Phylogeny and molecular evolution of green algae - Phycology ...

28 CHAPTER 2
Progress and perspectives
The slow progress in green algal phylogenetics can in part be attributed to the limited availability of
genomic data and the difficulties in consistently amplifying single-copy nuclear markers over the
entire spectrum of a group of organisms that dates back well into the Proterozoic. Our results
indicate that advanced phylogenetic analyses of multiple markers from different genomic
compartments show great promise in resolving ancient divergences within the green algae. The
present phylogeny has provided us with a framework to advance our understanding of the nature of
the morphological, cytological and ecological diversification of the Chlorophyta. Additional insight
would be gained from a dated phylogeny, which would allow an assessment of the timing of these
evolutionary events and, by consequence, the global ecological circumstances under which this
radiation took place.
Material and methods
Algal strains
Algal strain information is provided in Table S2. Cultures were grown under cool white fluorescent
lights at 18°C, with a 12/12h light/dark cycle. Dasycladales, Cladophorales and Derbesia were grown
at 23°C. Bold's basal medium and f/2 medium was used for freshwater and marine species,
respectively (Andersen 2005).
RNA isolation
Total RNA was extracted with a RNeasy Plant Mini Kit (Qiagen) or a NucleoSpin® RNA Plant kit
(Machery-Nagel) according to the manufacturer’s instructions, including a DNAse step to eliminate
genomic DNA contamination. RNA quality was checked on a 1% agarose gel and with
spectrophotometric analysis (Sambrook et al. 1989).
cDNA library construction and primer design
A standard cDNA library was constructed from 30 µg of total RNA of Cladophora coelothrix by VERTIS
Biotechnologie AG (Germany) and screened for nuclear genes potentially useful for green algal
phylogeny. After the cDNA library had been plated, 200 clones were randomly picked and sequenced
with M13 reverse primer (Table S3). Clones with a positive blastx hit to the Swiss-Prot database were
also sequenced with the M13 forward primer (Table S3).
For each recovered gene, an alignment including GenBank sequences from other green plants was
made. Primers were designed for sufficiently long genes that contained conserved regions and tested
on a variety of green algae, leading to successful amplification of 4 nuclear genes across a wide range
of green algae: 40S ribosomal protein S9, 60S ribosomal proteins L3 and L17 and oxygen-evolving
enhancer protein (OEE1). Actin and some glyceraldehyde-3-phosphate dehydrogenase (GapA)