A morphological and molecular study of Euglena anabaena

Prinze C. Mack1* and Richard E. Triemer2
Cook College, Rutgers University
Department of Cell Biology and Neuroscience Science
Piscataway, New Jersey

*Rutgers Undergraduate Research Fellow


Abstract

     Euglenoids represent one of the earliest divergences from the eukaryotic tree of life leading to higher plants and animals. Yet despite their evolutionary importance as early eukaryotes, euglenoids are poorly studied in terms of their own evolutionary relationships and morphological idiosyncracies. The focus of this project is to establish the phylogenetic relationships of one species of euglenoid, Euglena anabaena. The method for diagnosing this euglenoid species will incorporate morphological observations with the molecular sequence data. The combination of both sets of data strives to establish a congruence between the visible characteristics of the organism and its genomic information. As a result, inferences can be made as to where Euglena anabaena falls on the evolutionary tree and its relationship to other euglenoids.


Introduction


     Euglenoids are among the most ancient of the free-living eukaryotic protists, and are one of the earliest lineages to contain mitochondria. Although possessing the unique characteristic of being neither plants nor animals, euglenoids are poorly known despite being ubiquitous in fresh water, marine, and soil habitats. An increased knowledge of euglenoids is crucial because of the ecological importance of these organisms. Euglenoids are indicators of ecological well-being; favoring waters rich in organic materials (Wolken, 1967), and can be used to indicate the health of their environment.

     The organism under examination, Euglena anabaena, will be studied using morphological and molecular data, in hopes of establishing its phylogenetic history. Most prior attempts to establish a taxonomy of euglenoids have not used cladistic models, nor have they incorporated molecular data. Our method of establishing a taxonomy assesses a morphological and molecular congruence that may be relevant to most protists. Such an analysis method provides a less biased method of species diagnosis for euglenoids.

     Our phylogenetic analysis includes two euglenoids that are not members of the genus Euglena, namely, Astasia longa and Khawkinea quartana. They are included because they are believed to have lost the ability to photosynthesize. Astasia lacks chlorophyll and an eyespot while Khawkinea lacks only chlorophyll (Leedale, 1967). Analysis of their SSU rDNA (small subunit ribosomal DNA) suggests that Astasia and Khawkinea should be included in the genus Euglena. This conclusion is in agreement with one reached in a study by Montegut-Felkner and Triemer (1997).

     Although shared morphological features among euglenoids have been used to infer species relationships, the number of characters available for species diagnosis is limited. In this work, to increase the resolution of our species diagnosis, the small subunit ribosomal DNA (SSU rDNA) molecules of Euglena anabaena will be isolated, sequenced, and used for comparison; the use of rDNA to resolve taxonomic differences among euglenoids has been established (Montegut-Felker and Triemer, 1997). Therefore, a phylogenetic analysis of the 18S rDNA sequence of Euglena anabaena will be used to infer its evolutionary position among other euglenoids. The omnipresence of rRNA among organisms makes it an excellent indicator of evolutionary relationships (Frisvad et al., 1998). Likewise, convenient techniques for primary sequence determination add to the convenience of using the rRNA gene. Before the advent of the PCR, the rRNA gene was extracted and sequenced. However, PCR-amplified rDNA regions are preferred for phylogenetic analysis and generate higher yields during nucleotide extraction.

     The classification of euglenoids is controversial. Euglenoids reproduce asexually, so they cannot be distinguished according to reproductive compatibility, as can higher animals. As a result, "arbitrary distinctions ultimately are the criteria for species determination in asexually reproducing organisms such as Euglena" (Johnson, 1944). Past classifications of euglenoids have not accounted for the plasticity of the morphological traits. For example, Zakrys (1986) challenges the distinctions between Euglena anabaena var. minor and Euglena anabaena var. minima, stating that the observed size difference (Figure 1a) is due to the variability of the strains, and not to evolutionary distinction.

     Historically, euglenoids have belonged to the kingdom Protista. However, controversy surrounds this assignment of euglenoids and a sixth kingdom, the Euglenozoa, has been proposed to house them (Cavalier-Smith, 1981).

     About one third of all euglenoids are photosynthetic, whereas the other two thirds are either phagotrophic or osmotrophic. The most conspicuous feature of the photosynthetic euglenoids is their chloroplasts; many previous taxonomic studies have been based upon this character (Pringsheim, 1956; Zakrys, 1986). Euglenoid chloroplasts assume a particular shape; for instance, the Euglena anabaena chloroplast assumes a shield-shaped appearance (Figure 1b). Oftentimes, as with Euglena anabaena, the chloroplasts contain a proteinaceous center called the pyrenoid. Pyrenoids are localized concentrations of ribulose 1-5 bisphophate carboxylase-oxygenase (rubisco), the enzyme that catalyzes the reduction of carbon dioxide to glucose. Pyrenoids may be capped with plates of paramylon (Figure 1b). Paramylon is a beta 1,3 glucan that can be hydrolyzed to d-glucose and is found only in euglenoids (Godjics, 1953). The body of Euglena anabaena is spindle-shaped, as is the case for many of the Euglena species, and is covered by an elastic membrane complex called the pellicle. (Godjics, 1953). With the aid of this pellicle complex, many species of Euglena are able to engage in a unique form of wiggling movement known as metaboly. In addition, nearly all euglenoids can swim using one or more flagella for locomotion. All Euglena species bear a single emergent flagellum, which exits subapically at the anterior of the body (Figure 1c).


Materials and methods


Culture conditions

     Euglena anabaena was obtained from the culture collection at University of Texas (UTEX #373) and maintained in EG medium at 20oC under a 12 h light/12 h dark cycle.

DNA isolation

     Total genomic DNA was extracted using the Chelex procedure as described by Goff and Moon (1993). This process utilized Biotechnology grade Chelex 100 resin by Bio-Rad.

Amplification of SSU rDNA fragments

     The SSU rDNA coding sequence was amplified via polymerase chain reactions (PCR) using 10 microL of the DNA-containing solution from the Chelex extraction. The procedure used was that recommended by Promega biochemicals (Taq polymerase, buffer, MgCl2 at 2.5 mM, catalogue #PR-M1861; dATP, dTTP, dCTP, dGTP, catalogue #PR-U1240). Oligonucleotide primers were used to initiate DNA synthesis in the PCR. After a 3-minute pre-incubation at 95oC, thirty amplification cycles were carried out as follows: denaturing at 94oC for 2 min, then annealing at 37oC for 2 min, then 72oC for 4 min, followed by one cycle at 72oC for 11 min. To increase amplification yields, three sets of forward/reverse primer pairs were chosen to amplify three individual regions of the SSU rDNA coding sequence (Table 1).

Purification and extraction of rDNA fragments

     The amplified regions of SSU rDNA were purified by gel electrophoresis. The rDNA band was cut out of the gel, followed by a Qiaex Gel Extraction procedure as described by Qiagen.

Sequencing of rDNA: Linear amplification sequencing was performed on the purified template as defined by the manufacturer in the Stratagene Cyclist DNA sequencing kit (Cat No. 200325). The radioactive label used was alpha-[33P] -dATP (DuPont NEN, Boston, MA). The sequencing cycles were performed as follows: 30 cycles of denaturing at 94oC for 1.5 min, annealing at 55oC for 1.5 min, and extension at 72oC for 1.5 min. Gel electrophoresis was carried out on a 6.0% and 8.0% (19:1 acrylamide: bisacrylamide) gel (National Diagnostics' Sequagel System, Atlanta, GA). In separate experiments, the purified template was also sequenced by using the ABI automated sequencer at Robert Wood Johnson Medical School per their protocol.

Alignment of rDNA

     The sequence of Euglena anabaena was added to the aligned sequences obtained from the SSU rDNA databank at the University of Antwerp (maintained by Van de Peer, Nicolai, De Rijk, and De Wachter). The sequences were maintained on the GDE package software (Smith et al., 1994) on a Sparc 2 workstation. The primary sequence was aligned based on the secondary structure model of Euglena gracilis provided by University of Antwerp (Figure 2).

     Phylogenetic analysis of sequence data: Using a set of 800 unambiguously aligned positions, a parsimony analysis of 18S sequence data was performed using The Phylogeny Inference Program 3.5 (Phylip 3.5) on the Sun 2 workstation and with PAUP* 4.0d63 (Swofford 1996) on a Powermac 7600/132. Parsimony analysis attempts to explain the given data using the fewest number of steps possible.

     The data from Euglena anabaena were matched against data for nine other euglenoids; Astasia longa, Euglena viridis, Euglena pisciformis, Euglena gracilis, Euglena sp., Khawkinea quartana, Euglena acus, Euglena spirogyra, and Eutreptiella sp. as the outgroup (Figure 3). The data set was scored with a binary code, depending on the alignment of the primary structure of all 10 organisms. A score of "1" was given to each unambiguously aligned position. Scores of "0" were given where alignment was impossible. A majority rule analysis was performed on the data set as well, using 500 replicates, to assess the robustness of the tree.


Results and discussion

Morphological analysis

     The taxon E. anabena has been treated differently by various authors. Mainx (1926/27) described three variants of Euglena anabaena: E. anabaena typ., E. anabaena var. minor, and E. anabaena var. minima. Pringsheim (1956) and Johnson (1944) acknowledge the three varieties of E. anabaena, however Zakrys (1986) asserts that E. anabaena var. minor and E. anabaena var. minima are synonymous. Pringsheim (1956) also concludes that E. anabaena is closely related to E. thinophila based on measurements taken by Skuja (1939), and in fact may be considered to be the same.

     The dimensions given by Johnson (1944) were 36-45 microm by 16-22 microm. However, my observations of E. anabaena var. minor (UTEX # 373) yielded dimensions of 27.5-30 microm by 15-20 microm. One visible flagellum, located at the anterior end of the cell, is approximately one-half of the body length. There is also one prominent eyespot (Figure 1c).

     The shape of the cell is fusiform with a weakly spirally striated pellicle; this pellicle allows slight metabolic movement. Euglena anabaena is an active swimmer, and moderately quick in relation to others within the genus; when stationary, there is moderately active metaboly.

     The chloroplasts (chromatophores) are 6-10 in number and assume a "shield" shape. All of the chloroplasts contain central pyrenoids; in all observed cases, paramylon grains cap the pyrenoids (Figure 1b). However, paramylon grains were not observed freely floating in the cytoplasm.

Molecular data analysis

     The size of the 18S SSU rDNA gene of Euglena anabaena is estimated to be about 2.3 Kb. In this study, 800 bases were aligned and used in the analysis. Of these, 138 were parsimony informative. Parsimony analysis of the data matrix yielded two trees of 484 steps. In both trees, Euglena acus and E. spirogyra form a single clade. Khawkinea quartana, Euglena gracilis, E. species, E. pisciformis, and Astasia longa consistently group together. The trees vary only in the positioning of E. viridis and E. anabaena. In one case, E. anabaena diverges prior to E. viridis and in the second tree the opposite was true.

     A majority rule consensus tree was run with 500 bootstrap datasets (Figure 3). Two major clades exist within this group of organisms, one containing Euglena acus and Euglena spirogyra, and a second containing the remainder of the taxa. The Euglena acus and Euglena spirogyra clade is strongly supported. Strong support is also found for the Euglena gracilis, E. species, E. pisciformis, and Astasia longa clade. The exact position of E. viridis and E. anabaena remains unresolved, but there is strong support for their inclusion into the clade containing Khawkinea quartana, Euglena gracilis, E. species, E. pisciformis, and Astasia longa.

     The parsimony analysis tree illustrates congruence between the molecular and morphological data for these organisms. Euglena acus and Euglena spirogyra both have small disk-shaped chloroplasts that lack pyrenoids and paramylon caps. Euglena anabaena is consistently placed with Astasia longa, E. pisciformis, E. gracilis, E. sp., E. viridis and Khawkinea quartana. All of these organisms also share similar chloroplast features. Their chloroplasts all contain pyrenoids and are capped with paramylon grains. With the exception of E. viridis, all of these chloroplasts are shield-shaped and have a single, centrally located, capped pyrenoid. In Euglena viridis, the chloroplast is a stellate structure with a single pyrenoid capped by numerous paramylon grains. The chloroplast forms many ribbon-like extensions that radiate out from the pyrenoid. The morphological data would suggest that the E. anabaena should be more closely related to the former taxa than to E. viridis. This interpretation is consistent with the majority rule tree but lacks strong bootstrap support.

     In summary, morphological features of the chloroplast have been extensively used to diagnose Euglena species. The molecular data are congruent with the chloroplast data and support the use of the chloroplast as a key morphological character for distinguishing Euglena species.


Acknowledgments

     I would like to thank Dr. Aurea Vasconcelos and Dr. James French for sponsoring my George H. Cook Project. My most sincere gratitude goes to Carole Lewandowski, who spent countless hours teaching me and observing my progress. My achievements in the laboratory are largely indebted to her. Many thanks and best wishes go to Eric Linton; I greatly appreciate the dedication that he demonstrated in helping me learn. I wish lots of luck to Stacy Zimmermann, who has been a wonderful friend throughout the year and never hesitated to be there for me. I would also like to thank Dr. Barbara Goff, the Director of the George H. Cook Honors Project, for her assistance and dedication to the program. It is people like her that earn Cook College its reputation as a top professional institution.


References

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Ehrenberg, C. G. 1830. Neue Beobachtungenüber blutartige Erscheinungnen in Ägypten, Arabien, und Sibirien, nebst einer Übersicht und Kritik der früher bekannten. Pogg. Ann. Physik Chem. 94:477-514.

Frisvad, Jens C., Bridge, Paul D., Arora, Dilip K. 1998. Chemical Fungal Taxonomy. Marcel Dekker. Inc., New York. pp. 51-76.

Godjics, M. 1953. The Genus Euglena. The University of Wisconsin Press, Madison. pp.268.

Goff, L. and Moon, D. 1993. PCR amplification of nuclear and plastid genes from algal herbarium specimens and algal spores. J. Phycol. 29:381-4

Johnson, L.P. 1994. Euglena of Iowa. Trans. American Microscopical Soc. 43: 97-135

Leedale, Gordon F. 1967. Euglenoid Flagellates. Prentice-Hall Biological Series. pp. 27-33.

Lemmermann, E. 1913. Eugleninae. Die Susswasserflora Deutschlands, Osterreichs und der Schweiz, ed. A. Pascher, Vol. 2, 115-174. Jena : Gustav Fischer Verlag.

Mainx, F. 1926. Einige neue Vertreter der Gattung Euglena Ehrenberg mit Unterstützung der Gesellschaft zur Förderung deutschen Wissenschaft, Kunst und Literatur in Böhmen. Arch. Protistenk. 54:150-160.

Mainx, F. 1927. Beiträge zur Morphologie und Physiologie der Euglenen. I. Morphologische Beobachtung, Methode, und Erfolge der Reinkultur. II. Untersuchung über die Ernährungs- und Reizphysiologie. Arch. Protistenk. 60:305-414.

Montegut-Felkner, A.E. and Triemer, R.E. 1997. Phylogenetic relationships of selected euglenoid genera based on morphological and molecular data. J.Phycol. 33:512-519.

Pringsheim, E. G. 1956. Contributions towards a monograph of the genus Euglena. Nova Acta Leopoldina 125:1-168.

Skuja, H. 1939. Beiträge zur Algenflora Lettlands II. Materiali Latvijas alga florai II. Acta Horti. Botan. Univ. Latviensis, Riga11/12:41-169.

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Swofford, D.L. 1996. PAUP*: Phylogenetic analysis using parsimony (and other methods), version 4.0. Sinauer Associates, Sunderland, Massachusetts.

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Table 1. PCR Primers


Forward

Sequence 5?to 3?

Length


1(A) ZB ACC CTG GTT GAT CCT GCC AGT 21
467 (300) AGG GGT CGA TTC CGG AG 17
1055 GGT GGT GCA TGG CCG 15

 

 

 

Reverse Sequence 5?to 3? Length


516 ACC AGA CTT GCC CTC C 16
1913 Eug CAT CTG GGA GCA TCA CAG AC 20
1520 (B) ZB TGA TCC TTC TGA AGG TTC ACC TAC 24


Table 2. Interleaved data matrix used for sequence analysis



Figure 1a. Although both organisms belong to Euglena anabaena, there is a significant size difference. This is due to culture and/or light conditions.


Figure 1b. An illustration of the shield-shaped chloroplast with a paramylon cap in Euglena anabaena.


Figure 1c. The emergent flagellum(a) and eyespot (b) of Euglena anabaena.


Figure 2. The secondary structure of the 18S SSU rDNA of Euglena gracilis



Figure 3. The Majority Rule tree created from 500 bootstrap values. CONSENSUS TREE: The numbers at the forks indicate the number of times the group consisting of the species which are to the right of that fork occurred among the trees, out of 500 trees.



Copyright 1999 by Richard E. Triemer
Current URL: http://rutgersscholar.rutgers.edu/volume01/triemack/triemack.htm