Phylogeny and molecular evolution of green algae

176 Pages·2009·11.87 MB·English

Phylogeny and molecular evolution
of green algae
Ellen Cocquyt
Universiteit Gent
Faculteit Wetenschappen, Vakgroep Biologie
Onderzoeksgroep Algologie

Phylogeny and molecular evolution of green algae
Fylogenie en moleculaire evolutie van groenwieren

Ellen Cocquyt

Proefschrift voorgelegd tot het behalen van de graad van
Doctor in de Wetenschappen: Biologie
Academiejaar 2008-2009
Promotor: Prof. Dr. O. De Clerck (Universiteit Gent)
Co-promotor: Dr. H. Verbruggen (Universiteit Gent)

Leden van de leescommissie:
Prof. Dr. K. Hoef-Emden (Universität zu Köln, Germany)
Dr. P. Rouzé (Universiteit Gent)
Prof. Dr. A. Vanderpoorten (Université de Liège)

Overige leden van de examencommissie:
Prof. Dr. K. Sabbe (Universiteit Gent)
Prof. Dr. Erik Smets (Katholieke Universiteit Leuven and National Herbarium of the Nederlands)
Prof. Dr. W. Vyverman (Universiteit Gent)

Photographs cover: Frederik Leliaert and Heroen Verbruggen
Photographs upper band, from left to right and from top to bottom:
Boodlea, Phyllodictyon, Dictyosphaeria, Ulva, Cladophora and Valonia
Photographs lower band, from left to right:
Boergesenia, Codium, Halimeda, Dictyosphaeria and Cladophora
Photographs at the back, from left to right and from top to bottom:
Blastophysa, Trentepohlia, Ignatius and Chlamydomonas

The research reported in this thesis was funded by the Special Research Fund (Ghent University,
DOZA-01107605) and performed in the Research Group Phycology and the Center for Molecular
Phylogenetics and Evolution, Biology Department, Ghent University, Krijgslaan 281-S8, B-9000,
Ghent, Belgium. www.phycology.ugent.be
Dankwoord

Vooreerst zou ik mijn promotor Olivier willen bedanken. Bijna negen jaar geleden stapte ik op het
vliegtuig richting Zuid-Afrika. Ik mocht gedurende 2 maanden wieren inzamelen langsheen de Zuid-
Afrikaanse kust en zou leren ‘kijken’ naar roodwieren, samen met Olivier die daar toen gedurende
een jaar aan de Universiteit van Kaapstad werkte. Op de luchthaven van Kaapstad aangekomen vroeg
Olivier verwondert: “Is dat alles wat je mee hebt?”, wijzend naar mijn klein rugzakje. Tja, er zat een
rugby team op het vliegtuig en niet alle bagage was meegeraakt. De mijne stond nog in Londen. ’t Is
gelukkig allemaal goed gekomen en ik heb daar een fantastisch tijd gehad!
Eenmaal ik mijn licentiaatdiploma behaalde, ging ik nog even langs het labo om goedendag te
zeggen. Olivier stelde toen voor om met een Marie Curie beurs een tijdje bij Christine Maggs aan de
Queen’s University of Belfast te gaan werken. Hm, ik zou eigenlijk net gaan samenwonen met Toon.
Uiteindelijk ben ik toch vertrokken voor een half jaartje. Daar heb ik voor het eerst DNA
geëxtraheerd en PCR’s gedaan, en eveneens een fantastisch tijd beleeft. Terug in België mocht ik
onder voorwaarde dat ik een IWT beurs zou aanvragen, beginnen als laborante bij onze
onderzoeksgroep. Die IWT beurs werd niets, maar na anderhalf jaar kon ik dan toch beginnen aan
een doctoraat met een BOF beurs. Het resultaat daarvan is dit doctoraat!
Olivier, bedankt om me te begeleiden doorheen al die jaren.
Ten tweede, zou ik mijn co-promotor Heroen willen bedanken. Als laborante heb ik voor zijn
doctoraat veel praktisch werk gedaan, maar het laatste anderhalf jaar heb ik ontzettend veel hulp
van hem gekregen. Heroen, bedankt voor de hulp bij het analyseren van mijn gegevens en het snel
en grondig nalezen en verbeteren van de teksten.
Ten derde, zou ik Olivier, Heroen en Frederik willen bedanken om me te steunen. Zonder de talloze
brainstormmomenten en de hulp van jullie alle drie bij het verwerken en uitschrijven van de
resultaten, was ik nooit tot dit resultaat gekomen.
Caroline, het was leuk om gedurende drie jaar bureau en labo met je te delen. Eveneens bedankt
voor de hulp bij het praktisch werk. Andy en Renata, bedankt voor al het sequentiewerk.
Kadriye thanks to help me with PCR’s and cloning of some of the nuclear genes. It was often a
frustrating job, with a lot of trial and error.
Koen Sabbe, Ann Willems en Paul De Vos, bedankt voor de tijd die jullie hebben vrij gemaakt om,
vooral in het begin, te luisteren naar mijn vorderingen en me met jullie suggesties telkens een stapje
vooruit te helpen. Ook Steven Robbens en Yves van de Peer hielpen me door me in het begin de kans
te geven over het nog niet gepubliceerde Ostreococcus genoom te beschikken.
Klaus Valentin, thanks for the cDNA service. The generation of this cDNA library was a big step
forward during this PhD study. The people from VERTIS Biotechnologie AG (Freising , Germany) also
helped a lot to solve the problems I encountered during the screening of the cDNA library.
Aan de mensen van de plantkunde in de Ledeganckstraat, het was altijd leuk en gezellig tijdens de
middag of aan de koffietafel. Liesbeth, op het bankje aan het kleine vijvertje was het ook steeds
gezellig vertoeven. Ik heb daar goeie herinneringen aan!
Het laatste anderhalf jaar was het met de mensen van op de Sterre minstens even gezellig tijdens de
middagen in de Resto.
Katrien en Elke, bedankt voor het nalezen van een stukje Nederlandstalige tekst.
Eric, al wist je nooit goed waar ik nu precies mee bezig was, toch zou ik je willen bedanken om me
warm te maken voor de algologie, en om je vlucht naar Sri Lanka te verzetten zodat je aanwezig kunt
zijn op mijn publieke verdediging.
Ook mijn ouders en vrienden zou ik willen bedanken om me steeds te blijven steunen doorheen de
jaren.
En tenslotte, Toon die nu al bijna negen jaar lang mijn vriend is… nog vele fijne jaren voor ons!

Ellen
juni 2009
Contents

Chapter 1 General introduction and thesis outline 1
Chapter 2 Ancient relationships among green algae inferred from nuclear and 19
chloroplast genes
Chapter 3 Gain and loss of elongation factor genes in green algae 43
Chapter 4 Complex phylogenetic distribution of a non-canonical genetic code in green 69
algae
Chapter 5 Codon usage bias and GC content in green algae 81
Chapter 6 A multi-locus time-calibrated phylogeny of the siphonous green algae 91
Chapter 7 Systematics of the marine microfilamentous green algae Uronema curvatum 115
and Urospora microscopica (Chlorophyta)
Chapter 8 General discussion 129
References 143
Summary 161
Samenvatting 165

1
Introduction

Algae
Algae are a large and diverse group of eukaryotic photosynthetic organisms occurring in almost every
habitat. They exhibit a huge morphological diversity, ranging from tiny unicells to huge kelps over 50
m long. The first algal groups arose between 1 and 1.5 billion years ago (Douzery et al. 2004, Yoon et
al. 2004) after the symbiogenesis of a heterotrophic eukaryotic organism with a photosynthetic
cyanobacterium. This event gave rise to the primary plastids which are still present in the
Glaucophyta, red algae and green lineages including land plants (Reyes-Prieto et al. 2007). These
three lineages are collectively called Plantae or Archaeplastida (Cavalier-Smith 1981, Adl et al. 2005).
The other photosynthetic protists arose through secondary endosymbiosis of either a green or a red
alga. The euglenids and chlorarachniophytes are thought to have acquired their plastids from a green
alga in two separate secondary endosymbiotic events, while molecular evidence suggests that the
red algal plastid of cryptomonads, heterokonts, haptophytes, apicomplexans and dinoflagellates was
acquired by a single secondary endosymbiosis in their common ancestor (Archibald 2005, Archibald
2008). This process of serial cell capture and subsequent enslavement explains the diversity of
photosynthetic eukaryotes. Endosymbiosis forms the landmark evolutionary event, responsible for
the spread of photosynthesis through the Eukaryotic tree of life. Photosynthesis occurs in four of the
six supergroups: Archaeplastida (Glaucophyta, red algae, green plants), Chromalveolata
(cryptophytes, Stramenopila or heterokonts including diatoms and brown algae, haptophytes and
dinoflagallates), Rhizaria (Chlorarachniophyta) and Excavata (euglenoids) (Fig. 1).

Figure 1. Eukaryotic tree of life. The first
algae arose after the symbiogenesis of a
heterotrophic eukaryotic organism with a
photosynthetic cyanobacterium, giving rise
to the Archaeplastida. The other
photosynthetic protists arose through
secondary endosymbiosis of either a green
or a red alga and occur in four of six
supergroups (marked with respectively
green and red circles). The monophyly of
the Archaeplastida is well-supported and
most recent evidence favours the
Glaucophyta as earliest diverging lineage
within the Archaeplastida (modified after
Baldauf 2008, Lane and Archibald 2008).
2 CHAPTER 1

Archaeplastida
The monophyly of primary plastids has long been suggested by several features, such as a similar
gene content of plastid genomes, the presence of plastid-specific gene clusters that are distinct from
those found in Cyanobacteria, the conservation of the plastid-protein import machinery and protein-
targeting signals, and phylogenies based on plastid and cyanobacterial gene sequences (Palmer
2003). Nevertheless, several single-gene phylogenies and a few multigene phylogenies have
challenged this hypothesis (e.g., Stiller et al. 2001, Nozaki et al. 2003a, Nozaki et al. 2003b, Stiller and
Harrell 2005). Conclusive evidence for the monophyly of the Glaucophyta, red algae and green plants
was provided only relatively recently by Rodriguez-Ezpeleta et al. (2005) based on: (1) chloroplast
gene phylogenies showing the monophyly of primary plastid and (2) a phylogenomic dataset
containing 143 nuclear genes, ca. 30,000 amino acid positions which show the monophyly of all
organisms with a primary plastid (Fig. 1). The latter study, however, could not reveal the relation
among the three major lineages. Several nuclear genes suggest that red algae are the earliest
diverging Archaeplastida, but such results are inconsistent with many plastid gene trees that identify
glaucophytes as the earliest divergence. Most recent evidence favours the early divergence of
glaucophytes, as demonstrated by Reyes-Prieto et al. (2007) using a concatenated dataset of
conserved nuclear-encoded plastid targeted proteins of cyanobacterial origin. The latter evolutionary
scenario corroborates with two important putatively ancestral characters shared by glaucophyte
plastids and the cyanobacterial endosymbiont that gave rise to this organelle: the presence of
carboxysomes and a peptidoglycan deposition between the two organelle membranes. Both traits
were apparently lost in the common ancestor of red and green algae after the divergence of
glaucophytes.

Figure 2. The green algae exhibit a remarkable cytological diversity ranging from unicellar organisms (coccoid
or flagellates), over multicellular filaments and foliose blades, to coenocytic and siphonous life forms that are
essentially composed of a single giant cell containing countless nuclei (after Coppejans 1998). Arrows indicate
trends in morphological complexity rather than evolutionary hypotheses. For example, green algae are thought
to have evolved from a unicellular flagellate (the Ancestral Green Flagellate, AGF) rather than a coccoid life
form.
INTRODUCTION 3

Green lineage or Viridiplantae
Green algae are distributed worldwide and can be found in almost every habit ranging from polar to
tropical marine, freshwater and terrestrial environments and as symbionts (Pröschold and Leliaert
2007). They exhibit a remarkable cytological diversity ranging from the world’s smallest free-living
eukaryote known to date Ostreococcus taurii (Derelle et al. 2006), over multicellular filaments and
foliose blades, to siphonous life forms that are essentially composed of a single giant cell containing
countless nuclei (Fig. 2). Together with land plants, green algae form the green lineage or
Viridiplantae (also written as Virideaplantae or known as green plants, Chlorobionta, Chloroplastida
or Chlorophycophyta). Morphological and molecular studies have identified a major split within the
Viridiplantae giving rise to two monophyletic lineages, the Chlorophyta and the Streptophyta
(Pickett-Heaps and Marchant 1972, Lewis and McCourt 2004) (Fig. 3). The streptophytes comprise
several lineages of predominantly freshwater green algae (often called charophytes or charophyte
green algae) and the land plants (Embryophyta) which evolved roughly 470 million years ago from a
charophyte ancestor. The majority of green algae, however, belong to the Chlorophyta.

Streptophyta
When motile cells are present, the Streptophyta are characterized by biflagellate cells with
asymmetrically flagellar roots including a multilayered structure or MLS (a distinct parallel
arrangement of microtubules) and a smaller root. In all representatives the nuclear envelope breaks
down before the chromosomes separate (open mitosis) and the mitotic spindle is persistent which
helps to keep the daughter nuclei separate until cytokinesis has been accomplished. Biochemical
characters such as photorespiratory enzymes are different from those found in most chlorophyte
green algae (Figs. 4 and 5; Table 1).
Several phylogenetic studies have tried to determine the origins of the land plants, focussing on the
green algal progenitors of the Streptophyta. Recent studies have indicated the scaly green flagellate
Mesostigma as the earliest diverging streptophyte. Initially, the scaly flagellate was placed within the
prasinophytes (Mattox and Stewart 1984), later ultrastructural investigations (e.g. a flagellum which
is anchored in the cell by means of an asymmetric root) revealed the association with the
Streptophyta (Melkonian 1989). Molecular phylogenies also showed conflicting results regarding the
phylogenetic relationships of this enigmatic species: Mesostigma either diverges before the
Chlorophyta/Streptophyta split (Lemieux et al. 2000, Turmel et al. 2002a, Turmel et al. 2002b) or as
an early diverging flagellate within the Streptophyta (Bhattacharya et al. 1998, Marin and Melkonian
1999, Karol et al. 2001). Increasing taxon and gene sampling and the use of more realistic models of
evolution provide evidence that Mesostigma is an early diverging lineage within the Streptophyta
(Petersen et al. 2006, Lemieux et al. 2007, Rodriguez-Ezpeleta et al. 2007). The colonial soil alga
Chlorokybus diverges after Mesostigma in most phylogenies (Karol et al. 2001), although more recent
studies united Mesostigma and Chlorokybus as the earliest diverging branch of the Streptophyta
(Lemieux et al. 2007). Unbranched filaments that form the class Klebsormidiophyceae diverge next,
followed by the Zygnematophyceae clade, which includes unicells and unbranched filaments with
isogamous sexual reproduction. The more complex charophytes are Coleochaetophyceae and
Charophyceae both consisting of branched filaments with oogamous sexual reproduction. It remains