The Chlamydomonas Sourcebook: Volume 1: Introduction to Chlamydomonas and Its Laboratory Use

By Ursula Goodenough

The Chlamydomonas Sourcebook, 3rd Edition

Introduction to Chlamydomonas and Its Laboratory Use (Volume 1)

 

The gold-standard reference?covering the basic biology of the Chlamydomonas alga and techniques for its laboratory analysis  

 

Originally published as the standalone Chlamydomonas Sourcebook, then expanded as the first volume in a three-part comprehensive gold-standard reference,?The Chlamydomonas Sourcebook: Introduction to Chlamydomonas and Its Laboratory Use?has been fully revised and updated to include a?wealth of new resources for the Chlamydomonas community. Early chapters cover current understandings of its taxonomy, ultrastructure, cell and life cycles, and nuclear and organelle genomes, followed by technique-oriented chapters covering such topics as cell culture, mutagenesis, genetic analysis, construction of mutant libraries, and protein localization using immunofluorescence.  

 

This volume presents the latest in research and best practices, making it a must-have resource for researchers and students working in plant science and photosynthesis, fertility, mammalian vision, and biochemistry; crop scientists; plant physiologists; and plant, molecular, and human disease biologists.

• Remains the only complete reference to provide both the historical background and the most up-to-date information and applications on Chlamydomonas
• Includes best practices for applications in research, including methods for culture, genetic analysis, genomic and transcriptomic analysis, and mutant screening
• Helps researchers solve common laboratory problems, provides details on the properties of particular strains, and offers a comprehensive survey of molecular approaches
• Provides a broad perspective for studies in cell and molecular biology, genetics, plant physiology, and related fields

• Language: English
• Publisher: Academic Press
• Release date: Feb 15, 2023
• ISBN: 9780128224588

Book preview

Front Cover for The Chlamydomonas Sourcebook - Volume 1: Introduction to Chlamydomonas and Its Laboratory Use - 3rd Edition - by Ursula Goodenough

The Chlamydomonas Sourcebook

Volume 1: Introduction to Chlamydomonas and Its Laboratory Use

Third Edition

Edited by

Ursula Goodenough

Department of Biology, Washington University, St. Louis, MO, United States

Table of Contents

Cover Image

Title page

Copyright

List of contributors

Preface

Introduction

Chapter 1. The genus Chlamydomonas

Abstract

1.1 Introduction

1.2 Historical overview

1.3 Polyphyly of the genus and revised generic and species concept

1.4 Chlamydomonas reinhardtii as a model organism

1.5 Other wild-type isolates of Chlamydomonas reinhardtii

1.6 Phylogeny and ecology of Chlamydomonas-like organisms

1.7 Other biciliated and quadriciliated unicellular genera

1.8 Conclusions

References

Chapter 2. Cell ultrastructure

Abstract

2.1 Introduction

2.2 Early ultrastructural studies

2.3 The nucleus

2.4 Endoplasmic reticulum and Golgi

2.5 Contractile vacuoles

2.6 Acidocalcisomes

2.7 Microbodies

2.8 Cytoplasmic vacuoles

2.9 Mitochondria

References

Chapter 3. Cell walls

Abstract

3.1 Introduction

3.2 Wall composition: early studies

3.3 Vegetative wall ultrastructure

3.4 Fractionation and identification of vegetative wall components

3.5 Properties of volvocine HRGPs

3.6 Vegetative wall synthesis and assembly

3.7 The ciliary collar

3.8 Zygote walls: structure and composition

3.9 Zygote walls: expression of wall-related genes

3.10 Wall-defective mutants

3.11 Lytic enzymes

3.12 Palmelloid colonies

References

Chapter 4. Functional genomics of Chlamydomonas reinhardtii

Abstract

4.1 Introduction

4.2 The Chlamydomonas genome: structural genomics

4.3 An introduction to functional annotation

4.4 State of functional annotations in Chlamydomonas

4.5 Functional genomics of Chlamydomonas: overview

4.6 Transcriptomics

4.7 Proteomics

4.8 Phylogenomics

4.9 Genome-wide phenotype screens

4.10 Use of mutant collections for reverse-genetics

4.11 Databases and resources for Chlamydomonas genomics and functional genomics

4.12 Outlook and future directions

Acknowledgments

References

Chapter 5. The Chlamydomonas nuclear genome

Abstract

5.1 Introduction

5.2 The Chlamydomonas reference genome

5.3 Structural annotations and gene organization

5.4 Genome architecture

5.5 Genome evolution

5.6 Online resources for the Chlamydomonas genome

5.7 Future perspectives

Acknowledgments

References

Chapter 6. Gene expression: from transcription to alternative splicing

Abstract

6.1 Introduction

6.2 Differential gene expression

6.3 Splicing and alternative splicing

6.4 Nonsense-mediated decay

6.5 Other processes and genes involved in transcription

6.6 Future questions

References

Chapter 7. Organelle heredity

Abstract

7.1 Introduction

7.2 Structure, maintenance, and transmission of organellar genomes in vegetative cells

7.3 Uniparental inheritance during the sexual mating

References

Chapter 8. Cell cycle and circadian rhythms

Abstract

8.1 Introduction

8.2 Multiple fission

8.3 Cytology of the Chlamydomonas cell cycle

8.4 Regulatory logic and modeling

8.5 Cell cycle regulators

8.6 Cell cycle inhibitor and mutant studies

8.7 Omics and single-cell studies

8.8 Meiosis

8.9 Circadian cycles and diurnal rhythms

References

Chapter 9. The sexual cycle

Abstract

9.1 Introduction

9.2 Types of sexual reproduction within the genus Chlamydomonas

9.3 Stages in the reproductive process

9.4 Vegetative diploids and cytoduction

9.5 Genetic control of sexuality

References

Chapter 10. The multicellular relatives of Chlamydomonas

Abstract

10.1 Introduction

10.2 Diversity, habitat, and evolution

10.3 Asexual development

10.4 Sexual reproduction and evolution of the sex-determining locus

10.5 Insights from genome comparisons and from transcriptome analyses

10.6 Molecular genetic tools

References

Chapter 11. Growth techniques

Abstract

11.1 Culture media for Chlamydomonas reinhardtii

11.2 Growth vessels

11.3 Growth conditions

11.4 Culturing techniques

11.5 Presenting and documenting Chlamydomonas growth and fitness

11.6 Histological techniques

Acknowledgements

References

Chapter 12. Practical aspects of mating and tetrad analysis

Abstract

12.1 Practical considerations for mating

12.2 Zygote maturation and germination

12.3 Meiotic progeny

12.4 Tetrad analysis

12.5 Random progeny analysis

12.6 Dikaryons

12.7 Vegetative diploid cells

References

Chapter 13. Genetic transformation of Chlamydomonas nuclear, chloroplast, and mitochondrial genomes

Abstract

13.1 Historical perspectives

13.2 Adoption of molecular biology techniques in Chlamydomonas research

13.3 Development of techniques for the genetic transformation of the nuclear genome

13.4 Chloroplast and mitochondrial genome transformations and modifications

References

Chapter 14. Methods for the localization of cellular components in Chlamydomonas

Abstract

14.1 Introduction

14.2 Localizing proteins/polysaccharides in fixed cells

14.3 Live cell imaging

14.4 Localizing DNA/RNA

14.5 Localizing inorganic molecules

References

Chapter 15. Mutagenesis and genome resequencing

Abstract

15.1 Ultraviolet light as a mutagen

15.2 Alkylating agents

15.3 Conditional and dominant alleles

15.4 Other mutagens

15.5 Identifying causative mutations

15.6 Insertional mutagenesis

15.7 Estimating the number of colonies needed for saturation

15.8 Mutant screens and collections

15.9 Choice of starting strains

15.10 Genetic tests for inheritance and allelism

15.11 Genome resequencing

References

Chapter 16. Generation, storage, and utilizations of mutant libraries

Abstract

16.1 Introduction

16.2 Library generation and mutation mapping

16.3 Library storage

16.4 Forward genetic screens using mutant libraries

16.5 The Chlamydomonas Library Project library: an indexed and mapped library

16.6 Perspectives on pooled screens enabled by CRISPR technologies

Acknowledgments

References

Chapter 17. Reverse genetics

Abstract

17.1 Introduction

17.2 DNA repair systems

17.3 Targeted gene editing with engineered endonucleases in Chlamydomonas reinhardtii

17.4 Conclusions

References

Index

Copyright

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List of contributors

Olga Baidukova,     Institute of Biology, Experimental Biophysics, Humboldt University of Berlin, Berlin, Germany

Ian K. Blaby

US Department of Energy Joint Genome Institute, Lawrence Berkeley National Laboratory, Berkeley, CA, United States

Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA, United States

Crysten E. Blaby-Haas

US Department of Energy Joint Genome Institute, Lawrence Berkeley National Laboratory, Berkeley, CA, United States

The Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, CA, United States

Michal Breker-Dekel,     Department of Plant and Environmental Sciences, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel

Rory J. Craig

California Institute for Quantitative Biosciences, UC Berkeley, Berkeley, CA, United States

Institute of Evolutionary Biology, University of Edinburgh, Edinburgh, United Kingdom

Tatyana Darienko

Research Department for Limnology, University of Innsbruck, Mondsee, Austria

Institute of Microbiology and Genetics, Department of Applied Bioinformatics, University of Göttingen, Göttingen, Germany

Susan K. Dutcher,     Department of Genetics, Washington University in St. Louis, St. Louis, MO, United States

Benjamin D. Engel,     Biozentrum, University of Basel, Basel, Switzerland

Anne G. Glaesener,     QB3, University of California, Berkeley, CA, United States

Ursula Goodenough,     Department of Biology, Washington University, St. Louis, MO, United States

Peter Hegemann,     Institute of Biology, Experimental Biophysics, Humboldt University of Berlin, Berlin, Germany

Colleen Hui

Department of Chemistry and Biochemistry, University of California, Los Angeles, CA, United States

QB3, University of California, Berkeley, CA, United States

Sunjoo Joo,     Department of Botany, University of British Columbia, Vancouver, BC, Canada

Simon Kelterborn,     Institute of Biology, Experimental Biophysics, Humboldt University of Berlin, Berlin, Germany

Yusuke Kobayashi,     Department of Science, Graduate School of Science and Engineering, Ibaraki University, Bunkyo, Ibaraki, Japan

Jae-Hyeok Lee

Department of Botany, University of British Columbia, Vancouver, BC, Canada

Department of Biological Sciences, University of Manitoba, MB, Canada

Xiaobo Li

Key Laboratory of Growth Regulation and Translational Research of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, Zhejiang, P.R. China

Institute of Biology, Westlake Institute for Advanced Study, Hangzhou, Zhejiang, P.R. China

Dianyi Liu

Donald Danforth Plant Science Center, St. Louis, MO, United States

Department of Biology, University of Missouri, St. Louis, MO, United States

Luke C.M. Mackinder,     Centre for Novel Agricultural Products (CNAP), Department of Biology, University of York, York, United Kingdom

Stephen M. Miller,     Department of Biological Sciences, University of Maryland, Baltimore County, MD, United States

Yoshiki Nishimura,     Department of Botany, Kyoto University, Oiwake-cho, Kyoto, Japan

Hisayoshi Nozaki

Department of Biological Sciences, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo, Japan

Biodiversity Division, National Institute for Environmental Studies, Tsukuba, Ibaraki, Japan

Thomas Pröschold,     Research Department for Limnology, University of Innsbruck, Mondsee, Austria

Stefan Schmollinger,     QB3, University of California, Berkeley, CA, United States

Carolyn D. Silflow,     Department of Plant and Microbial Biology, University of Minnesota, St. Paul, MN, United States

Irina Sizova,     Institute of Biology, Experimental Biophysics, Humboldt University of Berlin, Berlin, Germany

William J. Snell,     Department of Cell Biology and Molecular Genetics, University of Maryland College Park, MD, United States

Maria J. Soto,     US Department of Energy Joint Genome Institute, Lawrence Berkeley National Laboratory, Berkeley, CA, United States

Gary D. Stormo

Department of Genetics, Washington University in St Louis, St. Louis, MO, United States

Center for Genomic Sciences and Systems Biology, Washington University in St Louis, St. Louis, MO, United States

Frej Tulin,     Department of Plant Biology, Carnegie Institution for Science, Stanford, CA, United States

James Umen,     Donald Danforth Plant Science Center, St. Louis, MO, United States

Olivier Vallon,     CNRS, Sorbonne Université, UMR7141, Institut de Biologie Physico-Chimique, Laboratory of Chloroplast Biology and Light-Sensing in Microalgae, Paris, France

Yulong Wang,     Key Laboratory of Growth Regulation and Translational Research of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, Zhejiang, P.R. China

Donald P. Weeks,     Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, NE, United States

Jenna Wingfield,     Department of Neuroscience, University of Florida Scripps Biomedical Research, Jupiter, FL, United States

Yuqing Yang,     Key Laboratory of Growth Regulation and Translational Research of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, Zhejiang, P.R. China

Preface

Francis-André Wollman

The name Chlamydomonas first appeared in descriptions of several flagellate species by Ehrenberg and by Dangeard in the last decades of the 19th century. In the middle of the 20th century, one of these species, Chlamydomonas reinhardtii—named after Ludwig Reinhardt, a Ukrainian botanist from the 19th century—became an invaluable biological resource for those who looked for genetic approaches to better understand integrated cell biology processes like motility or photosynthesis. Chlamydomonas research thus developed steadily over the past 70 years. The diversity of issues that could be best addressed with Chlamydomonas attracted an international community of scientists who were eager to share methodological advances that benefited researchers in numerous areas of biology.

Still this research community had to wait until 1989, before the first edition of the Chlamydomonas Sourcebook was published. The subtitle of the first edition was: A Comprehensive Guide to Biology and Laboratory Use. This most impressive achievement was undertaken by a single scientist, Elizabeth Harris, who at that time was the Director of the Chlamydomonas Genetics Center at Duke University, United States. Because she had gathered a huge collection of mutants over the years, from all major Chlamydomonas research laboratories throughout the world, she acquired a unique knowledge of the diversity of Chlamydomonas biological traits. Twenty years later, the development of new molecular biology tools applied to studies of Chlamydomonas led Elizabeth Harris to deliver a fully updated, second edition of the Chlamydomonas Sourcebook. As she noted in her preface to this second edition, more than 4000 papers referring to Chlamydomonas had been published in those 20 years (1989–2009). To handle the plethora of new information that had arisen from Chlamydomonas research, she partnered with two leading figures in the field, an expert in chloroplast gene expression, David Stern from the Boyce Thompson Institute, Unites States, and an expert in flagellar/ciliary biology, George Witman from the University of Massachusetts Medical School, Unites States. The second edition of the Sourcebook thus spanned three volumes covering the spectrum of Chlamydomonas research from an Introduction to Chlamydomonas and Its Laboratory Use (volume 1, E. Harris, ed.) to Organellar and Metabolic Processes (volume 2, D.B. Stern, ed.) and Cell Motility and Behavior (volume 3, G.B. Witman, ed.).

Over the next 13 years (2009–22), more than 11,000 papers that refer to Chlamydomonas were published. Part of the increasing attraction and success of Chlamydomonas as a model organism has come with the advent of the -omics age, with the constant efforts of many investigators to produce new, widely available resources, such as improved genome sequences and annotation and genome-wide or selected mutant libraries. Producing a third edition of the Chlamydomonas Sourcebook was thus a timely initiative. For this third edition, we kept the same three volume format as the previous edition, updating many chapters and introducing new chapters when needed. This up-to-date compendium of articles is meant to serve as a detailed resource used by established members of the Chlamydomonas research community, those who have recently initiated studies on this alga because of its diverse and dynamic biology and those who just want to understand more about the biology of this organism.

I was lucky enough to get on board, as editors for this new adventure, three prominent scientists who had pioneered several areas of Chlamydomonas research over the past decades. Ursula Goodenough, who knows the cell biology of Chlamydomonas as if she had lived within this cell for ages is the editor of volume 1. Arthur Grossman, who has long studied the dynamic flexibility of Chlamydomonas metabolism that reflects the intracellular integration of organelles is, with me, the coeditor of volume 2. Susan Dutcher, who has been instrumental in the development of many new tools to study cell motility in Chlamydomonas, is the editor of volume 3. The four of us were thrilled with an enthusiasm that was shared by more than a hundred (co)authors who immediately accepted a role in contributing to these three volumes.

I do think that the four editors originally chose to work with Chlamydomonas for the power of its genetics. As we have moved into the age of genomics, some may think that genetics has become less critical. However, the options of combining various genetic backgrounds and of screening for unique genetic phenotypes in analyzing biological processes continue to confer a unique appeal to Chlamydomonas, especially in the context of the highly curated Chlamydomonas genome sequence. While the third edition of this major effort supports this notion, I would bet that the fourth edition will establish the power of this pairing even more!

Introduction

Ursula Goodenough

As summarized in the preface to these volumes, the indomitable Elizabeth Harris pioneered this project in 1989 and wrote the introductory volume 1 of the second edition. Her retirement, and the burgeoning flow of new information on Chlamydomonas, signaled the need for an edited first volume, where her eight chapters have been expanded to 18 chapters written by experts in particular fields. Notably, the topics covered by her 50-page Chapter 8, called Chlamydomonas in the Laboratory, are now covered by 9 chapters that detail our current knowledge of and practices in growth protocols, functional genomics, genome sequencing, nuclear and organelle heredity and transformation, microscopic localization of cellular components, mutagenesis and genome resequencing, library construction and utilization, and reverse genetics. These technique-focused chapters are joined by extensively updated chapters on core features of the organism: its phylogeny, ultrastructure, cell walls, regulation of gene expression, cell cycle and circadian rhythms, and sexual cycle. A chapter is also included on its close multicellular relatives such as Volvox. Collectively, this volume should provide both initiates and experienced Chlamydomonas researchers with an up-to-the-minute overview of how this remarkable organism operates and with specific approaches for obtaining broader and deeper understandings of its capabilities.

Chapter 1

The genus Chlamydomonas

Thomas Pröschold¹ and Tatyana Darienko¹, ²,    ¹Research Department for Limnology, University of Innsbruck, Mondsee, Austria,    ²Institute of Microbiology and Genetics, Department of Applied Bioinformatics, University of Göttingen, Göttingen, Germany

Abstract

The genus Chlamydomonas has been known for almost 200 years and represents one of the largest green algal genera with more than 500 described species. This historical overview highlights the difficulties in the identification of Chlamydomonas-like organisms at the genus and species level. Phylogenetic analyses have revealed the polyphyly of the genera Chlamydomonas and Chloromonas. Molecular data reduce the genus Chlamydomonas to only three species: Chlamydomonas reinhardtii and its closest relatives. These join other genera, some of which continue to be called Chlamydomonas but are in the process of being re-named and entered into several families or orders among the Chlorophyceae and Chlamydophyceae. An overview of these additional genera is given in this chapter. C. reinhardtii is used as model organism in many laboratories because of its easy cultivation and genetic manipulability. The origin of the standard laboratory strains has been obscured by multiple exchanges among research laboratories and culture collections without clear documentation. New wild-type isolates of C. reinhardtii have provided information about natural genetic variability. The phylogeny and ecology of Chlamydomonas-like organisms are briefly discussed.

Keywords

Chlamydomonas; Chloromonas; genealogy; phylogeny; integrative approach

1.1 Introduction

The genus Chlamydomonas represents one of the largest green algal genera, with more than 500 described species. These have traditionally been designated as biflagellates, but current practice, adopted in this edition of the Chlamydomonas Sourcebook, is to refer to the eukaryotic organelle as a cilium and the bacterial organelle as a flagellum; hence they are designated as biciliates. This historical overview highlights the difficulties in identification of Chlamydomonas-like organisms at the genus and species level. Phylogenetic analyses have revealed the polyphyly of the genera Chlamydomonas and Chloromonas. As consequence of molecular data, the genus Chlamydomonas is reduced to only three species, Chlamydomonas reinhardtii and its closest relatives. These join other genera, some of which continue to be called Chlamydomonas but are in the process of being re-named in several families or orders among the Chlorophyceae or Chlamydophyceae. The classification of green algae into families, orders, and classes has a long history and is still under discussion. Traditionally, Chlamydomonas-like organisms were grouped to Chlamydomonadales, Volvocales, or simply classified as chlorophyte.

C. reinhardtii is used as model organism in many laboratories because of its easy cultivation and genetic manipulations. The origin of the standard laboratory strains has been obscured by multiple exchanges among research laboratories and culture collections without clear documentation. New wild-type isolates of C. reinhardtii have provided information about natural genetic variability. The phylogeny and ecology of Chlamydomonas-like organisms are briefly discussed.

1.2 Historical overview

Fig. 1.1 diagrams the ultrastructure of a typical Chlamydomonas cell, which displays the core features of Chlamydomonas-like organisms: a single cell surrounded with a cell wall containing a single green chloroplast with pyrenoid(s) (Ettl, 1976). Variations include the absence of a wall, variations in pyrenoid size and number, and the presence of four rather than two cilia.

Figure 1.1 Cell structure of a typical Chlamydomonas cell. BB, Basal body; CP, chloroplast; CV, contractile vacuoles; CW, cell wall; CY, cytoplasma; ER, endoplasmatic reticulum; ES, eyespot; F, flagella; G, Golgi apparatus; L, lipid drops; M, mitochrondria; N, nucleus; OG, osmophilic globuli; S, starch grains; P, cell wall papilla; PM, plasma membrane; PY, pyrenoid; V, vacuole. Modified after Ettl, H. (1976). Die Gattung Chlamydomonas Ehrenberg (Chlamydomonas und die nächstverwandten Gattungen II). Beihefte zur Nova Hedwigia, 49, 1–1122.

The genus Chlamydomonas was named by Ehrenberg (1833, 1838) for a green algal biciliate, which was originally called Monas by Müller (1786). Müller’s Monas pulvisculus differed from other species of Monas by the presence of a cell wall (Greek: chlamys, a cloak or mantle). Therefore, Ehrenberg transferred M. pulvisculus to his newly erected genus Chlamydomonas and provided illustrations showing biciliated cells with a single chloroplast containing pyrenoids. Unfortunately, it is not possible to ascertain which species of Chlamydomonas Müller investigated on the basis of his provided figures (Fig. 1.2A). The figures of Chlamydomonas pulvisculus by Ehrenberg (1838) showed many cells, which could belong to different species of Chlamydomonas or Chloromonas (Fig. 1.2B–D).

Figure 1.2 (A) Original colored lithograph of Monas pulvisculus of Müller (1786), and (B–D) original colored lithograph of Chlamydomonas pulvisculus of Ehrenberg (1838), containing several organisms, which can be identified as different species of Chlamydomonas (B: species of the section Amphichloris containing two pyrenoids, C: Chlamydomonas reinhardtii with a smooth chloroplast and Chlamydomonas noctigama with flagellated sporangia, and D: Chloromonas with perforated chloroplast without pyrenoids and several other species of Chlamydomonas).

Traditionally, Chlamydomonas comprises all biciliated green algae with a cell wall, two cilia of equal length, and a single chloroplast with pyrenoid(s) (Fig. 1.1) (Ettl, 1976). Since the first description, many species of Chlamydomonas have been described. Dangeard (1888) described C. reinhardtii as C. pulvisculus sensu Reinhardt (1876) and was the first to observe sexual reproduction. The number of Chlamydomonas species has dramatically increased since. Goroschankin (1890, 1891, 1905), Dill (1895), and other authors established additional species, so that in the first compendium provided by Pascher (1927), 146 species were already included. Species without pyrenoids were separated into the section Chloromonas by Pascher (1927) or even described as separate genus Chloromonas by Gobi (1899/1900), which was emended by Wille (1903). Detailed information about all described species was given in Gerloff (1940) and Ettl (1965, 1970, 1976, 1983). Despite the large number of species (421 Chlamydomonas, 139 Chloromonas, Ettl, 1983), no morphological synapomorphy was identified because characters can also be found in species of other genera. Ettl (1976) already considered the classification of Chlamydomonas as artificial.

To get a better overview of the diversity of Chlamydomonas-like species, Pascher (1927) and Ettl (1976) subdivided them into nine sections based on number and position of pyrenoid(s) in the chloroplast (Fig. 1.3). All species were differentiated by morphological features such as cell shape and -size, shape of chloroplasts, cell wall papilla, position and shape of the eyespot and the position of the nucleus and pyrenoid(s). In addition, the types of asexual and sexual production were used for classification of Chlamydomonas and Chloromonas (Ettl, 1970, 1976).

Figure 1.3 Scheme of the genera Chlamydomonas and Chloromonas (top row) and its subdivision into sections/subgenera (bottom rows) color-coded according to Ettl (1976). Pyrenoids are highlighted in red.

The relationship among Chlamydomonas species was investigated by Schlösser (1976) by testing sensitivity to sporangium autolysins, currently termed vegetative lytic enzymes (VLE), which release daughter cells from the mother wall at the conclusion of mitosis (Chapter 3, Section 3.11). In the life cycle of C. reinhardtii (Fig. 1.4), two different lytic enzymes have been experimentally observed: VLE, and gamete autolysins (GLE; see review of Matsuda, 1988 and Chapter 3, Section 3.11). A third autolysin is probably responsible for the release of the meiospores after the meiosis in the zygote (zygote lytic enzyme: ZLE, C in Fig. 1.4). VLEs are species or species-group specific, and can be used for classification. Schlösser (1976, 1984) used CO2-enriched aerated cultures for synchronization of the life stages and distinguished 15 VLE groups from 65 strains of different Chlamydomonas species.

Figure 1.4 Life cycle of Chlamydomonas reinhardtii. Modified after Schlösser, U. G. (1976). Entwicklungsstadien- und sippenspezifische Zellwandautolysine bei der Freisetzung von Fortpflanzungszellen in der Gattung Chlamydomonas. Berichte der Deutschen Botanischen Gesellschaft, 89, 1–56.

GLEs are produced during gametogenesis of C. reinhardtii and released after ciliary agglutination of two gametes to dissolve their cell walls before gamete fusion (Claes, 1971). This enzyme is not stage-specific in its activity: it dissolves not only the cell walls of gametes, but also can release daughter cells from vegetative mother walls, and can be used for isolation of protoplasts from vegetative cells (Schlösser, Sachs, & Robinson, 1976). Matsuda, Musgrave, Van Den Ende, and Roberts (1987) demonstrated that GLE digested cell walls of several Chlamydomonas species (i.e., C. reinhardtii, C. incerta, C. globosa, C. debaryana) as well as taxa belonging to the colonial families Tetrabaenaceae and Goniaceae sensu Nozaki, Yamada, Takahashi, Matsuzaki, and Nakada (2014) of the Volvocales. The genera Basichlamys and Tetrabaena, which were originally designated as four-cell Gonium species, were assigned to the Tetrabaenaceae, and the genera Gonium and Astrephomene were assigned to the family Goniaceae. These results indicate a close relationship between the Chlamydomonas species and the colonial families, which was confirmed by Matsuda (1988).

1.3 Polyphyly of the genus and revised generic and species concept

Phylogenetic analyses of small subunit of the nuclear ribosomal operon (SSU) rDNA sequences have revealed that the genera Chlamydomonas and Chloromonas are polyphyletic and can be subdivided into eight clades (Fig. 1.5, blue) within the Chlamydophyceae (clockwise group of the Chlorophyceae; Pröschold, Marin, Schlösser, & Melkonian 2001, and references therein). Some of these taxa belonged to clades which contained species of other biciliated and coccoid (nonciliated) algae. Therefore, the species of Chlamydomonas needed to be taxonomically revised, a process that was initiated by Pröschold et al. (2001). In that first contribution, the oogamous species of Chlamydomonas were transferred to the new genus Oogamochlamys, others to the genus Lobochlamys, and the genus Chloromonas was emended. Because of the difficult nomenclatural situation of the traditional type species (C. pulvisculus), C. reinhardtii was proposed as the conserved type of the genus Chlamydomonas, replacing C. pulvisculus (Pröschold & Silva, 2007), a proposal which was accepted by the International Botanical Congress in 2011 in Melbourne (Australia).

Figure 1.5 Molecular phylogeny of the Chlamydophyceae (CW-group of the Chlorophyceae) based on SSU rDNA sequences. The eight clades containing Chlamydomonas-like organisms (CD, Chlamydomonas; CM, Chloromonas) are highlighted in blue. The colored circles after these clades mark the presence of Chlamydomonas species belonging to the sections/subgenera according to their morphology (see Fig. 1.3). The phylogenetic positions of the members belonging to the vegetative lytic enzyme groups sensu Schlösser (1976, 1984) are given in green boxes.

As a result of these phylogenetic studies, the number of species remaining within the genus Chlamydomonas was drastically reduced, and only includes the model organism C. reinhardtii P.A. Dangeard and its close relatives C. incerta Pascher and Chlamydomonas schloesseri Pröschold & Darienko. C. schloesseri, the latter described by Pröschold, Darienko, Krienitz, and Coleman (2018) based on morphology, autolysin cross experiments, and multiple gene analyses. The taxonomic status of the closest related strains Culture Collection of ALgae (Sammlung von Algenkulturen) at the University of Göttingen, Germany (SAG) 7.73 (originally designated as C. incerta) and SAG 81.72 (C. globosa) was clarified by Pröschold and Darienko (2018). The three Chlamydomonas species belong to the VLE group 1 sensu Schlösser (1976, 1984). The strains belonging to VLE group 2 showed a one-sided cross reactions in cross experiments to group 1 and had been identified as C. debaryana. This species was transferred to the newly erected genus Edaphochlamys by Pröschold et al. (2018). Edaphochlamys showed a closer affiliation to the colonial Tetrabaenaceae, whereas Chlamydomonas represents its own lineage or is more closely related to the Goniaceae, which was suggested by Matsuda et al. (1987) based on the cross reactions of GLE (Fig. 1.6).

Figure 1.6 Molecular phylogeny of Chlamydomonas and representatives belonging to the Tetrabaenaceae, Goniaceae, and Volvocaceae based on SSU and ITS rDNA sequence comparisons.Unicellular taxa are highlighted in green boxes. Modified after Pröschold, T., Darienko, T., Krienitz, L., & Coleman, A. W. (2018). Chlamydomonas schloesseri sp. nov. (Chlamydophyceae, Chlorophyta) revealed by morphology, autolysin cross experiments, and multiple gene analyses. Phytotaxa, 362, 21–38.

Phylogenetic analyses of SSU rDNA sequences have revealed that strains belonging to the same VLE group are closely related. For example, the taxonomic placement of Chlamydomonas applanata, for example, Pringsheim (VLE group 7) was revised by Ettl and Schlösser (1992) and later confirmed by Gordon, Rumpf, Shank, Vernon, and Birky (1995). Buchheim, Buchheim, and Chapman (1997) demonstrated that the strains belonging to VLE group 14 (Chlamydomonas noctigama Korshikov) are almost identical in their SSU rDNA sequences. Several strains belonging to VLE groups 9 and 10 were identified as Chlamydomonas segnis Ettl and Chlamydomonas culleus Ettl, respectively. Both species are closely related and were transferred to the genus Lobochlamys by Pröschold et al. (2001) using an integrative approach (Pröschold & Leliaert, 2007). The strains of the VLE groups sensu Schlösser belong to the Reinhardtii, Oogamochlamys, Moewusii, and Polytoma clades (see Fig. 1.5). These examples demonstrate that VLE sensitivity is a good biochemical marker for classification of species in this radiation.

1.4 Chlamydomonas reinhardtii as a model organism

The ease of cultivation and genetic transformation made C. reinhardtii to a model organism for aspects of cell and molecular biology, physiology, and biotechnology. Despite the original description by Dangeard (1888) who found C. reinhardtii in Europe, most strains and mutants used for genetic and biochemical analyses derived from a single collection made by G.M. Smith in the United States (Fig. 1.7). All have been presented in the literature as descendants of a mating pair (plus and minus) from his collection, supposedly derived from a single zygote in a soil sample taken from a potato field near Amherst, Massachusetts, in 1945. Sequence analysis supports the presumption of a common origin, but is inconsistent with the hypothesis that all three sublines (Sager, Cambridge, and Ebersold/Levine lines) derive from clonal propagation of individual progeny of a single zygote (Kubo, Abe, Saito, & Matsuda, 2002; Pröschold et al., 2005). Probably they are descendants from crosses made among F1 or subsequent progeny of a single zygospore in the Smith laboratory, but we cannot exclude the possibility that they derive from different zygotes collected from the same site. In any case, the three lines have been separated since at least the early 1950s (Fig. 1.7).

Figure 1.7 Genealogy of standard Chlamydomonas reinhardtii laboratory strains. Only records from the literature and the data summarized in the catalogs of the culture collections were included. Discrepancies to genetic data were explained in the text. Modified from Harris, E. H. (1989). The Chlamydomonas sourcebook. San Diego, CA: Academic Press; and Pröschold, T., Harris, E. H., & Coleman, A. W. (2005). Portrait of a species: Chlamydomonas reinhardtii. Genetics, 170, 1601–1610.

Kubo et al. (2002) and Pröschold et al. (2005) demonstrated that the three major sublines of the standard C. reinhardtii strains differed at five unlinked loci: (1) the mating type, found on linkage group VI (Harris, 1989; Chapter 8); (2 and 3) the genes nit1 and nit 2 (linkage groups IX and III, respectively) which, singly or together, prevent growth on nitrate; (4) the nucleolar organizer repeats (unmapped); and (5) the autolysins (see details in Pröschold et al., 2005). The genealogy of the strains belonging to the three sublines presented in Fig. 1.7 was reconstructed using the literature and the records in the catalogs of various culture collections.

In an independent study, Gallaher, Fitz-Gibbon, Glaesener, Pellegrini, and Merchant (2015) reinvestigated 39 commonly used laboratory strains of C. reinhardtii by genome sequencing. They found some discrepancies compared to those presented in Fig. 1.7 and provided a revised genealogy of five sublines: (1) CC-1691 (Sager subline), (2) CC-1009 (Cambridge subline), (3) CC-1690 and CC-1010 (Sager and Cambridge sublines), and (4) CC-124 and CC-125 (both Ebersold/Levine sublines). The genome sequences revealed several differences in haplotype patterns among all three sublines. Most of them appeared in the Sager subline. CC-1690, CC-1010, and SAG 73.72 (supposed to be identical with IAM-C8) had the same haplotype pattern, whereas CC-1691 differed significantly. However, the IAM-C9 and CC-1009 were also identical, indicating that mistransfers happened in culture collections. To clarify this situation, the strains of the Sager subline need to be reinvestigated by sequencing of all descendants deposited in the different culture collection such as Culture collection of the University of Texas at Austin, USA (UTEX), Microbial Culture Collection at the National Institute for Environmental Studies, Tsukuba, Japan (NIES), and SAG.

Within the Ebersold/Levine subline, it is very interesting that the cell wall–less strain CC-503 (which served as the original genome reference strain) showed the same haplotype pattern as the strains CC-125 and CC-620, whereas the strains CC-124 and CC-621, which should be identical according to the records, differed in their pattern. The origins of the standard laboratory strains still remain incomplete and need to be resolved by further studies.

1.5 Other wild-type isolates of Chlamydomonas reinhardtii

The standard laboratory strains as well as several thousands of mutants are available at the Chlamydomonas Resource Center (https://www.chlamycollection.org). All these strains originated from G.M. Smith as mentioned above. An additional strain from the Smith collection, SAG 54.72 (=CC-1373,=UTEX 1062), was described as separate species, Chlamydomonas smithii, by Hoshaw and Ettl (1966) based on slightly different morphology. However, this isolate is interfertile with standard laboratory strains (Hoshaw, 1965) and belongs to VLE group 1 (Pröschold et al., 2005, 2018; Schlösser, 1976, 1984).

Despite intensive searches in environmental samples, only ~30 natural isolates of C. reinhardtii are available, found in several culture collections (Table 1.1). These new wild-type strains were isolated from soil samples collected from different places in North America and Japan (Craig et al., 2019; Gross, Ranum, & Lefebvre, 1988; Nakada, Shinkawa, Ito, & Tomita, 2010; Nakada, Tsuchida, Arakawa, Ito, & Tomita, 2014; Sack et al., 1994; Spanier, Graham, & Jarvik, 1992).

Table 1.1

Interestingly, no isolate of C. reinhardtii from Europe is available despite the type locality of the original description by Dangeard (1888), which was probably in Europe. The origin of a few strains (SAG 11-31, SAG 11-32c, SAG 18.79, and SAG 77.81) could not be clarified, but some data indicate that these isolates derived from the Smith’s standard laboratory strains (Ferris, 1989; Pröschold et al., 2005). The same is possible for the strain NIVA-CHL13, which needs to be verified by genome sequencing.

The genetic diversity of most of available strains was demonstrated by sequencing of the chloroplast and mitochondrial (Gallaher, Fitz-Gibbon, Strenkert, Purvine, & Merchant, 2018) as well as of the nuclear genomes (Craig et al., 2019; Flowers, Hazzouri, Pham, Rosas, & Bahmani, 2015; Gallaher et al., 2015).

1.6 Phylogeny and ecology of Chlamydomonas-like organisms

As demonstrated in Fig. 1.5, Chlamydomonas-like organisms are distributed into eight lineages of the Chlorophyceae, which are designated here as clades based on the study of Pröschold et al. (2001). Nakada, Misawa, and Nozaki (2008) proposed slightly different naming for these clades according to the PhyloCode, which has been partially used in several publications. Monadoid samples identified as Chlamydomonas species and biciliated stages (zoospores, gametes) of coccoid genera and species are often not easy to distinguish from each other. For a clear identification, knowledge of the whole life cycle is necessary for systematics and nomenclature. The systematics of green algae and consequently of Chlamydomonas-like organisms is controversial and the use of traditional families and orders is dependent on the concepts used for classification (for details about the different concepts, see Pröschold & Leliaert, 2007). Ettl (1981) proposed the class Chlamydophyceae for Chlamydomonas-like organisms and their relatives based on morphological features and subdivided this class into four orders based on the organization level of vegetative cells/colonies. This subdivision represents the Chlamydomonadales (unicellular bi- and quadriciliates), Volvocales (colonial biciliates), Chlorococcales (zoosporine coccoids, Chlorococcum, and similar taxa), and Tetrasporales (biciliated palmelloid taxa). Phylogenetic analyses revealed that these orders are polyphyletic, which was already mentioned by Ettl (1981). However, the Chlamydophyceae as class correspond well with the clockwise group of Chlorophyceae, but the formal description needs emendations, which will be proposed soon (Pröschold et al., in preparation).

As consequence of the current status of the systematics, the clades containing Chlamydomonas-like organisms will only be named by a representative (Fig. 1.5, blue highlight). These clades probably represent separate orders of Chlamydophyceae.

1.6.1 Stephanosphaera clade

This lineage is one of the most diverse groups of the Chlamydophyceae. It contains monadoid and coccoid taxa with unicellular, sarcinoid (cell clusters), and colonial organization. To this clade belong several unidentified Chlamydomonas strains. It is uncertain if these strains can be assigned to described species of Chlamydomonas and Chloromonas or if they are only stages of coccoid or sarcinoid genera. Kawasaki, Nakada, and Tomita (2014) and Watanabe, Mitsui, Nakayama, and Inouye (2006) demonstrated that many of them are closely related to Chlorococcum and Chlorosarcinopis, respectively, two genera that are also polyphyletic. The volvocalean genera Balticola, Pascherina, Pyrobotrys, and Stephanosphaera also belong to this clade (Buchheim, Sutherland, Buchheim, & Wolf, 2013; Munakata, Nakada, Nakahigashi, Nozaki, & Tomita, 2016). All strains were isolated from aquatic and terrestrial habitats.

1.6.2 Tetragama clade

Only one strain, NIES-446, originally assigned as Chlorogonium metamorphum, forms this lineage, which was transferred to Chlamydomonas tetragama by Nozaki, Aizawa, and Watanabe (1996). Its phylogenetic position was revealed by SSU and rbcL sequences (Hepperle et al., 1998; Nozaki, Ohta, Morita, & Watanabe, 1998).

1.6.3 Polytoma clade

The genus Polytoma represents the nonphotosynthetic equivalent of Chlamydomonas. Phylogenetic analyses of SSU rDNA sequences have revealed that Polytoma uvella, the type species of the genus, and a few unidentified strains are closely related to the green species Chlamydomonas applanata, C. pulsatilla, and C. subcaudata (Pocock, Lachance, Pröschold, Kim, & Huner, 2004; Rumpf, Vernon, Schreiber, & Birky, 1996). Ettl and Schlösser (1992) studied the morphology of several strains assigned as different species of Chlamydomonas, which all belong to the VLE group 7. They concluded that they all belong to C. applanata, which was later confirmed by SSU rDNA sequences (Gordon et al., 1995).

1.6.4 Moewusii clade

The other Chlamydomonas species often used in genetics, C. moewusii (also known as Chlamydomonas eugametos) and C. noctigama (also known as Chlamydomonas monoica) belong to this clade. Closely related to these taxa are several species originally assigned to the coccoid genera Chlorococcum and Tetracystis (Watanabe & Lewis, 2017). Several other Chlamydomonas species originated from freshwater, marine, terrestrial, and acidophilic habitats (e.g., Chlamydomonas acidophila, Chlamydomonas raudensis, Chlamydomonas pitschmannii, or symbiotic Chlamydomonas hedleyi) are members of this clade, mixed with coccoid species (Gerloff-Elias, Spijkerman, & Pröschold, 2005; Nakada, Tomita, Wu, & Nozaki, 2016; Pocock et al., 2004; Pollio et al., 2005; Barcyte, Pusztai, Škaloud, & Eliás, 2022; Tesson & Pröschold, 2022). The taxonomic revision of this clade is in progress.

1.6.5 Microglena clade

Several cold-adapted and polar strains as well as few brackish and marine species assigned as Chlamydomonas are members of this clade, which was taxonomically revised using an integrative approach by Demchenko, Mikhailyuk, Coleman, and Pröschold (2012). Chlamydomonas monadina, a species reproducing by anisogamous sexual reproduction, the marine species Chlamydomonas uva-maris, and Chlamydomonas reginae were transferred to the genus Microglena. Nakada, Nozaki, and Tomita (2012), Nakada and Tomita (2014), and Nakada, Takahashi, and Tomita (2018) added a few more species (Microglena media, Microglena redcarensis) to this genus.

1.6.6 Chloromonas clade

This species-rich clade contains the taxonomically revised genus Chloromonas (Hoham, Bonome, Martin, & Leebens-Mack, 2002; Matsuzaki, Nozaki, & Kawachi, 2018; Pröschold et al., 2001 and references therein; Hoham & Remias, 2020 and references therein), Clainomonas, a quadriciliated genus with a distinct cell wall (Novis, Hoham, Beer, & Dawson, 2008), and Gloeomonas, a biciliated genus with distinctive ciliary insertions (Nakada, Matsuzaki, Krienitz, Tomita, & Nozaki, 2015; Nozaki, Nakada, & Watanabe, 2010). Most of these biciliates are widely distributed in snow and ice environments, others in freshwater habitats, and some in soil samples, which are periodically flooded by rain.

1.6.7 Oogamochlamys clade

The genus Oogamochlamys was established by Pröschold et al. (2001) for oogamous-reproducing Chlamydomonas species. The sister group of this genus is Lobochlamys, which comprises Chlamydomonas species with vegetative cells surrounded by a mucilage layer (L. segnis and L. culleus). The species concept of Lobochlamys was confirmed by autolysin data of Schlösser (1976, 1984). Closely related to both genera are the pheromone-producing Chlamydomonas allensworthii (Coleman, Jaenicke, & Starr, 2001; Starr, Marner, & Jaenicke, 1995) and the tetrasporalean genus Asterococcus (Nakazawa & Nozaki, 2004). Most of the species are widely distributed in terrestrial habitats.

1.6.8 Reinhardtii clade

As noted above, C. reinhardtii, C. incerta, and C. schloesseri are closely related to various colonial volvocalean genera (Fig. 1.6) (see Chapter 10). Further Chlamydomonas species such as Chlamydomonas asymmetrica, Chlamydomonas baca, Chlamydomonas rapa, and the Chlamydomonas-like genera Heterochlamydomonas (Watanabe, 2020) and Vitreochlamys (Nakazawa, Krienitz, & Nozaki, 2001) also belong to this clade, but represent own genera. Closely related are the genera Paulschulzia and Tetraspora, originally classified as Tetrasporales, and the sarcinoid genus Neochlorosarcina (Watanabe et al., 2006). All these species are widely distributed in freshwater and terrestrial habitats.

1.7 Other biciliated and quadriciliated unicellular genera

In addition to Chlamydomonas-like organisms, which are distributed in the eight clades described above, several biciliated and, occasionally, quadriciliated unicellular genera form their own lineages within the Chlamydophyceae (clockwise group of the Chlorophyceae [CW-group] of the Chlorophyceae; see Fig. 1.5).

The fusiform genus Chlorogonium is also polyphyletic. The type species, Chlorogonium euchlorum, and its relatives Chlorogonium capillatum and Chlorogonium elongatum, is sister of the genera Haematococcus, an important genus in biotechnology (Allewaert et al., 2015; Buchheim et al., 2013), and Brachiomonas, a genus widely distributed in rock pools of almost all coast lines (Buchheim et al., 2013), and form the so-called Chlorogonium clade (Nakada, Nozaki, & Pröschold, 2008; Nozaki et al., 1998). Other Chlorogonium species (Chlorogonium neglectum and Chlorogonium kasakii) belong with the genera Asteromonas and Dunaliella to the Dunaliella clade and were therefore transferred to the genus Gungnir (Nakada, Nozaki, et al., 2008).

Dunaliella species are widely distributed in marine, saline, and hypersaline environments in high abundances (Massjuk, 1973) and are of great importance in biotechnology. The genus is monophyletic (Assunção et al., 2012; Emami et al., 2015).

Phylogenetic analyses of Chlamydomonas-like organisms that form calcified and organic loricas, traditionally summarized in the family Phacotaceae, have revealed that they mostly belong to the Phacotus clade (Hepperle et al., 1998). Only the genus Dysmorphococcus represents its own lineage (Dysmorphococcus clade).

The investigated species of the quadriciliated genus Carteria (for details of all described species, see Ettl, 1979) are split into two lineages, Radiosa and Crucifera clades (Nozaki, Ito, Watanabe, Takano, & Kuroiwa, 1997), while another quadriciliated genus Hafniomonas represents its own lineage (Nakada, Suda, & Nozaki, 2007). Like Hafniomonas, the genus Polytomella, nonphotosynthetic quadriciliates, also forms its own clade closely to the Reinhardtii clade (MacDonald & Lee, 2015; Smith, Hua, & Lee, 2010).

Summarizing, existing studies (only key papers are cited here) have demonstrated the high morphological and genetic diversity among the Volvocales. The morphotype Chlamydomonas can be linked to almost every other lineage among the Chlamydophyceae. Therefore the taxonomy and nomenclature are still in progress and are often challenging because of the rules of International Code for Nomenclature of Algae, Fungi and Plants.

1.8 Conclusions

The taxonomic revision of Chlamydomonas-like organisms is in progress and the use of an integrative approach has been proposed. This polyphasic approach includes morphological characters, life history, biochemical features, and molecular signatures for classification into genera and species. Analyses of secondary structures (SSU, interval transcribed spacer [ITS]) are of great value for species delimitation. Coleman (2000, 2003, 2009) demonstrated that the presence of at least one compensatory base change (CBC) in the conserved region of ITS-2 between two specimens is correlated with mating incompatibility and hence speciation. The ITS-2/CBC approach has been successfully applied to Chlamydomonas-like organisms (Demchenko et al., 2012; Pröschold et al., 2018). The combination of traditional (morphology, biochemistry) and contemporary (molecular) characterization leads to a clear identification of species and genera and formed the basis for the selection of strains, from which the genomes will be sequenced (i.e., Hirashima, Tajima, & Sato, 2016; Leebens-Mack, Barker, & Carpenter, 2019).

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