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Supplementary material
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Isolation of Asterionella genotypes
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For each year, single Asterionella colonies were isolated from samples at the start and at the peak of the
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Asterionella spring-bloom in Lake Maarsseveen (The Netherlands). Single colonies were isolated and
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transferred to modified CHU-10 medium (Stein, 1973) enriched with twofold concentrations of PO4 and
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FeCl3. As Asterionella colonies consist of uniclonal cells only, isolating single colonies is a simple way to
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achieve uniclonal cultures. Culture establishment from isolated colonies was fairly unbiased with a
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larger than 95% success rate. The isolates were maintained in uniclonal batch cultures in Sanyo versatile
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environment test chambers (Sanyo Electrics Co., Ltd., Japan) at 18°C ± 1°C and discontinuous
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illumination on a 14:10h light:dark cycle at 50µmol quanta s-1 m-2 provided by cool white fluorescent
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lamps (Master TL-D 30W/830, Philips, The Netherlands). All genotype cultures had slight bacterial
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contaminations.
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DNA extraction
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Asterionella DNA was extracted with a Qiagen DNeasy Plant Mini Kit (Qiagen BV., The Netherlands) with
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a modification of the lysis step. Fifty mL of dense Asterionella cultures were centrifuged at 740g for 10
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min. The supernatant was discarded and the remaining pellet was transferred into a fresh tube with 0,5g
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of 0.1mm zirconium beads and 300µl lysis buffer which consisted of 10 mM Tris 1M, 1 mM EDTA pH8
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and 0.5% SDS. Then the sample was bead-beaten three times with a mini bead beater (Biospec Products,
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Bartlesville, USA) at 840g for 30 sec per round and cooled on ice between each round. After adding
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100µg mL-1 Proteinase K and short vortexing, the sample was incubated at 60°C for one hour and then
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processed further following the Qiagen DNeasy Plant Mini Kit protocol from the first precipitation step
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onwards.
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AFLP analysis
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To assess the genetic similarity among Asterionella genotypes they were fingerprinted by AFLP
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(amplified fragment length polymorphism). AFLPs are sensitive, reproducible markers (Rademaker et al.,
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2000) well able to provide useful information on population genetic structure such as the clonal
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structure of a single algal bloom sample (John et al, 2004). The AFLP analysis was performed by
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Keygene® (Wageningen, The Netherlands) using the same four EcoRI/Msel AFLP primer combinations as
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reported in De Bruin et al (2004), a) Eco+GA & Mse+AT, b) Eco+GA & Mse+CC, c) Eco+GA & Mse+CG, d)
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Eco+GC & Mse+AC. In the AFLP analysis each locus was treated as a separate character and scored for
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presence or absence, yielding a binary data matrix for each early and late bloom subset of genotypes
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separately. All loci with missing data were excluded from further analysis. The matrices were used to
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estimate genetic similarity of all possible pair-wise combinations of the tested genotypes using Jaccard’s
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similarity coefficient (Jaccard, 1901). Jaccard’s similarity ignores shared absences, and the coefficient
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ranges from one (complete similarity) to zero (no similarity) (Bonin, Ehrich & Manel, 2007). Genotypes
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are assumed to be closely related if their fingerprint patterns differ only in a single genetic event, which
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usually results in differences in two to three loci (Tenover et al, 1995).
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Literature supplementary material
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Bonin A., Ehrich D. & Manel S. (2007) Statistical analysis of amplified fragment length polymorphism
data: a toolbox for molecular ecologists and evolutionists. Mol Ecol, 16, 3737-3758.
De Bruin A., Ibelings B. W., Rijkeboer M., Brehm M. & van Donk E. (2004) Genetic variation in
Asterionella formosa (Bacillariophyceae): Is it linked to frequent epidemics of host-specific
parasitic fungi? J Phycol, 40, 823-830.
Jaccard P. (1901) Étude comparative de la distribution florale dans une portion des Alpes et des Jura.
Bull Soc Vaud Sci Nat, 37, 547-579.
John U., Groben R., Beszteri N. & Medlin L. (2004) Utility of amplified fragment length polymorphisms
(AFLP) to analyse genetic structures within the Alexandrium tamarense species complex. Protist,
155, 169-179.
Rademaker J., Hoste B., Louws F. J., Kersters K., Swings J., Vauterin L., et al (2000) Comparison of AFLP
and rep-PCR genomic fingerprinting with DNA-DNA homology studies: Xanthomonas as a model
system. Int J Sys Evol Microbiol, 50, 665-677.
Stein J. (1973) Culture methods and growth measurements. In Handbook of phycological methods. UK:
Cambridge University Press.
Tenover F. C., Arbeit R. D., Goering R. V., Mickelsen P. A., Murray B. E., Persing D. H., et al (1995)
Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel-electrophoresis
- criteria for bacterial strain typing. J Clin Microbiol, 33, 2233-2239.
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