Hunting for nuclear markers in green algal lineages: Molecular evolution
of glucose-6-phosphate isomerase
Ellen Cocquyt, Frederik Leliaert, Heroen Verbruggen & Olivier De Clerck
[email protected]
Research Group Phycology and Center for Molecular Phylogenetics and Evolution (CeMoFE), Biology Department, Ghent University, K.L. Ledeganckstraat 35, 9000 Gent, Belgium
Introduction
• The Chlorophyta exhibit a remarkable cytological diversity
Fig 1
• Unravelling the evolutionary history of this diverse group is a
difficult task, due to:
• antiquity of the major defining lineages
• considerable rate variation among lineages
• scarcity of useful molecular phylogenetic markers
 examination of several independent nuclear markers is
needed, therefore:
• different genes with known function and sequence data
available are tested
• a cDNA library is screened to search for new useful genes
Chlorophyceae
Prasinophytes
1 Chlamydomonas reinhardtii
Fig 1: Remarkable cytological diversity in the Chlorophyta
ranging from unicellular microscopic algae with a single
nucleus, over multicellular filaments and foliose blades, to
coenocytic and siphonous life forms that are essentially
composed of a giant cell containing thousands of nuclei.
Chlorococcum
scabellum1
Halosphaera
cf. minor2
Pediastrum
duplex2
Ulva sp.
Anadyomene
wrightii4
Phyllodictyon
anastomosans4
Boodlea
composita4
Boergesenia
forbesii
Siphonocladus
tropicus4
Photographe r s : 1. www.bio.utexas.edu/research/utex/
2. Lewis and McCourt 2004
3. www.Algaebase.org
4. Frederik Leliaert
Glucose-6-phosphate isomerase (G6PI)
1 Dryopteris caudipanna
• Enzyme of the carbohydrate biosynthesis pathway, after the
Calvin cycle, in photosynthetic organisms and of the glycolysis in
all living organism
1 Arabidopsis thaliana
2 A. halleri ssp gemnifera
1 or 2 Clarkia
• Genome sequences of C. reinhardtii and O. tauri revealed that
both have a single copy of G6PI  Fig 2
1 Dioscorea
2 Leavenworthia at least
Results
3 Zea mays
• G6PI phylogenetic tree of several green algae
1 or 2 Festuca ovina
Land plants
Ulothrix
sp2
Phycopeltis
arundinacea3
1 Ostreococcus tauri
1 Actinidia normally
2 Actinidia chinensis
Ferns
Klebsormidium
flaccidum2
Hydrodictyon
reticulatum2
2 Oryza sativa
Fig 2: Evolution and copy number of G6PI in the Chloroplastida Tree after D. Mandoli et al.
CMO124C Cyanidioschyzon merolae
Toward resolution of the “Fuzzy Nodes” in green plant phylogeny (http://faculty.washington.edu/mandoli/fuzzy.ph)
CMT497C Cyanidioschyzon merolae
80
Embryophyta
Arabidopsis thaliana 5g42740
95
Closterium sp. c R4 3
• Identification of nuclear genes useful for phylogenetic
reconstruction in the Chlorophyta
Chlorokybus atmophyticus R99
Chlamydomonas reinhardtii CC1690 R96
95
Tetraselmis straita R76
Bolbocoleon piliferum R95
Halochlorococcum tenius R102
66
Acetabularia accetabulum AAL00001487
76
Ulvales
Ulotrichales
Dasycladales
Ostreobium quekettii c R1 12
• Primer design based on the G6PI genome sequences of
Arabidopsis thaliana, Oryza sativa, Chlamydomonas reinhardtii
and Ostreococcus tauri
• DNA extraction PCR Cloning  Sequencing
• RNA extraction RT-PCR PCR Sequencing
Derbesia teniussima R98
Bryopsidales
Ulvophyceae
93
Cladophoropsis herpestica c R9 3
56
Boergesenia forbessii c R21 2
Siphonocladus tropicus R84
Cladophora catenata c R34 2
100
Cladophorales
Boodlea composita c R38 6
NJ tree with bootstrap support
Excluding 3 rd codonposition
100
Cladophora coeothrix c R35 12
89
Struvea sp. R85
Valonia utricularis R86
Conclusions
• Epiphytic or endophytic bacteria interfere with direct DNA amplification of nuclear genes
• Information content of G6PI depends on taxonomic level
deep phylogenies require exclusion of 3rd codon pos
 species level relationships are generally well-supported
 introns at fixed positions may offer opportunities towards studies at the intraspecific level
• Most likely only one (functional) copy of G6PI gene is present in the taxa tested
Acknowledgements: This research is funded by a BOF-project of the Ghent University
Chlorophyta
Methods
Chlorophyceae
Trebouxiophyceae
Acrochaete repens R94
89
87
Charaphyta
Scenedesmus obliquus SOL00005809
55
• Investigation of the G6PI gene(s) useful as a phylogenetic
marker in the Chlorophyta
Streptophyta
Oryza sativa 2
100
Aims of the study
Rhodophyta
Oryza sativa 1
100