMO, SOUTH DAKOTA

Perry H. Rahn and Christopher S. Johnson Department of Geology & Geological Engineering South Dakota School of Mines & Technology Rapid City, SD 57701

ABSTRACT

In 1996 low concentrations of ethylene dibromide (EDB) were found in water wells at Nemo, South Dakota. EnviroSearch International, a consultant hired by the U.S. Forest Service, reported concentrations up to 13 ug/L in do- mestic wells. The source of the contaminant has not been firmly documented, but is believed to be the result of left-over pesticides disposed at the USFS Work Station area in the 1970’s. EDB was detected in nine domestic wells. Monitoring wells were installed. Sixteen additional wells within 3 km of Nemo were sampled but EDB was not detected. Water in two wells 1 km south- southeast of town (Kaberna and Weston residences) had high concentrations: 13 and 2.2 ug/L EDB respectively. The impacted landowners were initially pro- vided bottled water by the USFS. The USFS then drilled new wells outside of the contaminant plume and now provide water to the school and impacted landowners. A map showing concentrations of EDB shows a plume extending towards the south-southeast. This direction is the same as the jointing in the Precambrian schist and quartzite found at Nemo. This structural anisotropy strongly influences the direction of contaminant migration.

Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 157

DIRECT ELECTROCHEMICAL OXIDATION OF AMINO ACIDS AT DIAMOND THIN-FILM ELECTRODES

Eric Hammond and Miles Koppang Department of Chemistry University of South Dakota Vermillion, SD 57069

Greg M. Swain Department of Chemistry and Biochemistry Utah State University Logan, UT 84322

ABSTRACT

Of the 20 amino acids found in proteins, only tyrosine, and tryptophan and cysteine can be electrochemically oxidized in aqueous solutions at conven- tional carbon-based electrodes such as glassy carbon. As such, liquid chro- matographic analysis with electrochemical detection (LCEC) of amino acids re- quires precolumn derivatization with an electrochemically active tag. Recent- ly, electrode surfaces capable of catalytic oxygen transfer have been developed allowing for amino acid oxidation without derivatization. We have studied the unique properties of diamond thin-film electrodes for use in electroanalysis. These materials are especially attractive for electroanalysis due to their wide working potential window for aqueous solvents, low background currents and increased signal to background ratios as compared to conventional carbon- based electrodes. In prior studies, we observed direct oxidation at diamond electrodes of aliphatic polyamines including ethylenediamine, putrescine, ca- daverine, spermine and spermidine without derivatization. Using cyclic voltammetry, well-resolved cyclic voltammetric oxidation waves, with respect to the background signal, were observed for all five polyamines at the 1 mM level with E1/2’s of ~+0.9 V vs. Ag/AgCl. A reaction mechanism for aliphatic polyamine oxidation was proposed whereby the polyamine adsorbs at surface boron-sites, OH• is generated at nearby non-diamond carbon impurity sites, the radical attacks the coordinated amine followed by slow desorption of the oxidization product. We have observed similar voltammetric results for 15 of the 20 amino acids. All 15 amino acids produced oxidation waves with E1/2’s of ~+0.9 V vs. Ag/AgCl. The invariance of oxidation potential with changes in amino acid structure is consistent with an electrode reaction mechanism where- by oxidation of the amino acid is controlled by surface oxidation of hydroxide to hydroxy radical (OH∑). The hydroxy radical then oxidizes the amino acid which is bound to the surface through coordination of the amine functionality to surface boron-sites. By varying carbon to hydrogen ratios during film prepa- 158 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) rations, the levels of non-diamond impurities can be varied. Electrode surfaces, prepared using conditions to maximize the amount of non-diamond carbon on the surface, produced the largest oxidation signals for amino acids. Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 159

EXPERIENCES IN CREATING AN INTERNET CHEMISTRY COURSE AND STUDENTS’ ATTITUDES TOWARDS THE COURSE

Dwayne W. Rehfeld Northern State University Aberdeen, SD

ABSTRACT

The delivery of information to students and the approach to teaching has been undergoing tremendous changes in the past few years with the Internet becoming so easily accessible. There is also becoming a growing population of students who would prefer an asynchronous approach to course material. Thus a one-semester non-science major chemistry course was developed using WebCT, an authoring tool for placing course material on the Internet, and ChemSkill Foundations electronic homework. The WebCT chemistry course consisted of the following: a) a brief outline of topics that students were ex- pected to know and understand, b) a calendar to list class activities and as- signment due dates, c) the course quizzes and exams, d) a bulletin board for student discussions and announcements, e) student grades and f) a private e- mail system for members enrolled in the course. The exam portion of WebCT permitted random generation of numbers for mathematical problems and per- mitted random selection of exam questions so each student received a unique examination. For drill and practice type of assignments, WebCT allowed stu- dents to repeat the activities to perfect their skills and knowledge. The learn- ing curve for utilizing WebCT to create the course was relatively short. The newly designed WebCT chemistry course was taught for four semesters and one summer session. The first semester the newly developed course was taught, it was taught as an asynchronous course. The class period was strictly used as a help session for those students having difficulty with the assignments and thus attendance was not required. Of the 120 students enrolled in the class, 20 usually attended the class period help session. The students com- plained they had to learn the material on their own and they preferred a “lec- ture” course. The students did like the flexibility of no deadlines for assign- ments. This feature also removed from the instructor the decision of whether an excuse for late material was valid. Each succeeding semester the course was modified to include more “lecture” and more credit was given for in-class activities. Class attendance improved so attendance averaged about 90 stu- dents. Most students liked the Internet course and found the ChemSkill Foun- dations electronic homework helpful. The laboratory portion of the course was required of all students and was performed in the traditional manner. The laboratory made use of computer based labs (CBL) including graphing and t- test analysis.

Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 161

WORKING WITH DIHYDROGENATEDTALLOW DIMETHYL AMMONIUM METHYLCARBONATE

Jenny Bjergaard and Dr. Gary Earl Augustana College Sioux Falls, SD

ABSTRACT

One project goal for this research included determination of the critical mi- celle concentration of the surfactant dihydrogenatedtallow dimethyl ammoni- um methyl carbonate using laser light scattering. The light scattering was mea- sured at an angle of 900 for solutions ranging in concentration from approxi- mately 5E-05M to 1.0M. A critical micelle concentration (CMC) was found at 1.4E-02M. Furthermore, data indicated that a second critical micelle concen- tration may be present at approximately 1E-05M. However, future research is needed to confirm or disprove the existence of this CMC. Another project goal for this research was the development of chromate and molybdate quaternar- ies from dihydrogenatedtallow dimethyl ammonium methyl carbonate using anion exchange. FT-IR spectroscopy was used to confirm the formation of these compounds. However, further research is needed to determine the best method of preparation and to determine the physical properties of these new compounds.

Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 163

SYNTHESIS OF DIMETHYL DIHYDROGENATEDTALLOW AMMONIUM COMPOUNDS EXHIBITING AROMATIC ANIONS

Steven R. Moeckly and Gary W. Earl Department of Chemistry Augustana College Sioux Falls, SD 57197

ABSTRACT

Previous research under the NSF/EPA grant referrenced below resulted in the synthesis of dimethyl dihydrogenatedtallow ammonium methylcarbonate under moderate conditions. This synthesis involved the use of relatively harm- less reagents compared to those currently used in the industrial syntheses of similar quaternary ammonium compounds. In terms of novel synthesis, it would be desirable if one would could completely convert this weakly basic quaternary ammonium compound to its highly basic hydroxide analog so that simple strong base/(weak or strong) acid reactions could be performed to quantitatively produce the quaternary ammonium salt of the acid. Moreover, it would be even more highly desirable if this process was simple and inex- pensive. The quite involved and expensive contemporary methods for pro- ducing quaternary ammonium compound derivatives of varying anions are salt precipitation reactions involving silver, electrolytic reactions, and ion exchange reactions via an ion exchange resin. Therefore, research was undertaken to implement a simple and cost-effective procedure resulting in quantitative yields which could be adapted to produce quaternary ammonium compounds with various different anions. Simple, effective, and time-efficient methods for the production and anal- ysis of dimethyl dihydrogenatedtallow ammonium hydroxide, and its benzoate and p-toluate analogs were devised. It was found that the quaternary ammo- nium methylcarbonate could be converted to its hydroxide analog in a two- phase alcohol/water system. Sulfamic acid titration of the quaternary ammo- nium hydroxide product confirmed over 90% completion of reaction. The qua- ternary hydroxide was then reacted with either benzoic acid or p-toluic acid to produce the corresponding benzoate and p-toluate analogs, respectively. Pro- ton NMR of these products confirmed high product purity.

ACKNOWLEDGEMENTS

The National Science Foundation and Environmental Protection Agency, NSF/EPA grant CHE 9613052, Witco Chemical Company, and Augustana Col- lege.

Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 165

SYNTHESIS OF A BIODEGRADABLE SURFACTANT FROM STARCH

Levi M. Stanley and Gary W. Earl Department of Chemistry Augustana College Sioux Falls, SD 57197

ABSTRACT

This research involved the initial steps in the synthesis of a biodegradable surfactant molecule based on starch. The emphasis was on synthesizing a more biodegradable molecule than that which is currently used in chemical in- dustry. The research included the processes for synthesizing: ethylene glycol glycosides(EGG), a butylene oxide(BO) adduct, and a lauroyl chloride adduct. The final product should be a di or tri-saccharide with hydroxybutyl ethers and lauric esters. The EGG synthesis, outlined in Tenside Detergents. “New Biodegradable Surfactants Derived from Starch.” P. E. Throckmorton et. al. January/February. 1973., was monitored for completion by gas chromatography, using the disap- pearance of ethylene glycol as an indicator of completion. The reaction was analyzed using an FT-IR spectrometer and the Gardner color scale. The BO adduct synthesis was also monitored by gas chromatography. The

BO adduct was analyzed by FT-IR spectra for the presence of a CH3 stretch. The water solubility was tested and found to be water soluble. The lauric ester synthesis was performed to develop a more acceptable “hydrophilic hydrophobic” balance for the molecule. The lauroyl chloride adduct was analyzed by FT-IR for the presence of a CH2 stretch of increased intensity and for a C=O stretch. The lauroyl chloride adduct was found to form an emulsion in water. Further reactions with epichlorohydrin and triethyl amine were considered and provide a possibility for the quaternization of the lauroyl chloride adduct.

ACKNOWLEDGMENTS

This research was carried out at Augustana College thanks to NSF/EPA grant #CHE 9613052 and ARAF.

Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 167

SYNTHESIS OF QUATERNARY AMMONIUM COMPOUNDS FROM 2-AMINOGLUCOSE

John D. Gilbertson and Gary Earl Department of Chemistry Augustana College Sioux Falls, SD 57197

ABSTRACT

This research dealt with the development a method for preparing quater- nary ammonium compounds from 2-aminoglucose, which is a natural molecule found in the exoskeletons of crayfish, lobsters, insects, etc. These quaternary ammonium compounds are potentially more biodegradable than the ones used in chemical industry today. The method involved placement of a protecting group, such as anisaldehyde, on the nitrogen of the amino group, producing an imine, via a procedure outlined in J. Chem. Ed., vol 76, no.1, Jan. 1999. The protected 2-aminoglucose ring was then treated with a 2:1 ratio of acid chlo- ride to substitute the ring with 2 C12 ester groups and remove the protecting group. The nitrogen was then quaternized with a 3:1 ratio of ethyl iodide. The reactions all proceeded at room temperature to protect the integrity of the monomer. The product from the “protecting” step was analyzed 60 MHz H1 FT-NMR looking for the 4 to 1 integration ratio of the benzene ring:imino group H’s. The product from the esterification was analyzed for the C=O peak using a KBr pellet on a Nicolet FTIR. Due to the placement of the C=O peak at 1700cm-1, it was indicative that the esterification was a success. The 1o and 2o H’s of the

C12 ester groups were also confirmed by the FT-NMR. The product from the esterification step was also analyzed by using pH and a Cl- test. The positive Cl- test and basic pH suggested that the product was actually the tertiary amine salt. Once the esterified 2-aminoglucose salt was quaternized, it was analyzed in the FT-NMR. The presence of the quaternary ammonium peak from 3-4 ppm suggested that the quaternization was a success. pH and I- tests were al- so used to confirm identity. Conductance measurements of the Adogen 442 quat(dimethyl ditallow methyl carbonate quaternary ammonium compound) were also made to at- tempt to find the Critical Micelle Concentration(CMC). The equivalent con- ductance was plotted vs. concentration to analyze for breaks in the curve. A maximum was obtained at ~9E-05M. This maximum is indicative of the CMC.

ACKNOWLEDGMENTS

This research was carried out at Augustana College thanks to NSF/EPA grant #CHE 9613052 and ARAF.

Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 169

VIRTUAL GEOLOGY FIELD TRIPS OF SOUTH DAKOTA

Howard J. Woodard and Mike Hildreth South Dakota State University Brookings SD

ABSTRACT

A website “Virtual Geology Field Trips of South Dakota” has been con- structed to display images of geologic formations and natural landscape fea- tures of South Dakota in one of three regions of the state: eastern, central-west- ern, and the Black Hills. In addition, the site depicts fossils, aspects of eco- nomic geology, environmental issues, and features impacted by the processes of glaciation and erosion. To access the images, topics are previewed by ex- amining text descriptions of one of three regions. Over 200 digital images are embedded in the text that includes explanations and locations of the picture. Maps, graphs, and other associated web sites are linked to the text. Digital im- ages can also be referenced by accessing a regional tour map with symbols as- sociated with a particular feature. The web site is useful for students pre- viewing aspects of the field trip course (PS-244) offered by SDSU, and as a sub- stitute for parts of this field trip that are jeopardized because of inclement weather. This teaching tool will also enhance the field experience of students in other courses and should increase their appreciation for the beauty and nat- ural resources of the state. The WWW URL of the site is: http://www.abs.sd- state.edu/plantsci/nat_res_field_trips/index.htm. The site is accessible to the public and is an excellent tool to demonstrate images of geography, natural re- sources, and ecology to primary and secondary school students throughout the state.

Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 171

ISOLATION, CLONING, AND SEQUENCING OF THE PORCINE AGOUTI-RELATED PROTEIN GENE (agrp)

Michael Halverson, Nels Granholm and Carl Westby Department of Biology/Microbiology and Donald Marshall Department of Animal and Range Sciences South Dakota State University Brookings, SD 57007

Agouti-related protein (AGRP), an orexigenic agent and an antagonist of hypothalamic melanocortin receptors (MC3-R and MC4-R) is a naturally occur- ring neuropeptide that regulates overall feeding behavior and weight home- ostasis of mammals. In mice, AGRP and agouti protein (AP) act as ligands for melanocortin receptors on hypothalamic neurons (MC4-R) and melanocytes (MC1-R), respectively. The way in which AGRP interacts with MC4-R is strik- ingly similar to the interaction of AP with MC1-R within murine hair bulb melanocytes. We isolated, subcloned, and sequenced the ORF of the porcine agrp gene (GenBank Accession No. AF177762) using PCR primers designed from bovine and human agrp cDNA sequences. A BLAST search of the ampli- fied putative porcine agrp nucleotide sequence of 978 base pairs revealed strik- ing similarity to human, cow, and mouse agrp genes. These data along with sequence analysis using the computer software Gene Jockey and Gene In- spector suggest that we have isolated an authentic porcine agrp homologue. Continued sequence analysis of nucleotides of the porcine agrp gene may re- veal breed-specific polymorphisms important in feeding behavior, feed effi- ciency, carcass quality, and energy metabolism.

Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 173

BIOSYSTEMATIC EVALUATION OF SOUTH DAKOTA POPULATIONS OF BECKWITH’S CLOVER USING RAPDS

Jeffrey D. Noll and Gary E. Larson Department of Biology and Microbiology South Dakota State University Brookings, SD 57007

Melvin R. Duvall Department of Biological Sciences Northern Illinois University DeKalb, IL 60115

ABSTRACT

Beckwith’s clover (Trifolium beckwithii Brewer ex S. Wats.) is a low grow- ing rhizomatous perennial clover endemic to the western United States. It typ- ically occurs in moist streamside meadows at elevations of 1,200 to 2,125 me- ters above mean sea level (msl) (Gillet, 1972). Unexpectedly, at least five dis- junct populations of T. beckwithii also occur in extreme eastern South Dakota in moist tallgrass prairies located in the upper Big Sioux River watershed. These sites are only 500 to 600 meters above msl and are about 1200 km east of the nearest populations which are in southwestern Montana. The earliest report of Beckwith’s clover in South Dakota extends back over 100 years (Saunders, 1899). Random Amplified Polymorphic DNA (RAPD) profiles from T. beckwithii were compared between South Dakota, Idaho, and northern Cal- ifornia in an attempt to evaluate the biosystematics of Beckwith’s clover in South Dakota. Five individuals from each one of the South Dakota, Idaho, and northern California populations were sampled and analyzed through RAPD technology. Total cellular DNA was extracted from each accession, combined with a ten-base primer, and subjected to PCR which amplified random genomic fragments. These fragments were separated electrophoretically on 0.8% agarose gels, stained in ethidium bromide, visualized under short wavelength UV, and photographed. Photographed bands were scored as binary pres- ence/absence data and Jaccard coefficients were calculated for each pairwise comparison of individuals and populations as a measure of genetic similarity. Unweighted pair group method of analysis (UPGMA) cluster analysis was per- formed on the binary data as implemented in PAUP*4.0 (Swofford, 1998). Mean genetic measures among the three populations displayed a pattern indi- cating the presence of Beckwith’s clover in South Dakota may be the result of a long distance dispersal event from northern California. The South Dakota and northern California populations displayed over twice the genetic similari- ty of the South Dakota and Idaho populations (Anova; P<0.001). Additional- ly, the South Dakota accessions clustered with the northern California acces- 174 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) sions in UPGMA. These data appear to support an original dispersal event, sometime prior to 1899, from a parent population genetically similar to the one sampled in northern California.

LITERATURE CITED

Gillet, J. 1972. Taxonomy of Trifolium (Leguminosae). IV. The American species of section Lupinaster (Adanson) Seringe. Canadian Journal of Botany 50: 1975-2007. Saunders, D. A. 1899. Ferns and flowering plants of South Dakota. Experi- ment Station Bulletin 64, South Dakota Agricultural College, Brookings, SD. Swofford, D. L. 1998. PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods). Version 4. Sinauer Associates, Sunderland, Mas- sachusetts. Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 175

NATIVE PLANTS OF THE NORTHERN GREAT PLAINS: ETHYLENE INDUCTION OF ASTERACEAE SEED GERMINATION

April Stahnke and R. Neil Reese Biology/Microbiology Department South Dakota State University Brookings, SD 57007

As part of our efforts to develop alternative agricultural systems utilizing plants native to the Northern Great Plains, methods for overcoming seed dor- mancy are being tested. Seeds of Achillea millefolium L., Antennaria neglecta Greene, Chrysopsis villosa (Pursh) Nutt., Echinacea angustifolia DC, He- lianthus maximilianii Schrad, Liatris punctata Hook., Ratibida columnifera (Nutt.) Woot. & Standl., and Townsendia exscapa (Richards.) Porter were col- lected from several populations within the state of South Dakota. Initial ger- mination tests showed varying degrees of seed dormancy between populations and species. Liatris, Achillea, Echinacea and Ratibida showed germination rates of ~10 to 20 %, while the other four species showed almost no germina- tion without extensive periods of stratification. Seed source and seed maturi- ty have been shown to significantly affect germination rates. Therefore, only well developed seeds (relatively high dry weight) taken from flower heads that readily released the seeds, were used for this experiment. To improve germi- nation without months of stratification, the seeds were treated using the ethep- hon protocol developed for Echinacea (Feghahati and Reese, 1994). Briefly, the seeds were exposed to two weeks of continuous light (40 W cool white fluorescent tubes), in the cold (5 C), and in the presence of the phytohormone ethylene, applied as ethephon (1mM). This treatment has been shown to elim- inate the need for months of stratification. Germination was significantly in- creased for all seed sources (with final germination ranging from ~30 to 85%) except for those of Liatris and Ratibida, which showed no improvement. Tetrazolium tests were conducted to allow calculation of germination rates of viable seeds. The results indicated that the variability in the increased rates of germination, in response to the ethephon treatment, reflected the variability in seed viability and that germination of live seeds ranged between 70- 85%. Fur- thermore, the lack of response by Liatris and Ratibida seeds to the treatment reflected the fact that most of the viable seeds germinated without any need of stratification or ethephon treatment. Factors affecting seedling emergence from soil were also examined during these experiments. Preliminary observation suggest that many of these factors may need study to ensure the success of agronomic protocols for successful plant production.

Feghahati and Reese (1994). J. Amer. Soc. Hort. Sci. 119(4):853-858.

Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 177

INHERITANCE OF GLUME LENGTH IN OATS

Mei Shen and Dale Reeves Plant Science Department South Dakota State University Brookings, SD 57007

ABSTRACT

Glume length is one of the most obvious panicle characteristics. Modern oat varieties are usually associated with short glumes. However, there are few reports about the inheritance of glume length. The objectives of this experi- ment were to determine the genetic pattern of oat glume length and to esti- mate simple correlation coefficency of glume length to groat length, kernel weight, panicle grain weight, plant height, and heading days. Seven selections or lines with long or short glumes were used to make five reciprocal and two single crosses. Data were collected from F1 in greenhouse and F2 in fields of Brookings, SD and Aberdeen, SD. The results showed there was no cytoplas- mic and environment effects on the inheritance of glume length. The distri- bution of glume length in F2 is close to normal distribution. Chi-square test indicated glume length is controlled by more than three nuclear loci. There was no significant correlation between glume length and tested traits.

Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 179

PRODUCTION OF CELL WALL DEFICIENT FORMS (L-FORMS) FROM SELECTED BACILLUS STRAINS

Nichole Baye, YongMei Luo, and Bruce Bleakley Biology/Microbiology Department South Dakota State University Brookings, SD 57007

ABSTRACT

The relationship formed between bacterial L-forms and green plants was relatively unheard of until research by A.M. Paton was published several years ago. Paton and others at the University of Aberdeen in Scotland showed that stable cell-wall-deficient (CWD) cells (L-forms) of Bacillus strains may be in- troduced into and remain viable in plants. Theoretically, the small CWD bac- terial cells may readily pass through the plant’s cell membrane and remain healthy in the osmotically stable cytoplasm. The CWD bacterial cells are in- troduced into the seed of the plant. This is done by soaking the seeds in a sus- pension of healthy L-forms at pre-radicle emergence. This relationship be- tween plants and bacteria has been observed in many cases. For this experi- ment Russ spring wheat and four native strains of bacteria were used. Soil microbes from South Dakota wheat foliage, or residue, which antago- nize two fungal pathogens of wheat—Pyrenophora tritici-repentis, and Fusar- ium graminearum—were used. These four Bacillus strains were designated 1BE, 1BA, 1BC, and 1D3. The 1B strains have uncertain species affiliation. The 1D3 strain is Bacillus amyloliquifaciens. All four strains are Gram positive rods. L-Phase Medium (LPM) was used to induce the L-form. Cultures were maintained by weekly sub-cultures. Before sub-culturing, cells were studied with a phase-contrast microscope at 1000x magnification. During the fifth and sixth weeks photomicrographs were taken of the cells. For the disease trial, rods from the 1BE strain were protoplasted. The cells were then placed in containers with seeds of Russ Spring Wheat showing pre- radicle emergence. After three hours of soaking in the bacterial cells the seeds were planted in large pots. When the plants reached flowering stage they were inoculated with the scab fungus. After the heads of the plants matured, they were checked for the extent of disease development. The induction of L-forms for Bacillus strains 1BE and 1BC was successful. Strains 1BA and 1D3 appeared to form syncytia. The concentration of peni- cillin used in this trial was slightly lower than that used by Paton. Also, Paton added another antibiotic—phosphomycin—in order to slow the reversion of L- forms to cells with walls. One disease trial was performed using Paton’s meth- ods. The results from this disease trial showed that the controls proved more effective against wheat scab than the test treatments. More disease trials should be performed to either repudiate or substantiate these results. Further studies 180 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) of the interaction between L-form and host plant should be conducted. Also, antibiotic production capabilities of these L-forms should be studied. Perhaps the L-forms do not produce the same substances in the same quantities as the parent strains. Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 181

ELECTROANALYSIS OF BIOGENIC AMINES AND AMINO ACIDS AT DIAMOND THIN-FILM ELECTRODES

Miles Koppang and Eric Hammond Department of Chemistry University of South Dakota Vermillion, SD 57069

Malgorzata A. Witek and Greg M. Swain Department of Chemistry and Biochemistry Utah State University Logan, UT 84322

ABSTRACT

Diamond thin-film electrodes have gained increased attention in recent years for electroanalysis due to their wide working potential window for aque- ous solvents, low background currents and increased signal to background re- pines as compared to conventional carbon-based electrodes. However, the re- lationship between the physical, chemical and electronic properties of dia- mond, and the observed electrochemical performance remains incompletely understood. For polycrystalline films, the electrochemical properties appear to be influenced in a complex manner by various factors which include, (i) the dopant type and concentration (i.e., electronic properties), (ii) morphological features such as grain boundaries, extended and point defects, and (iii) the non-diamond or amorphous carbon impurity content. The applicability of electroanalysis for biogenic amines and amino acids was investigated in alka- line media using cyclic voltammetry, and FIA-EC and LC-EC in the ampero- metric detection mode. Well-resolved cyclic voltammetric oxidation waves, with respect to the background signal, were observed for five aliphatic polyamines (ethylenediamine, putrescine, cadaverine, spermine and spermi- dine) and 17 of the 22 naturally-occurring amino acids at the 1 mM level with

E1/2’s of ~+0.9 V vs. Ag/AgCl. These analytes produced voltammetric waves consistent with a limited number of active microsites supporting electron-trans- fer. The proposed reaction mechanism for aliphatic biogenic amine oxidation involves adsorption of the polyamine at surface boron-sites, generation of OH• at nearby non-diamond carbon impurity sites, attack of the radical at the coor- dinated amine, and slow desorption of the product.

Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 183

AVIFAUNA OF AGRICULTURAL WETLANDS ON THREE FARMS IN EASTERN SOUTH DAKOTA

Thomas R. Kirschenmann Pheasants Forever Worthing, SD 57077

Daniel E. Hubbard Department of Wildlife and Fisheries Sciences and Diane H. Rickerl Department of Plant Science South Dakota State University Brookings, SD 57007

In this study we evaluated the avian use of wetlands on three farms each using a different agricultural management system: a conventional system (CON), an organic system (ORG), and transitional no-till system (TNT). Wa- terfowl pair abundance varied for individual species on individual wetland classes (temporary, seasonal, and semipermanent water regimes) both between systems and between years in 1993 and 1994. Total waterfowl pair abundance was generally higher in temporary wetlands on the ORG and/or TNT farming systems depending on year, however it was higher in seasonal and semiper- manent wetlands on the CON farming system. Species richness was typically higher on the ORG system for individual wetland classes and when all wetland classes were combined. Abundance of non-waterfowl breeding birds, both in- dividual species and total birds was typically higher on the ORG and/or TNT farming systems. We conclude that we could not detect any consistent trends in avian use between systems. Due to the inherent variation between the wet- land numbers, sizes and habitat conditions among the farms, the influence of the type of systems, if any, were obscured.

Abstracts of Invited Symposium Papers

presented at The 85th Annual Meeting

of the South Dakota Academy of Science

Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 187

ELECTROCHEMISTRY AND CHARACTERIZATION OF COMPLEX SURFACES

Royce Engstrom, Organizer University of South Dakota Vermillion, SD

IODINE FILMS AND ELECTROCHEMICAL OSCILLATIONS

Joseph E. Vitt and Ling Ma Department of Chemistry University of South Dakota Vermillion, SD 57069

- A thick I2 film deposits during the electrochemical oxidation of I when the concentration of I- exceeds about 5 mM in aqueous solution. The nature of this film has been studied extensively, with many reports in the electrochemi- cal literature over the past 30 years. However, an oscillating reaction that oc- curs for I- in the presence of this film has not been investigated thoroughly.

For potential values from about 0.6 V to 1.0 V in 1 M HNO3, a constant current was observed at a Au rotating disk electrode, which corresponded to the situ- ation where the rates of dissolution and formation of the I2 were equal. For a narrow range of potentials around 1.1V vs. SCE, sustained, periodic oscillations in current were observed. The ring electrode of a Au-Au rotating ring-disk - electrode was used to monitor the concentrations of I and I2 during the oscil- lations in current that occur during I- oxidation at the disk electrode. With the ring electrode poised at a potential sufficient to oxidize I-, the anodic current at the ring decreased while the anodic current at the disk increased, as was ex- pected for a typical shielding experiment. However, when the ring was poised - at a potential sufficient to reduce I2 (or I3 ) in a collection experiment, the ca- thodic current also decreased simultaneously with the increase in anodic cur- rent at the disk. These results demonstrate convincingly that the oscillating cur- rent involves the periodic formation and dissolution of the I2 film.

THE SCANNING VIBRATING ELECTRODE TECHNIQUE AND ITS APPLICATION TO THE STUDY OF CORROSION PROCESSES

Dennis E. Tallman Department of Chemistry North Dakota State University Fargo, ND 58105

The scanning vibrating electrode technique (SVET), also known as the cur- rent density probe, is capable of providing a spatially (and temporally) re- solved mapping of current density in an electrolyte immediately above a sub- strate surface. A microelectrode probe is vibrated in two dimensions (parallel 188 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) and perpendicular to the surface) as the tip is rastered above the surface of the substrate at a height of ca. 100-200 microns. Current flow in the electrolyte due to electrochemical processes occurring on the substrate surface results in a volt- age (IR) drop within the electrolyte. This voltage drop is sensed by the vi- brating probe and resolved by two phase-sensitive detectors into a voltage vec- tor having magnitude and direction. The voltage vector is converted into a cur- rent density vector using calibration data generated in a separate experiment. In our laboratory, the SVET is being used to study corrosion processes on steel and aluminum alloy surfaces. In particular, we are trying to understand the na- ture of the electrochemical interactions between electroactive conducting polymers and these active metal substrates. The instrumentation will be de- scribed and selected examples from our research will be discussed to illustrate the capabilities of the technique.

FIA STUDY OF ION PARTITIONING INTO ULTRA-HIGH SURFACE AREA CARBON FIBER ELECTRODES

Theodore Kuwana, Brian Coleman and Tina Huang Department of Chemistry University of Kansas Lawrence, KS 66045

Rick Kelly Department of Chemistry Merrimack College North Andover, MA 01845

An observation that 10-um diameter carbon fibers (DuPont E120) could be electrochemically fractured to produce ultra-high surface areas [1,2], as a con- sequence of opening micro- to nano-porous interior, prompted the current study. That is, the interior behaves like a membrane that has the ability to ex- change ions and molecules from solution. It is convenient to study the parti- tioning phenomenon by flow injection analysis (FIA) methods, where the frac- tured fibers are affixed in a micro-flow channel and the electrochemical (charg- ing) current measured at various fixed potentials during successive injections of known amounts of ions and molecules. With cations such as Li+, Na+, Mg+2, Ca+2, Ba+2 and Cd+2, the extent of partitioning increased with larger charge-to- size (hydrated) ratios. Thus, Li+ is partitioned the least while Ba+2 and Cd+2 are much greater. The extent of partitioning is also concentration dependent, as ex- pected. The peak-shaped cathodic currents are consistent with an interior that possesses deprotonated carboxylic acid sites, which would be negatively charged at pH > 4. However, the injection of KF, KCl, KBr and KI solutions at the same concentration as the background KNO3 gave anodic (oxidative) peaks, which are indicative of anion partitioning. In some cases, initial peaks are followed by reverse cathodic peaks, which are indicative of the direction of ion flow as the fiber comes to equilibrium with the background electrolyte. Molecules are more complicated to understand as the extent of partitioning ap- pears to depend on several factors including charge (+ or -), size, configura- Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 189 tion (shape) and hydrophobicity. The currents are much larger, of course, if the ion or molecule undergoes an electron transfer reaction at the applied po- tential. The FIA partitioning results will be compared to our earlier experi- mental and simulated cyclic voltammetric studies [3,4] in the case of redox molecules.

1. Swain, G. M.; Kuwana, T. Anal. Chem. 1991, 63, 517-519. 2. Swain, G. M.; Kuwana, T. Anal. Chem. 1992, 64, 565-568. 3. Kelly, R. S., Weiss, D. J.; Chong, S. H., Kuwana, T. Anal. Chem. 1999, 71, 413-418. 4. Weiss, D. J.; Kelly, R. S.; Cumaranatunge, M.; Kuwana, T. Anal. Chem. 1999, 71, 3712-3720.

ELECTROANALYSIS OF BIOGENIC AMINES AND AMINO ACIDS AT DIAMOND THIN-FILM ELECTRODES

Miles Koppang and Eric Hammond Department of Chemistry University of South Dakota Vermillion, SD 57069

Malgorzata A. Witek and Greg M. Swaim Department of Chemistry and Biochemistry Utah State University Logan, UT 84322

Diamond thin-film electrodes have gained increased attention in recent years for electroanalysis due to their wide working potential window for aque- ous solvents, low background currents and increased signal to background re- pines as compared to conventional carbon-based electrodes. However, the re- lationship between the physical, chemical and electronic properties of dia- mond, and the observed electrochemical performance remains incompletely understood. For polycrystalline films, the electrochemical properties appear to be influenced in a complex manner by various factors which include, (i) the dopant type and concentration (i.e., electronic properties), (ii) morphological features such as grain boundaries, extended and point defects, and (iii) the non-diamond or amorphous carbon impurity content. The applicability of electroanalysis for biogenic amines and amino acids was investigated in alka- line media using cyclic voltammetry, and FIA-EC and LC-EC in the ampero- metric detection mode. Well-resolved cyclic voltammetric oxidation waves, with respect to the background signal, were observed for five aliphatic polyamines (ethylenediamine, putrescine, cadaverine, spermine and spermi- dine) and 17 of the 22 naturally-occurring amino acids at the 1 mM level with

E1/2’s of ~+0.9 V vs. Ag/AgCl. These analytes produced voltammetric waves consistent with a limited number of active microsites supporting electron-trans- fer. The proposed reaction mechanism for aliphatic biogenic amine oxidation involves adsorption of the polyamine at surface boron-sites, generation of OH• 190 Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) at nearby non-diamond carbon impurity sites, attack of the radical at the coor- dinated amine, and slow desorption of the product.

INFLUENCE OF LIGAND BULK ON COPPER(II/I) COORDINATION CHANGE

Richard J. Jaeger and David T. Pierce Department of Chemistry University of North Dakota Grand Forks, ND 58202

Richard J. Staples Department of Chemistry and Chemical Biology X-ray Crystallographic Laboratory Harvard University Cambridge, MA 02138

Pendent-thiaether derivatives of the macrocycle 1,4,7-triazacyclononane (TACN) were synthesized to determine how coordination change associated with the Cu(II/I) couple is affected by ligand bulk. Cu(II) complexes with these N3S3 ligands (Ln, n=1-6) were structurally characterized by electronic ab- sorption spectroscopy, electron-paramagnetic resonance spectroscopy, and x- ray crystallography. These methods indicated a preference for lower coordi- nate geometry (6- to 5- to 4-coordiante Cu(II)) as alkyl bulk of the thiaether pendent arms was increased (methyl to i-propyl to t-butyl). Cyclic voltamme- try of the [Cu(II/I)Ln]2+/+ couples (n=2-5) in acetone media consistently indicat- ed a square mechanism that appeared to involve 5- and 4-cordinate isomers of [Cu(II)Ln]2+ and [Cu(I)Ln]+. Thermodynamic and kinetic constants were ex- tracted from the voltammetric traces using digital simulation. These data re- vealed that, as alkyl bulk increases, the rate of [Cu(II)Ln] isomerization de- creases and 4-coordinate geometry becomes thermodynamically favored.

STRUCTURE/FUNCTION OF MEDIUM CHAIN Acyl-CoA DEHYDROGENASE: THE IMPORTANCE OF SUBSTRATE POLARIZATION

Marian T. Stankovich and Kim Sabaj University of Minnesota Department of Chemistry Minneapolis, MN 55455

Peter Tonge State University of New York, Stony Brook Chemistry Department Stony Brook, NY 11794.

In the proposed mechanism of the medium chain acyl-CoA dehydrogenase (MCAD), the substrate binding in the hydrophobic active site is accompanied Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 191 by the interaction of two hydrogen bonds of MCAD with the carbonyl group of the substrate, producing a polarized product-like transition state. The prod- uct-like transition state results in a positive charge being stabilized near the flavin, causing the redox potential of MCAD to shift positive, and a change in the pKa in the alpha protons of the substrate and the catalytic base of the MCAD. Our goal is to investigate the interaction (polarization) using a Raman active product-like probe (hexadienoyl-CoA) to obtain direct evidence of po- lariza-tion in the active site, but we first had to show that the probe binds in the active site and exhibits the same behavior as the product.

CORRELATION OF INTERFACIAL PROPERTIES DURING THE POTENTIAL-CONTROLLED DISSOLUTION OF MAGNETITE

Royce C. Engstrom and Julia S. Heaton Department of Chemistry University of South Dakota Vermillion, SD 57069

A complex geological surface has been studied with respect to redox-con- trolled dissolution behavior. The sample consisted of a matrix of fayalite, a min- eral that is non-conductive and that dissolves slowly in acidic media, penetrat- ed by pools and veins of magnetite, a conductive mineral that undergoes re- ductive and oxidative dissolution. In this study, the fayalite served as a bench- mark against which magnetite dissolution was measured upon application of an applied potential. The dissolution was measured using three different, but correlative, approaches: 1) cyclic voltammetry to determine the amount of charge injected during the dissolution process; 2) in situ atomic force mi- croscopy to measure physical retreat rates of the magnetite surfaces; and 3) scanning electrochemical microscopy to measure interfacial iron concentra- tions. A semi-quantitative comparison of the results from the three types of measurement showed good correlation, in that solution concentration of iron followed the charge injected during electrolysis, and the physical retreat rate was quantitatively consistent with those two measurements. The results pro- vide a thorough understanding of a complex geochemical interfacial process.

Proceedings of the South Dakota Academy of Science,Vol. 79 (2000) 193

NUTRITION SYMPOSIUM

Bonny Specker, Organizer E.A. Martin Program in Human Nutrition South Dakota State University

DIET AND CANCER Lawrence Kushi Columbia University

THE DIETARY FIBER HYPOTHESIS REVISITED Dennis Gordon North Dakota State University

DIETARY BORON AND ERYTHRITOL AFFECT REPRODUCTION AND FETAL DEVELOPMENT IN RATS Kay A. Keehr and Curtiss D. Hunt USDA, ARS, Grand Forks Human Nutrition Research Center

ADJUSTMENT TO A PEAK PHYSICAL WORK CAPACITY TREADMILL TEST William A. Siders, Henry C. Lukaski, and Clinton B. Hall USDA, ARS, Grand Forks Human Nutrition Research Center

LOW DIETARY ZINC AND COPPER NEGATIVELY AFFECTS PLASMA AND URINE INDICATORS OF BONE HEALTH Forrest H. Nielsen, Cindy D. Davis, and David B. Milne USDA, ARS, Grand Forks Human Nutrition Research Center

EVALUATION OF REGIONAL BIOELECTRICAL IMPEDANCE MEASUREMENTS TO ASSESS WHOLE BODY COMPOSITION H.C. Lukaski, W.A. Siders and C.B. Hall USDA, ARS, Grand Forks Human Nutrition Research Center

OSTEOPOROSIS: IS IT A DISEASE OF CHILDHOOD Bonny Specker, E.A. Martin Program in Human Nutrition South Dakota State University Brookings, SD 57007

THE PHYTOCHEMICAL RAGE: FAD OR FACT Karen Dorn and Dee Peterson Augustana College Sioux Falls, SD