Source: MICHIGAN STATE UNIV submitted to
RESTRUCTURING THE PHYLOGENY AND TAXONOMY OF THE EUGLENOPHYTA
Sponsoring Institution
State Agricultural Experiment Station
Project Status
TERMINATED
Funding Source
STATE
Reporting Frequency
Annual
Accession No.
0207527
Grant No.
(N/A)
Project No.
MICL03423
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Jun 1, 2006
Project End Date
May 31, 2011
Grant Year
(N/A)
Project Director
Triemer, R. E.
Recipient Organization
MICHIGAN STATE UNIV
(N/A)
EAST LANSING,MI 48824
Performing Department
PLANT BIOLOGY
Non Technical Summary
Despite their economic importance in both agriculture and medicine (fish kills in commercial ponds, potent source of [alpha tocopherol] vitamin E, antibiotic activity, antitumor activity), we understand little about these ancient protists. Perhaps the oldest of human traits is the desire to describe and understand the world around us. For most of mankind's history we have been completely unaware of the diverse world of microorganisms that dominates our planet. That perception changed dramatically in 1674 when the Dutch cloth merchant Antonie van Leeuwenhoek wrote to a friend about his discovery of a tiny wriggling creature that today, we know to be Euglena viridis, the first described microscopic protist. Yet in the 300+ years since its discovery, Euglena and its relatives remained largely unknown despite their economic importance (fish kills in commercial ponds, potent source of [alpha tocopherol] vitamin E, antibiotic activity). For a group of organisms that may have predated all animals and land plants by as much as a billion years, this seems unwarranted. The objective of this research was to evaluate and clarify the evolutionary relationships among taxa in the Euglenophyta using both molecular and morphological data.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20640501103100%
Knowledge Area
206 - Basic Plant Biology;

Subject Of Investigation
4050 - Protozoa;

Field Of Science
1103 - Other microbiology;
Goals / Objectives
The proposed project will generate a revised taxonomy for the photosynthetic euglenoids based on phylogenetic relationships while promoting the training of the next generation of algal systematists. The PIs will explore euglenoid samples from 100 to 200 years old using light and electron microscopy in a manner previously not attempted for euglenoids. The proposed research will provide a web-based resource for studying euglenoids which will include, digital identification keys, image libraries of the taxa, a searchable database of original scientific articles dating back to 1838, phylogenetic trees and molecular and morphological datasets. The proposed Euglenoid Project will provide the model upon which other protistan studies can be based. The results of the project will provide taxonomic tools that can be used not only by protistan specialists, but also by ecologists, limnologists, water quality monitors, and students and teachers at the K12 level to identify and study euglenoids. Rare taxonomic literature will now be readily available to the scientific community. The training program from the postdoctoral to undergraduate level, will include persons from underrepresented groups, and ensure that euglenoid taxonomists will be prepared to carry on this work for many years to come.
Project Methods
Phylogenetic analyses will be conducted on morphological and molecular data. For the morphological studies, a set of 37 characters with 97 associated states will be analyzed using parsimony. The starting list of characters will be updated and significantly expanded during the project. Trees will be generated using the Branch and Bound option of PAUP* (Swofford, 2002). Bootstrap resampling analysis, displayed as a 50% majority-rule consensus tree, will be used to provide an estimate of tree robustness. For molecular analyses, DNA will be isolated and sequenced as described in (Brosnan et al., 2003, Nudelman et al., 2003 and Linton et al., 1999). In short, genomic DNA was isolated from a centrifuged pellet of cultured cells using either the Chelex procedure (Goff & Moon, 1993) with Chelex 100 resin (Bio Rad, 143-2832), or the DNeasy Tissue Kit (Qiagen, 69504) following the animal tissues protocol. A 1.3-1.6 kb region of the LSU rDNA starting at stem 11 (Larsen, 1992) and ending at stem 35a will be amplified by PCR using 15-20 ng of total genomic DNA or 10 μL of DNA-containing solution from the Chelex extraction. Primers specific to the LSU rDNA in euglenoids were created by comparing the LSU rDNA of Euglena gracilis, which had previously been sequenced by M.N. Schnare et al. (GenBank X53361), with published LSU rDNA primers (Freshwater & Bailey, 1998; Palumbi, 1996). Primers used for sequencing are listed in Table 2 of Brosnan et al. (2003). The complete SSU rDNA gene will be amplified by PCR, purified, and sequenced as described by Linton et al. (1999). Primers used for sequencing are listed in Table 2 of Nudelman et al. (2003). Species of the biflagellate photosynthetic genera, Eutreptia and, Eutreptiella, and two species of phagotrophic euglenoids, Peranema and Petalomonas will be used as outgroups for establishing overall phylogenetic relationships among the photosynthetic euglenoids under consideration. Previous molecular studies (Brosnan et al. 2003; Nudelman et al., 2003) demonstrated the suitability of these taxa for this purpose. The sequences for the SSU rDNA and LSU rDNA will be manually aligned as suggested by Sogin & Gunderson (1987) using the genetic data environment (GDE 2.2) program (Smith et al., 1994). Secondary structure of Euglena gracilis is used as a guide in the alignment of the SSU rDNA as described by Linton et al. (2000). LSU rDNA sequences will be aligned with the published E. gracilis secondary structure model of Wuyts et al. (2001) and Schnare et al. (1996) as recommended by Kjer (1995). Regions of sequence where bases are not be reliably aligned due to insertions, deletions, and length variations will be removed from the analysis due to the inability to assess homology. Sequence data will be analyzed using distance, maximum likelihood (PAUP*4.0b10 Swofford, 2002) and Bayesian analyses (MrBayes 2.01 Huelsenbeck, 2000) recognizing the limitations and advantages of each. The evolutionary model and parameters for the analyses will be selected using the hierarchy likelihood ratio test (hLRT) as implemented in Modeltest 3.06 (Posada & Crandall, 1998).

Progress 06/01/06 to 05/31/11

Outputs
OUTPUTS: The project to revise the systematics of the Euglenophyta has continued to move forward. During the 2010 year, one graduate student, one postdoctoral fellow, one undergraduate student, one technician and one high school student have worked on the project under my direction. A major output was the publication of our book entitled " A Color Atlas of Photosynthetic Euglenoids" [ISBN 978-0-87013-879-9] by Ionel Ciugulea (postdoctoral fellow) and myself. This modern treatise will aid biologists, limnologists, ecologists, phycologists and water quality scientists to properly identify these organisms. As a result of our taxonomic studies, we identified a bioactive compound from a toxic euglenoid which was responsible for several fishkills in commercial fish ponds throughout the southern U.S. PARTICIPANTS: Dr. Richard E. Triemer, Project Director; Mr. Matthew Bennett, laboratory manager; Mr. Donovan Watza, Michigan State University sophomore; Ms. Krystle Wiegert, a second year Plant Biology Graduate Student; Ms. Louise Harder, an East Lansing High School student who worked on the project over the summer months TARGET AUDIENCES: biologists phycologists aquaculturists ecologists limnologists water quality monitors students and teachers at the K12 level PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The results of our phylogenetic studies (Protist, April 2010) have resulted in the description of one new genus, two new families of Euglenophyceae, several redescriptions of species and the establishment of epitypes where necessary. The studies have shown that some of the morphological characters formerly used to separate genera and species cannot be supported by the molecular data, but others are supported and should be used as delimiting criteria. The euglenophycin toxin has been characterized (Toxin, 2010), the data published and a patent is pending. The compound has potential use as an herbicide and as a potential anticancer agent. A grant has been obtained to have the compound synthesized and begin animal trials. A patent through USDA (serial no. PCT/US09/58552) identifying the toxin and potential uses of the toxin is pending.

Publications

  • Monfils, A. K., Triemer, R. E. and Bellairs, E. F. 2010. Characterization of paramylon morphological diversity in photosynthetic euglenoids (Euglenales, Euglenophyta). Phycologia. 49:XXX In press.
  • Zimba, P. V., Moeller, P. D., Beauchesne, K., Lane, H. E. and Triemer, R.E. 2010. Identification of Euglenophycin-a toxin found in certain euglenoids. Toxicon 55:100-104.
  • Ciugulea,I. and Triemer, R. E. 2010 A Color Atlas of Photosynthetic Euglenoids - Michigan State University Press, East Lansing, MI 204pp.
  • Linton, E.W., Karnkowska-Ishikawa, A., Kim, J-I., Shin,W., Bennett, M., Kwiatowski, J., Zakryś, B. and Triemer, R. E. 2010. Reconstructing Euglenoid Evolutionary Relationships using Three Genes: Nuclear SSU and LSU, and Chloroplast 16S rDNA Sequences and the description of Euglenaria gen. nov. (Euglenophyta). Protist. 161: 603,619.
  • Kim, J-I., Shin, W. and Triemer, R. E. 2010. Multigene analyses of photosynthetic euglenoids and new family, Phacusaceae (Euglenales). J. Phycol. In press. - published online Nov. 2, 2010. http://onlinelibrary.wiley.com/doi/10.1111/j.1529-8817.2010.00910.x/p df


Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: The project to revise the systematics of the Euglenophyta has continued to move forward. During the 2009 year, one graduate student, one postdoctoral fellow, one undergraduate student, one technician and one high school student have worked on the project under my direction. The number of taxa sequenced has now grown to over 200 and the new sequences for five different genes have or will be deposited in Genbank and as such are accessible to the scientific community. We developed new methods that allow us to amplify genomic DNA from single cell isolates using multiple displacement amplification (MDA). The success of the MDA was a critical development for it allowed us to analyze species from field samples which could not be brought into culture and sequenced. As a result of our taxonomic studies, a toxic euglenoid was identified which was responsible for several fishkills in commercial fish ponds throughout the southern U.S. A patent was filed through USDA (serial no. PCT/US09/58552) identifying the toxin and potential uses of the toxin. Our euglenoids website (http://euglena.msu.edu) continues to grow. In addition to having basic information on identifying euglenoids, an interactive digital key to the genera and a bibliographic library of original meta-tagged references in pdf format, we have added over 70 video clips showing the organisms "in action". The results of our data were presented at 3 scientific conferences. The plenary talk at the Microbial Tree of Life Workshop was designed specifically for graduate students and postdoctoral fellows working with euglenoids. Linton, E. and Triemer, R.E. 2009. Analyzing the Euglenoid Plastid Genome. 2009. Plant and Animal Genome XVII, San Diego, CA, January 10-14, 2009. Triemer, R.E. (Plenary speaker) 2009. Phylogeny of Photosynthetic Euglenoids - What Have We Learned Microbial Tree of Life Workshop, Virginia Commonwealth University, June 19, 2009. Bennett, M. S. and Triemer, R. E. 2009. A method for obtaining nuclear gene sequences from field samples. Phycological Society of America, Honolulu, Hawaii, July 18-22, 2009 Watza, D., Kim, J-I., Bennett, M. S., Lowery, C. and Triemer, R. E. 2009. Euglenoid phylogeny based upon the evaluation of protein and ribosomal coding genes. Phycological Society of America, Honolulu, Hawaii, July 18-22, 2009. Linton, E. W., Karnkowska, A., Kim, J-I., Ciugulea, I., Shin, W., Bennett, M., Kwiatowski, J., Zakryś, B. and Triemer, R. E. 2009. Reconstructing euglenoid evolutionary relationships using three genes: nuclear ssu and lsu, and chloroplast 16s rdna sequences and the description of Euglenaria gen. nov. (Euglenophyta). Phycological Society of America, Honolulu, Hawaii, July 18-22, 2009. PARTICIPANTS: Dr. Richard E. Triemer, Project Director; Mr. Matthew Bennett, laboratory manager; Mr. Donovan Watza, Michigan State University sophomore; Ms. Krystle Wiegert, a first year Plant Biology Graduate Student; Ms. Margaret Lindman, an East Lansing High School student who worked on the project over the summer months TARGET AUDIENCES: biologists, phycologists, aquaculturists, ecologists, limnologists, water quality monitors, students and teachers at the K12 level PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The results of our 2009 phylogenetic studies (submitted for publication) have resulted in the description of one new genus, two new families of Euglenophyceae, several redscriptions of species and the establishment of epitypes where necessary. The studies have shown that some of the morphological characters formerly used to separate genera and species cannot be supported by the molecular data, but others are supported and should be used as delimiting criteria. The successful application of the multiple displacement amplification method to euglenoids has allowed us to explore the phylogeny of taxa that we collect from the field but cannot culture in the lab. The technique has already been successfully applied to six species from field samples and the sequences are now being added to our molecular database. The euglenophycin toxin has been characterized, the data published and a patent is pending. We are now beginning to explore possible herbicidal and medicinal applications of the toxin.

Publications

  • Zimba, P. V., Moeller, P. D., Beauchesne, K., Lane, H. E. and Triemer, R.E. 2009. Identification of Euglenophycin-a toxin found in certain euglenoids. Toxicon (in press, published online July 15, 2009)


Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: The Project to restructure the phylogeny and taxonomy of the Euglenophyta continues to be productive. We now have SSU rDNA and complete LSU rDNA sequence for over 170 species, as well as psb0, chloroplast SSUrDNA, Hsp90 and elongation factor sequenced for many of these taxa. A combined multigene dataset for the nuclear and chloroplast ribosomal genes has been compiled and analyzed. A draft manuscript is in review. A manuscript on the molecular phylogeny of the loricate taxa, Trachelomonas and Strombomonas was published in 2008. A book project with Michigan State University Press entitled, A Color Atlas of Euglenoids, is nearly completed and will go to the publisher in January 2009. The book will provide information on the basic biology of euglenoids, a taxonomic key and full color plates for each taxon giving complete authorities and references. This text will serve as a major reference that will be used by biologists, ecologists, and water management scientists to identify euglenoids. We continue to make major strides in raising the visibility of euglenoids in the scientific community and in introducing teachers and students to euglenoids. Our research was presented at the annual meeting of the Phycological Society of America in August 2008. Our website, euglena.msu.edu,continues to serve as a major resource for scientists, teachers and students. An updated version of the website is now online with high resolution images available for more than 130 taxa. Additional images and video continue to be added to the site. The website also includes a searchable, meta-tagged, digital library housing all of the major taxonomic literature relating to euglenoids that was written prior to 1926 (copyright cutoff date). Four high school biology teachers (Ms. Lisa Weise- Holt High School-2004 and 2005; Mr. Drew Hall - Grand Ledge High School-2005; Ms. Colleen Palmer - Okemos High School - 2006; Mr. Mike Van Antwerp - Holt High School -2007) and two undergraduate teacher interns (Mr. Pete Kechele and Mr. Nate Powers 2008) have participated in the project to date. In addition to collecting algae, sequencing and learning phylogenetics, all of the biology teachers have developed classroom exercises which have been published in the Michigan Science Teacher's Association Journal. In the summer of 2008, Ms. Caitlin Lowery, a freshman at University of Michigan, continued to work on the project developing the website and sequencing several taxa for multiple genes. Caitlin joined the lab as a high school student and has continued to work on the project as an undergraduate. In the summer of 2008, Mr. Donovan Watza joined the lab as a high school student. He is enrolled as an undergraduate at Michigan State University in the Fall and continues to work on the project. During this period we also had a graduate student, Ms. JongIm Kim, visiting in the lab from Chungnam National University in Korea. She is a full time student with a former postdoc on the project, Dr. Woongghi Shin. PARTICIPANTS: Dr. Richard E. Triemer, Project Director Mr. Matthew Bennett laboratory manager Mr. Pete Kechele Michigan State University teacher intern Mr. Nate Powers Michigan State University teacher intern Ms. Caitlin Lowery, a freshman at University of Michigan Mr. Donovan Watza joined the lab as a high school student and continues to work on the project as a freshman at Michigan State. Ms. JongIm Kim, visiting graduate student from Chungnam National University in Korea. TARGET AUDIENCES: biologists phycologists aquaculturists ecologists limnologists water quality monitors students and teachers at the K12 level PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Understanding of the evolutionary relationships among euglenoids has been extended in the scientific community and the taxonomy has been changed to reflect the new knowledge. Correct identification of algae is critical when making decisions regarding biological diversity, environmental impacts and water quality. Five students, including two women, have been provided with research experiences and training.

Publications

  • Ciugulea, I., Nudelman, M.A., Brosnan, S. and Triemer. R.E. 2008. Phylogeny of the Euglenoid Loricate Genera Trachelomonas and Strombomonas Inferred from Nuclear SSU and LSU rDNA (Euglenophyta). J. Phycol. 44:406 to 418.


Progress 01/01/07 to 12/31/07

Outputs
OUTPUTS: The phylogenetic relationships among over 120 taxa of euglenoids have been determined. This has resulted in a re-classification of several taxa and a redefinition of several genera including the establishment of epitypes to eliminate taxonomic confusion. PARTICIPANTS: Matthew Bennett - laboratory manager Ionel Ciugulea - postdoctoral fellow Caitlin Lowery - Holt High School student Peter Kechele - MSU undergraduate Nathan Powers - MSU undergraduate TARGET AUDIENCES: Phycologists, ecologists, water quality specialists, protozologists

Impacts
In the process of inferring phylogenies we have identified a new carotenoid pigment which can be used to identify euglenoid blooms in commercial fish ponds. We have also identified a toxin responsible for massive fish kills in commercial aqua culture ponds in over five southern states. A patent is pending for the toxin.

Publications

  • Triemer, R.E. and Farmer, M.A. (2007). A decade of euglenoid molecular phylogenetics. In: Unravelling the algae: the past, present and future of algal systematics. (Brodie, J. & Lewis, J. eds). Systematics Association Series, CRC Press.


Progress 01/01/06 to 12/31/06

Outputs
We have sequenced three genes and analyzed the data for over 100 taxa of photosynthetic euglenoids. The euglenoid toxin that produces fishkills has been chemically analyzed via HPLC and NMR. Final analyses should be completed early in 2007. Paramylon, the beta 1 3 glucan storage product of euglenoids, has been shown to have antitumor properties. Since the morphology of the paramylon grains varies across genera, the chemical properties, and hence the antitumor properties, may vary as well. We have begun morphological and chemical studies on isolated paramylon grains from diverse species.

Impacts
The project will elucidate the phylogeny of phtosynthetic euglenoids. We have shown that several related species of photosynthetic euglenoids priduce a toxin which causes fishkills. We are testing related species for the toxin and characterizing the chemical nature of the toxin. Understanding the nature of the toxin should provide insights in how to deal with the toxin.

Publications

  • Nudelman, M. A., Leonardi, P.I., Conforti, V. and Triemer, R. E. 2006. Fine Structure and Taxonomy of Monomorphina Aenigmatica comb. nov. (Euglenophyta). J. Phycol. 42:194-202.
  • Triemer, R. E., Linton, E., Shin, W., Nudelman, A., Monfils, A., Bennett, M. and Brosnan, S. 2006. Phylogeny of the Euglenales based upon combined SSU and LSU rDNA sequence comparisons and description of Discoplastis gen. nov. (Euglenophyta) J. Phycol. 42:731-740.