Submitted Abstracts

TALKS

NEW VOLVOCACEAN ALGAE PROGRESSIVELY IDENTIFIED

Hisayoshi Nozaki1, Toshihiro K. Yamada1, Fumio Takahashi2, 3 and Takashi Nakada4
1.    Dept. of Biol. Sci., Grad. Sch. of Sci., University of Tokyo, Tokyo, Japan
2.    Grad. Sch. of Life Sci., Tohoku University, Sendai 980-8577 Japan
3.    JST, PRESTO, Saitama 332-0012, Japan
4.    Inst. Adv. Biosci., Keio University, Yamagata 997-0052, Japan

The Volvocaceae includes several genera that are distinguished from one another by vegetative colonies and sexual reproduction. Since Nozaki in Nozaki and Kuroiwa (1992, Phycologia) described Yamagishiella, no additional genera have been described in this family. Furthermore, until recently, no new species have been described in the genus Volvox since Starr (1970, J. Phycol.) described V. pocockiae.
Our recent taxonomic studies based on the cultured material and molecular phylogenetic analyses delineated three new species in the genus Volvox: V. (sect. Merrillosphaera) ovalis Nozaki & Coleman (2011, J. Phycol.), V. (sect. Volvox) ferrisii Isaka et al. (2012, J. Phycol. in press) and V. (sect. Volvox) kirkiorum Nozaki et al. in Isaka et al. (2012, J. Phycol. in press). Each of these three species has its unique morphological attributes and distinct phylogenetic position within the Volvocaceae, but lack of living material in some described species (e. g. V. capensis, V. spermatosphaera) has still been problematic in the taxonomy of the genus Volvox.
Very recently, a possible new genus was found from a lake in Japan. This organism is similar to Yamagishiella and Eudorina in having 16- or 32-celled spheroidal colonies without differentiation of somatic cells. However, it differs from Yamagishiella by its anisogamous sexual reproduction and from Eudorina in distribution of contractile vacuoles on the surface of the protoplasmicht of vegetative cells (cf. Yamada et al. 2008, Eur. J. Phycol.). Phylogenetic analyses demonstrated that it is sister to the genus Platydorina within the Volvocaceae.

CRYPTIC NON-GREEN ENDOSYMBIOSIS IN THE SECONDARY PHOTOSYNTHETIC EUKARYOTES, CHLORARACHNIOPHYTA, BASED ON EXTENDED PHYLOGENETIC ANALYSES

Y. Yang1, S. Maruyama2, H. Sekimoto3, H. Sakayama4 and H. Nozaki1
1.    Department of Biological Sciences, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan
2.    Department of Biochemistry and Molecular Biology, Dalhousie University, Sir Charles Tupper Medical Building, 5850 College Street, Halifax, Nova Scotia, B3H 4R2, Canada
3.    Department of Chemical and Biological Sciences, Faculty of Science, Japan Women’s University, 2-8-1 Mejirodai, Bunkyo-ku, Tokyo 112-8681, Japan
4.    Department of Biology, Graduate School of Science, Kobe University, 1-1 Rokkodai-cho, Nada, Kobe-shi, Hyogo 657-8501, Japan

Chlorarachniophyta is a group of photosynthetic eukaryotes harboring secondary plastids of a distinct green algal origin. Although previous phylogenetic analyses of nuclear genes encoding Calvin cycle enzymes demonstrated the presence of genes apparently not derived from green algal endosymbionts in Bigelowiella natans (Chlorarachniophyta), the origins of these “non-green” genes in “green” secondary phototrophs were unclear due to the limited taxon sampling. Here, we sequenced five new phosphoribulokinase (PRK)genes (from one euglenophyte, two chlorarachniophytes, and two glaucophytes) and performed an extended phylogenetic analysis of the genes. Our phylogenetic analyses demonstrated that the PRK sequences from three genera of Chlorarachniophyta were placed within the red algal clade. These “non-green” affiliations were supported by the taxon-specific insertion/deletion sequences in the PRK alignment. Our results suggest that PRK genes may have been transferred from a “red algal” ancestor to Chlorarachniophyta before the divergence of the “green” secondary phototrophs. Similarly, the extended phylogenetic analysis of sedoheptulose-1,7-bisphosphate (SBP) shows the same affiliation between chlorarachniophytes and red algae.

COLONIAL REPRODUCTION AND CELL CYCLE REGULATION IN VOLVOCINE ALGAE

Deborah Shelton
Dept. of Ecology and Evolutionary Biology, University of Arizona, Tucson, AZ, USA
 
A life cycle consists of patterns of growth, division, and separation of cells. In unicellular species, these cell-level processes are typically regulated and deployed to maximize cell-level fitness. After an evolutionary transition to multicellularity, these same cell-level processes are presumably regulated so as to maximize colony-level fitness. Using the volvocine algae as a model system, we are investigating how the regulation of cell growth, division, and separation changes as a multicellular life cycle emerges. One fundamental aspect of cell cycle regulation in volvocine algae is the number of divisions per instance of multiple fission, n. In unicellular species, n is fecundity. In colonial species, n is both fecundity and colony cell number—a colony-level feature with potential effects on colony growth and viability. The fitness effects of regulating n in a particular way could be substantially different for unicellular compared to colonial species, and indeed “genetic modulation of cell number” has been proposed as an early step in the volvocine transition to multicellularity. However, there is little basic knowledge on patterns of regulation of n in simple colonial genera such as Basichlamys and Gonium. We think that such patterns should form the basis for detailed hypotheses concerning changes in cell cycle regulation during a major evolutionary transition. We are quantifying the relationship between n and parental cell size for a variety of relatively “simple” colonial Volvocales. We use a variety of culturing conditions to create parental cell size variation and light microscopy to assay the size of parental cells and number of offspring cells. These data complement a mathematical modeling approach designed to explore the ways in which growth, division, and separation of cells can be arranged into cycles capturing some fundamental features of simple colonial volvocine algae.

MATING TYPE LOCUS OF GONIUM PECTORALE

Takashi Hamaji1, Yuko Mogi2, Patrick Ferris3, James Umen4, Ichiro Nishii5, Yoshiki Nishimura1 and Hisayoshi Nozaki2
1.    Kyoto University, Kyoto, Japan
2.    University of Tokyo, Tokyo, Japan
3.    University of Arizona, AZ
4.    Donald Danforth Plant Science Center, MO
5.    Temasek Life Sciences Laboratory, Singapore

The volvocine lineage is well-suited for a comparative genomic approach to the evolution of sexual dimorphism for three reasons: 1) Volvocine algae have a range of species that include isogamous through oogamous species (Nozaki et al. 2000, MPE). 2) Genome sequences of oogamous Volvox carteri and isogamous, unicellular Chlamydomonas reinhardtii are available (Merchant et al. 2007 Science; Prochnik et al. 2010 Science). 3) The genetic and molecular basis of mating type differentiation in C. reinhardtii has been well-studied such as the mating type locus (MT). Key genes involved in mating type differentiation are encoded on MT loci in C. reinhardtii (Ferris and Goodenough 1994 Cell; Ferris et al. 2002 Genetics), the isogamous 8- or 16-celled colonial Gonium pectorale (Hamaji et al. 2008 Genetics; 2009 J. Phycol.), oogamous Pleodorina starrii (Nozaki et al. 2006 Curr. Biol.) and V. carteri (Ferris et al. 2010 Science). Because identifying key factor(s) toward oogamy requires information about MT of intermediate species close to oogamous organisms (Charlesworth and Charlesworth 2010 Curr. Biol.), we have sequenced BAC clones containing the MT region of G. pectorale. Draft contigs of ~500 kb from mating type minus and ~400 kb from mating type plus show that MT in G. pectorale is potentially larger than in C. reinhardtii . Gene composition of MT loci among C. reinhardtii, G. pectorale and V. carteri have been compared and characterized. Divergence between alleles of shared genes in the rearranged (R) domain of MT sheds light on their history and potential cooption into the sexual cycle.

MAT3 DIVERGENCE AFTER THE EVOLUTION OF ANISOGAMY IN THE COLONIAL VOLVOCALES

Rintaro Hiraide1, Hiroko Kawai-Toyooka1, Takashi Hamaji2, James Umen3 and Hisayoshi Nozaki1
1.    Dept. of Biol., University of Tokyo, Tokyo, Japan
2.    Dept. of Botany, Kyoto University, Kyoto, Japan
3.    Donald Danforth Plant Science Center, St. Louis, MO, USA

The Volvocaceae are believed to be the most ideal lineage in which to study the evolution of sexual reproduction (Kirk 2006, Curr. Biol.). The transition from isogamy to anisogamy might have occurred within the Volvocaceae after the split between the Yamagishiella and Eudorina sub-lineages (Nozaki et al. 2000, MPE). For this reason, Eudorina plays an extremely important role in investigating the origin of male–female dimorphism. MAT3 is a mating locus gene encoding a cell size regulatory protein that is a potential candidate responsible for the evolution of anisogamy. The male and female alleles of MAT3 from the oogamous species Volvox are highly diverged from each other, but they are not diverged in isogamous Chlamydomonas (Ferris et al. 2010, Science). However, sequences of MAT3 homologs have not been previously studied in the anisogamous members of the Volvocaceae. Therefore we sought to identify the MAT3 homolog of Eudorina. We isolated the MAT3 gene from male and female strains of Eudorina sp. and found out that there are just two base differences (including one nonsynonymous change) in the coding region (3519bp) of MAT3 between the two sexes. These results suggest that the divergence of MAT3 between male and female occurred after the evolution of anisogamy within the Volvocaceae. Our phylogenetic tree of MAT3 proteins also supports this scenario.

ESTABLISHMENT OF A VOLVOX CHROMATIN IMMUNOPRECIPITATION (CHIP) PLATFORM TO ANALYZE THE FUNCTION OF THE MASTER CELL-FATE DETERMINATION REGULATOR REGA

Alicia D. Howard1, Daniela Strenkert2, Michael Schroda2 and Stephen M. Miller1
1.    Dept. of Biological Sciences, University of Maryland Baltimore County, Baltimore, MD, USA
2.    Max Planck Institute for Molecular Plant Physiology, Potsdam-Golm, Germany

Volvox carteri possesses two cell types: large, reproductive gonidia and small, motile somatic cells that resemble Chlamydomonas unicells in morphology but do not divide. The regA gene is expressed only in somatic cells and is essential for maintaining them in a terminally differentiated state. regA encodes a putative transcriptional repressor (RegA) that possesses a VARL domain, which resembles the DNA-binding SAND domain of several animal and plant transcription factors. RegA is hypothesized to repress growth and reproduction in somatic cells by binding (and repressing) the promoters of nuclear-encoded chloroplast protein genes (NECPGs), since NECPG transcripts do not accumulate in wild type somatic cells but are synthesized in somatic cells of regA mutants. As a first step toward testing this hypothesis we have developed a chromatin immunoprecipitation (ChIP) platform for Volvox and are using it to assay occupancy of histones in the upstream regions of NECPGs and several other genes. We have optimized a number of experimental parameters, including DNA-protein crosslinking, chromatin shearing, antibody incubations, and real time PCR conditions. As a first test of our platform we examined occupancy of histone H3 at the hsp70A and control promoters under ambient and heat shock conditions. Consistent with previous results others have reported for heat shock gene promoters in other organisms, our results indicate that heat shock induces marked displacement of histones from the hsp70A promoter. We are now applying our ChIP protocol in a ChIP-seq experiment to determine RegA targets. Progress toward the goal of identifying these targets will be reported.

LIGHT RECEPTORS OF VOLVOX CARTERI

Arash Kianianmomeni1 and Armin Hallmann2
1.    Institute of Biology, Experimental Biophysics, Humboldt-University of Berlin Invalidenstr., Berlin, Germany
2.    Department of Cellular and Developmental Biology of Plants, University of Bielefeld Universitätsstr., Bielefeld, Germany

The photosynthetic organisms like green algae of the family Volvocaceae use light receptor system to find optimal environmental conditions for photosynthesis and to avoid light damage. Many other survival processes like gene expression, protein and lipid synthesis are initiated and regulated by the light. In Volvox carteri, three groups of light receptors have been identified based on genome sequence database of JGI: phototropin (VcPHOT1), two cryptochromes (VcCRYp and VcCRYa) and eight member of rhodopsin-like receptor family (Vop1 and Vop2, VchR1, VchR2, VcCop5, VcCop6, VcCop7 and VcCop8).
VcPHOT1, VcCRYp and VcCRYa are homolog to known plant blue light receptors. In contrast, there is no evidence for red light receptors, so called phytochromes, in Volvox. However, only VchR1 and VchR2 have been characterized as bleu light receptors and no data is available regarding to the light spectra properties of other six members of rhodopsin-like receptors.
The characterization study of VchR1 and VchR2 show that these two genes are extremely expressed in somatic cells of Volvox. The expression of VchR1 is very high at the points of cell differentiation and release during both asexual and sexual life cycles. Moreover, the expression is very sensitive to environmental factors like light and temperature. VchR2 is more expressed at the point of cell differentiation, especially by sexual life cycle. Since blue light is involved by regulation of sexual life cycle in green algae, VchR2 might play a key role in sexual development of Volvox. The characterization of other blue light receptors indicates that their expression is regulated by cell type and developmental stages in Volvox.

NEW INSIGHTS INTO THE REGULATION OF REGA EXPRESSION IN VOLVOX CARTERI

Stephan G. Koenig and Aurora M. Nedelcu
Biology Department, University of New Brunswick, Fredericton NB, Canada

Although most multicellular organisms comprise one or more somatic cell lineages that do not have reproductive potential, the mechanistic basis for somatic cell differentiation is poorly understood. Volvox carteri is a simple multicellular organism with only two cell types: ca. 2,000 small somatic cells and up to 16 large reproductive cells. In this species, somatic cell fate is determined by regA—a master regulatory gene that is believed to encode a transcription factor, which represses nuclear genes coding for chloroplast proteins. regA is expressed only in somatic cells and its induction is thought to be strictly dependent on the size of cells at the end of embryogenesis (i.e., only cells smaller than 8um express regA and differentiate into somatic cells). However, the mechanism responsible for translating differences in cell size into differential regA expression is not known. We used a RT-qPCR approach to investigate the expression pattern of regA in both wild-type and regenerating somatic cells (i.e., cells that regain reproductive potential) under various experimental settings. Our data provide new insights into the regulation of regA expression and the mechanistic basis of somatic cell differentiation in V. carteri, as well as (2) the evolution of somatic cell differentiation in this group of algae.

ELEMENTAL MAPPING OF VOLVOX DURING OXIDATIVE STRESS

Oana Marcu1,2, Matthew Lera1,3, Helen Nichol4 and Samuel Webb5
1.    Space Biosciences Division, NASA Ames Research Center, Moffett Field, CA, USA
2.    Carl Sagan Center, SETI Institute, Mountain View, CA, USA
3.    Lockheed Martin IS&GS Civil, Moffett Field, CA, USA
4.    Department of Anatomy and Cell Biology, University of Saskatchewan, SK, Canada
5.    Stanford Synchrotron Radiation Lightsource, Stanford Linear Accelerator Center, CA, USA

A common response of all cellular life to changes in environmental conditions is the intracellular production of reactive oxygen species (ROS). At significant levels these can damage biomolecules, yet they also act as important signaling cues that ensure adaptive responses and survival. A stable intracellular redox milieu is crucial for the structural integrity of cells and for cell fate decisions[1]. This balance can be maintained by intracellular elements (transition metals and salt ions[2,3]) that mitigate the reactivity of oxygen species.
We have shown using microprobe X-ray fluorescence imaging that in Volvox carteri the production of reactive oxygen species in response to heat stress correlates with the cellular transit of several elements. The presence and ratio of these elements may be responsible for the regulation of reactive oxygen species formed during heat stress[4].
The distinct signature of germ vs somatic cells in Volvox indicates differential accumulation of elements during development, and tests the hypothesis that intracellular ions regulate ROS and thus the downstream pathways specific to each cell type during changes in environmental conditions.
[1] Burhans, W.C. and N.H. Heintz. Free Radic Biol Med, 2009, 47(9):1282-93
[2] Daly MJ, et al., PLoSBiol, 2007, 5(4): e92. doi:10.1371/journal.pbio.0050092
[3] Kish, A., et al., Environ Microbiol, 2009. 11(5): p. 1066-78.
[4] Nedelcu, A.M., et al, Proc Biol Sci, 2004. 271(1548): p. 1591-6.


THE FIDELITY OF PHOTOTAXIS IN VOLVOX CARTERI

Knut Drescher1,2, Raymond E. Goldstein1 and Idan Tuval1
1.    Department of Applied Mathematics and Theoretical Physics, University of Cambridge
2.    Present Address: Department of Molecular Biology, Princeton University

Along the evolutionary path from single cells to multicellular organisms with a central nervous system are species of intermediate complexity that move in ways suggesting high-level coordination, yet have none. Instead, organisms of this type possess many autonomous cells endowed with programs that have evolved to achieve concerted responses to environmental stimuli. Here experiment and theory are used to develop a quantitative understanding of how cells of such organisms coordinate to achieve phototaxis, by using the colonial alga Volvox carteri as a model. It is shown that the surface somatic cells act as individuals but are orchestrated by their relative position in the spherical extracellular matrix and their common photoresponse function to achieve colony-level coordination. Analysis of models that range from the minimal to the biologically faithful shows that, because the flagellar beating displays an adaptive down-regulation in response to light, the colony needs to spin around its swimming direction and that the response kinetics and natural spinning frequency of the colony appear to be mutually tuned to give the maximum photoresponse. These models further predict that the phototactic ability decreases dramatically when the colony does not spin at its natural frequency, a result confirmed by phototaxis assays in which colony rotation was slowed by increasing the fluid viscosity.

WHY MOTILITY?

John O. Kessler
Department of Physics, University of Arizona, Tucson AZ, USA

Volvocales live in environments ranging from deep waters, quiescent or agitated, to puddles with depth of one or a few centimeters. While deep habitats present concentrations or intensities such as nutrients and illumination, ranging from marginal to optimum, shallow ones do not. Swimming occurs in all of them. “Reasons” for swimming -- getting someplace that is environmentally “better”, escaping predators, improving the rate of inward-and-outward molecular transport, generation of intracolony gradients, conservation of obsolete evolved properties, or “just can’t help it” -- will be discussed.
Support by DOE DEAC02-06CH11357 is gratefully acknowledged.

A GENERAL ALLOMETRIC AND LIFE-HISTORY MODEL FOR THE TRANSITION TO MULTICELLULARITY AND CELLULAR DIFFERENTIATION

Cristian A. Solari1, John O. Kessler2 and Raymond E. Goldstein3
1.    CONICET Researcher, Laboratorio de Biología Comparada de Protistas, Departamento de Biodiversidad y Biología Experimental (FCEyN), Universidad de Buenos Aires, Buenos Aires, Argentina
2.     Department of Physics, University of Arizona, Tucson, AZ, USA
3.    Department of Applied Mathematics and Theoretical Physics, University of Cambridge, Cambridge, UK

The transition from unicellular, to colonial, to larger multicellular organisms has benefits, costs, and requirements. Here we present a simple model that explains the dynamics involved in the unicellular-multicellular transition using life-history theory and allometry. We model the two fitness components (fecundity and viability) and compare the fitness of hypothetical colonies of different sizes with varying degrees of cellular differentiation to try to understand the general principles that underlie the evolution of multicellularity. We argue that germ-soma separation evolved as a means to counteract the increasing costs and requirements of larger multicellular colonies. The model shows first that the cost of investing in soma decreases with size. Secondly, as reproduction costs increase exponentially with size for undifferentiated colonies, soma specialization can give an indirect benefit to the colony by decreasing such costs and a direct benefit by helping reproductive cells acquire resources for their metabolic needs. Thirdly, germ specialization is favored once soma has evolved and takes care of vegetative functions. Some volvocine green algae allometric relationships are used to explain the model. Our model and analysis shows that the cost of reproducing an increasingly larger group likely played an important role in the transition to multicellularity and cellular differentiation.

RAPID TRANSITION TOWARDS THE DIVISION OF LABOR VIA EVOLUTION OF DEVELOPMENTAL PLASTICITY

Sergey Gavrilets
Department of Ecology and Evolutionary Biology, Department of Mathematics, National Institute for Mathematical and Biological Synthesis, University of Tennessee, Knoxville, TN, USA
A crucial step in several major evolutionary transitions is the division of labor between components of the emerging higher-level evolutionary unit. Examples include the separation of germ and soma in simple multicellular organisms, appearance of multiple cell types and organs in more complex organisms, and emergence of casts in eusocial insects. How the division of labor was achieved in the face of selfishness of lower-level units is controversial. I present a simple mathematical model describing the evolutionary emergence of the division of labor via developmental plasticity starting with a colony of undifferentiated cells and ending with completely differentiated multicellular organisms. I explore how the plausibility and the dynamics of the division of labor depend on its fitness advantage, mutation rate, costs of developmental plasticity, and the colony size. The model shows that the transition to differentiated multicellularity, which has happened many times in the history of life, can be achieved relatively easily. My approach is expandable in a number of directions including the emergence of multiple cell types, complex organs, or casts of eusocial insects.

COMPLEXITY AND INDIVIDUALITY IN THE VOLVOCINE ALGAE

Matthew D. Herron1,2
1.    Dept. of Ecology and Evolutionary Biology, University of Arizona, Tucson, AZ, USA
2.    Department of Zoology, University of British Columbia, Vancouver, BC, Canada

A single-celled protist such as Chlamydomonas is unambiguously an individual, as is a differentiated multicellular organism such as Volvox. By some criteria at least, multicellular organisms are more complex than unicellular ones. What are these criteria, and how does this increase in complexity take place? At what point during the evolution of multicellular organisms do groups of cells become individuals in their own right? I consider these questions in light of the developmental changes involved in the evolution of multicellularity in the volvocine algae.

VOLVOCALEAN ORGANELLE GENOME EVOLUTION

David Roy Smith
Canadian Institute for Advanced Research, Department of Botany, University of British Columbia, Vancouver, BC, Canada

Some of the most diverse and unusual mitochondrial and plastid DNAs from all eukaryotes come from green algae, particularly volvocalean algae. This talk explores the organelle genome architectural diversity within the Volvocales, and green algae as whole, and uses population genetic data from model algal species, including Volvox carteri, to address contemporary hypothesis on genome evolution. It is argued that the genomes of green algae are fashioned by nonadaptive forces.

NEXT GENERATION TRANSCRIPTOME SEQUENCING PROVIDES INSIGHTS INTO THE GENETIC CONTROL PROGRAM FOR SEXUAL DIFFERENTIATION IN VOLVOX CARTERI

Sa Geng1, Peter L. De Hoff2, Bradley J. S. C. Olson3, Rhitu Rai2 and James G. Umen1
1.    Donald Danforth Plant Science Center, St. Louis, MO, USA
2.    The Salk Institute for Biological Studies, La Jolla, CA, USA
3.    Division of Biology,Kansas State University, Manhattan, KS, USA
Volvox carteri makes sexually dimorphic gametes with large eggs and small sperm, and does so in response to a species-specific sex-inducer protein rather than in response to –N as occurs in Chlamydomonas and other Volvocine genera. The Volvox sex determining locus, MT, has two haplotypes, MTF and MTM, that are significantly expanded and diverged relative to MT+ and MT- in Chlamydomonas. We have used RNA-seq based transcriptome analysis to compare the program of sexual differentiation in Volvox from vegetative and sexual stages. These data identified at least five classes of autosomal genes whose expression is under control of the MTF and MTM haplotypes. These include i) genes that are regulated by sex inducer independent of gender, ii) genes that are co-regulated by both sex inducer and MTM, iii) genes that are coregulated by sex inducer and MTF, iv) genes that are female biased in expression independent of sex inducer, and v) genes that are male-biased in expression independent of sex inducer. Class I genes are likely involved in general aspects of sexual differentiation, while class II and III genes are likely to be involved in gender-specific functions. Interestingly, the number of genes in class II (male, sex specific) is greater than in class III (female, sex specific) and matches the observation that sperm are more highly differentiated cell types than eggs. The existence of Class IV and V genes was not anticipated, but they indicate that even in their asexual phase where they are morphologically identical, Volvox males and females have different autosomal gene expression profiles under the control of MTM and MTF. Together these data provide the basis for deciphering the network of gene expression involved in male and female sexual differentiation in Volvox.

THE VOLVOCALES GENOME PROJECT

Bradley J.S.C. Olson1, Pierre Durand2, Hisayoshi Nozaki3, Patrick Ferris4 and Richard E. Michod4
1.    Ecological Genomics Institute, The Division of Biology, Kansas State University, Manhattan, KS, USA
2.    Evolutionary Medicine Laboratory, Department of Molecular Medicine and Haematology, University of the Witwatersrand, Parktown, South Africa
3.    Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo, Japan
4.    Department of Ecology and Evolutionary Biology, The University of Arizona, Tucson, AZ, USA

The Volvocales and their unicellular relatives have long been viewed as an important model system for the evolution of multicellularity and cellular differentiation. Chlamydomonas and to a lesser degree Volvox are well developed molecular-genetic model systems, but other Volvocacean species representing important evolutionary steps toward multicellularity, have not been as well developed. The completion of the genomes of Chlamydomonas and Volvox has demonstrated that even though these organisms differ markedly in the morphology, their genomes are surprisingly similar. This suggests that the evolutionary path to multicellularity and cellular differentiation requires only a few genetics changes. With the availability of second-generation sequencing technology, the Volvocales Genome Project seeks to focus initially on sequencing the genomes of Gonium pectorale, Eudorina elegans and Pleodorina starii. The project goals, sequencing and annotation strategies will be presented as well as an initial analysis of the genome assemblies.

THE VOLVOX GENOME PROVIDES INSIGHTS INTO EVOLUTIONARY STRATEGIES FOR EVOLVING COMPLEXITY

Simon Prochnik1, James Umen2, Aurora M Nedelcu3, Armin Hallmann4, Stephen M Miller5, Ichiro Nishii6, Jeremy Schmutz7, Jane Grimwood7 and Daniel Rokhsar1,8
1.    DOE Joint Genome Institute, Walnut Creek, CA, USA
2.    Donald Danforth Plant Science Center, St. Louis, MO, USA
3.    University of New Brunswick, Biology Department, Fredericton, Canada
4.    University of Bielefeld, Plant Biology, Bielefeld, Germany
5.    University of Maryland, Biological Sciences, Baltimore, MD, USA
6.    Nara Women's University, Biological Sciences, Nara Prefecture, Japan
7.    HudsonAlpha, Institute for Biotech, Hunstville, AL, USA
8.    UC Berkeley, Centre for Integrative Genomics, Berkeley, CA, USA

Muliticellularity has evolved in half a dozen major eukaryotic groups. Outside the opisthokonts, the closely-related chlorophytes Volvox carteri and Chlamydomonas reinhardtii represent the first pair of sequenced species that span the evolution of multicellularity. Comparisons of the genome sequences and the ~15,000 predicted protein coding genes apiece reveal insights into the genetic changes that accompanied the evolution of multicellularity within the green algal lineage. A core set of nearly 2,000 genes is uniquely shared between the two chlorphyceae. Strikingly, those gene families associated with extra-cellular matrix are dramatically larger in Volvox than Chlamydomonas. These clade-specific gene families likely provided the raw materials for changes that accompanied the evolution of multicellularity, in contrast to the large-scale invention of proteins accompanying the appearance of metazoans or the signal transduction protein based evolutionary strategy for increasing complexity recently revealed by the sequencing of multicellular Ectocarpus (Phaeophytes). Directed genome finishing and deep transcript sequencing in both chlorophyte species has improved genome assemblies and annotations. For example the v.4 Chlamydomonas assembly is now chromosome-scale and 5.4 million 454 EST sequences have modified 48% of the current 17,114 gene models. These updated resources increase the resolution of genome-scale comparisons.
Molecular and Evolutionary Genetics (Chair: James Umen)

ELUCIDATING THE ORIGINS OF THE REGA GENE

P. Ferris, Q. Li, E. Hanschen, P. Durand and R. E. Michod
Dept. of Ecology and Evolutionary Biology, University of Arizona, Tucson, USA

Commentators often view the most significant event in the Volvocalean evolution as the appearance of a division of labor between reproductive and somatic cells that is most apparent in the largest colonials, the genus Volvox. Mutant searches in V. carteri identified only a single locus, regA, with a clear-cut effect on germ-soma determination: the apparent somatic cells of the mutant spheroids soon regenerate into reproductive cells, instead of undergoing cell death. The regA gene has been characterized (Kirk et al 1999), and shown to be a VARL (SAND) domain-containing transcription factor. A phylogenetic tree of all the predicted VARL domain genes in the Chlamydomonas and Volvox genomes has been constructed (Duncan et al. 2007) and shows that the regA gene is one of four paralogs whose only close Chlamydomonas relative is the RLS1 VARL gene.
We are currently completing the sequence of the regA gene from three species closely related to V. carteri f. nagariensis, identified using degenerate primer PCR. All of these species set aside their germ line by unequal cleavage during embryogenesis. We are now moving to the more interesting question of whether regA can be identified in Volvox species that do not have unequal cleavage and in Pleodorina. Attempts to isolate regenerator mutants in Volvox species other than V. carteri are also planned.

EVOLUTION OF THE VARL GENE FAMILY AND GERM/SOMA DIFFERENTIATION IN THE VOLVOCINE ALGAE

Erik R. Hanschen1, Jonathan Featherston1, Pierre Durand2 and Richard E. Michod1
1.    Dept. of Ecology and Evolutionary Biology, University of Arizona, Tucson, AZ, USA
2.    Evolutionary Medicine Laboratory, Department of Molecular Medicine and Haematology, University of the Witwatersrand, Parktown, South Africa

One of the defining characteristics of an evolutionary transition to multicellularity is the formation of terminal cellular differentiation. The evolution of specialized somatic and germ cells is almost universally the first cellular differentiation in multicellular species, reflecting its fundamental importance in the transition to multicellular life. Due to its importance in determining cellular differentiation in volvocine algae, understanding the evolution of the regA gene and the VARL (regA-like) gene family may elucidate the role of gene duplication in the evolution of multicellularity. Previous analyses have studied the relationship of VARL genes in Chlamydomonas reinhardtii and Volvox carteri, but it is difficult to resolve the evolutionary history with just two species. Newly sequenced Pleodorina starrii genomic data will be included into previous analyses of the VARL gene family. An analysis of gene synteny and gene phylogenies containing Chlamydomonas reinhardtii, Volvox carteri, and Pleodorina starrii provides insight into the role and timing of the VARL domain duplications in the evolution of cellular differentiation.

THE POPULATION GENETICS OF MT: RESOLVING THE DIVERGENCE PARADOX BETWEEN CHLAMYDOMONAS AND VOLVOX MT GENES

James Umen1, Peter De Hoff2, Bradley Olson2,3 and Patrick Ferris2,4
1.    Donald Danforth Plant Science Center, St. Louis, MO, USA
2.    The Salk Institute, La Jolla, CA USA
3.    Kansas State University, Manhattan, KS USA
4.    University of Arizona, Tucson, AZ USA

Sexual reproduction in Volvocine algae coevolved with the acquisition of multicellularity. Comparative analyses of Chlamydomonas reinhardtii and Volvox carteri mating loci provided insights into how the colonial Volvocine algae might have evolved sexual dimorphism (Ferris, Olson et al, Science 328 p351, 2010), but also raised questions about why the MT locus in Chlamyomonas shows less divergence between haplotypes than expected (Charlesworth, Current Biol. 20:12 p R250, 2010). The paradox of an unexpectedly youthful-looking MT locus in Chlamydomonas might be resolved if its evolution could be observed on longer time scales than allowed by laboratory crosses. Here we have used sequencing of MT genes from different geographic isolates of Chlamydomonas to examine the dynamics of MT evolution in this species. Our data reveal lower than expected nucleotide diversity in MT genes, and strong evidence of genetic interactions between shared genes in the plus and minus haplotypes. These interactions include gene conversion within the Rearranged (R) domain and crossover recombination in the flanking regions, none of which are observed in laboratory crosses. These data resolve the age paradox for Chlamydomonas MT and provide a model for how shared genes in the Chlamydomonas MT locus remain relatively homogenous and “youthful”. They also highlight the fundamentally different evolutionary trajectories of the C. reinhardtii and V. carteri MT loci.

Poster Session

EMBRYONIC INVERSION IN VOLVOCINE ALGAE

Stephanie Hoehn and Armin Hallmann
Dept. Cell & Dev. Biol. Plants, Bielefeld University, Bielefeld, Germany

One key step in the evolution from unicellular to multicellular organisms in volvocine algae is the so called "inversion", during which embryos turn themselves right-side out. In Volvox inversion is essential, as at the end of the embryonic cleavage, the flagellar ends of all cells point towards the interior of the embryo rather than toward the exterior where they are needed for locomotion [1], [2]. Earlier work in Volvox carteri showed that this morphogenetic process is driven by cell shape changes and cell movements relative to cytoplasmic bridges. [3]. However, not much is known about the underlying mechanisms and the purpose of inversion especially in related volvocine algae with a quite simple type of inversion (e.g. Gonium).
In order to learn more about this morphogenetic process and its evolutionary origins, inversion in several species was analyzed by light and electron microscopy.
[1] Viamontes, G. I., Kirk D. L. 1977. Cell shape changes and the mechanism of inversion in Volvox. J. Cell Biol. 75:719-730.
[2] Hallmann, A. 2006. Morphogenesis in the family Volvocaceae: different tactics for turning an embryo right-side out. Protist 157:445-461.
[3] Nishii, I., S. Ogihara and D. L. Kirk. 2003. A kinesin, invA, plays an essential role in Volvox morphogenesis. Cell 113:743-753.

MECHANICAL ASPECTS OF INVERSION IN VOLVOX CARTERI

Aurelia R. Honerkamp-Smith, Jocelyn Dunstan Escudero and Raymond E. Goldstein
Department of Applied Mathematics and Theoretical Physics, University of Cambridge

Volvox carteri embryos complete their development with a dramatic inversion of the hollow sphere of cells. This process has been extensively studied [1], and in particular, the recent work of Nishii et. al. [2,3] has shown that inversion is a two-stage process, comprising a traveling wave of microtubule and kinesin dependent cell rearrangement followed by an actin and myosin dependent contraction of the posterior half of the embryo. Our goal is to quantitatively measure the force generated by this actomyosin contraction and the elastic properties of embryonic cells in order to understand inversion from a mechanical standpoint.
References:
[1] Kirk, D. L. 1998. Volvox: molecular-genetic origins of multicellularity and cellular differentiation. Cambridge University Press.
[2] Nishii, I., S. Ogihara and D. L. Kirk. 2003. A Kinesin, InvA, plays an essential role in Volvox morphogenesis. Cell 113:743.
[3] Nishii, I. and S. Ogihara. 1999. Actomyosin contraction of the posterior hemisphere is required for inversion of the Volvox embryo. Development 126:2117.

PHOTOTAXIS IN THE MULTICELLULAR GREEN ALGA VOLVOX

Noriko Ueki1, Shigeru Matsunaga2, Isao Inouye3 and Armin Hallmann1
1.    Department of Cellular and Developmental Biology of Plants, University of Bielefeld, Germany
2.    The Promotion Center for Research and Education, Graduate University for Advanced Studies (Sokendai), Japan
3.    Institute of Biological Sciences, University of Tsukuba, Japan

Phototaxis is essential for volvocine green algae because they need to orientate themselves to receive sufficient light for photosynthesis. We analyzed the phototactic behavior in the spheroidal, multicellular volvocine green alga Volvox rousseletii (Volvocales, Chlorophyta) to address the question of how a multicellular organism accomplishes phototactic swimming without any known direct communication among cells. In response to light stimuli, not only did the flagella waveform and beat frequency change, but the effective stroke was reversed. Moreover, there was a photoresponse gradient from the anterior to the posterior pole of the spheroid, and only cells of the anterior hemisphere showed an effective response. The latter caused a reverse of the fluid flow that was confined to the anterior hemisphere. The responsiveness to light is consistent with an anterior-to-posterior size gradient of eyespots. At the posterior pole, the eyespots are tiny or absent, making the corresponding cells appear to be blind. Pulsed light stimulation of an immobilized spheroid was used to simulate the light fluctuation experienced by a rotating spheroid during phototaxis. The results demonstrated that in free-swimming spheroids, only those cells of the anterior hemisphere that face toward the light source reverse the beating direction in the presence of illumination; this behavior results in phototactic turning. On the basis of our results, we developed a mechanistic model that predicts the phototactic behavior in V. rousseletii. Our results also indicate how recently evolved multicellular organisms adapted the phototactic capabilities of their unicellular ancestors to multicellular life.

ANALYSIS OF VARL DOMAIN EQUIVALENCY AMONG THE REGA-GROUP VARL PROTEINS

Jose Ortega and Stephen Miller
Dept. of Biological Sciences, University of Maryland Baltimore County, Baltimore, MD, USA

Somatic cell fate in Volvox carteri is controlled by RegA, a putative transcription factor that shares a conserved domain (the VARL domain) with 13 other V. carteri and 12 Chlamydomonas reinhardtii proteins known as Rls (V. carteri) and RLS (C. reinhardtii) proteins. The VARL domain is related to the SAND domain, which in certain animal proteins has been shown to have DNA-binding and protein-binding activity. To determine whether the VARL domains of the Rls/RLS proteins most closely related to RegA (the RegA-group VARL proteins) might be functionally equivalent to that of RegA, we made chimeric regA genes encoding proteins with VARL domains from five different Rls/RLS proteins and tested their ability to rescue the phenotype of a regA mutant strain. Our results provide insights into the functional divergence of these VARL domains and into the evolution of the RegA-group proteins.

ABUNDANCE VARIATION OF VOLVOCALES IN RESPONSE TO TEMPERATURE CHANGE

V. J. Galzenati, V. Conforti and C.A. Solari.
DBBE, Facultad de Ciencias Exactas y Naturales, UBA, Buenos Aires, Argentina.

Temperatures are expected to increase by approximately 2-4.5°C over the next 100 years. Being able to understand the effects of this change on abundance is of high importance. Although physiologists have a well-developed theory for how the intrinsic rate of increase (r) changes with temperature (T), r has little predictive power about outcomes in competitive systems, and most populations do not experience exponential growth; they level off to a carrying capacity (K) or a stochastic equilibrium (N*). In short, the physiological predictions about r do not necessarily translate into predictions about K or N*. As far back as 1934, Gause suggested that N* shows systematic patterns as a function of T, but this has received little follow up work. To increase our scarce knowledge on how abundance responds to temperature we developed microcosm experiments with the volvocine green algae as a model system. We propose to measure in semi-continuous cultures r and N* as a function of T in single species systems, and will extend the experiments to multi-species competitive systems. r and N* was estimated as function of number of cells and chlorophyll content. Preliminary results of Chlamydomonas reinhardtii and Gonium pectorale show that these organisms follow patterns similar to the ones measured by Gause. When analyzing N* vs. r, Gonium shows a strong positive correlation in number of cells, but there is no clear correlation in Chlamydomonas. Volvocales characteristics allow studying differences in size and complexity in response to temperature.

DEVELOPMENTAL RESPONSE OF V. CARTERI TO NUTRIENT DEPRIVATION

Alexandra Y. Harryman and Stephen M. Miller
Department of Biological Sciences, University of Maryland, Baltimore County,
Baltimore, MD, USA

Phosphorus, sulfur, and light are essential for the development and growth of almost all photosynthetic organisms. While the effects of nutrient and light limitation on growth and development have been well studied in some green species, they are not well documented for Volvox carteri. To this end we are analyzing the response of V. carteri to deprivation of light, phosphorus, and sulfur to determine how and when growth and development are affected by these environmental perturbations. We cultured V. carteri in the absence of light, phosphorus, and/or sulfur and documented the effect on its growth and development over the course of several generations. The effects of light deprivation were obvious within 24 hours; adult spheroids did not expand after juvenile somatic cell differentiation, and juveniles failed to expand after hatching. Surprisingly, phosphorus-starved individuals developed normally for nearly two life cycles to produce nearly-normal looking young adults in the second generation, but the gonidia of these adults were aberrantly condensed and failed to undergo embryogenesis. The development of sulfur-deprived individuals also appeared nearly normal into the second generation, but unlike the phosphorus-starved individuals, many gonidia of the young adults of that generation remained arrested in an uncondensed state, and the somatic cells of many individuals became enlarged. Individuals grown without both phosphorus and sulfur were affected mid-way through the second life cycle; while the first-generation adult spheroids contained gonidia that completed embryogenesis, their juveniles expanded only slightly and the gonidia within those juveniles remained small and became moribund. Interestingly, under all of the starvation conditions analyzed, most spheroids continued to swim throughout the course of the time study. These studies will be used to complement expression analyses for genes suspected of mediating these deprivation responses and analyses on cell viability. Ultimately our goal is to better understand the effect of the environment on V. carteri development, and to better understand how the interplay between environment and genetics shaped Volvocine lineage evolution.