Exploring the Holocene through fossil cyanobacterial sequences from Antarctic core sediments

1. Exploring the Holocene through fossil cyanobacterial sequencesfrom Antarctic core sediments Rafael Fernandez-Carazo1, Krzysztof Waleron1,2, Dominic A. Hodgson3, Elie Verleyen4, Wim Vyverman4and Annick Wilmotte1 1CIP, Institute of Chemistry B6, University of Liège, B-4000 Liège, Belgium. 2 Department of Biotechnology, University of Gdansk & Medical School of Gdansk, 80-822 Gdansk, Poland. 3 British Antarctic Survey, NERC, Cambridge, UK. 4Laboratory of Protistology and Aquatic Ecology, Ghent University, B-9000 Ghent, Belgium Email: rfernandez@ulg.ac.be Introduction The HOLANT project aims to determine how the climate of coastal (Sub-)Antarctic regions has varied during the Holocene and study the ecosystem responses. We have tracked environmental changes from the cyanobacterial diversity on the basis of 16S rRNA genes extracted from sediment cores from 2 lakes (lakes BK1 and BK2) in Beak Island (Antarctic Peninsula). Lake BK2 was formed in the relict outflow streambed of BK1. Lake BK2 shows higher nutrient concentrations and lake primary production that might also be related to its closer proximity to the sea and thus the more frequent visits by penguins which lead to natural nutrient enrichment (eutrophization). However, the dominant benthic phototrophs in these systems, the cyanobacteria, do not leave recognizable fossils, making it difficult to assess their response to past climate variability. Material & Methods We have analysed 2 sediment cores taken from lakes BK1 and BK2 in Beak Island (Fig.1). DGGE was performed using specific primers for cyanobacteria. The sequenced bands were compared to public databases to find their closest relatives (BLAST, RDPII). Lost on ignition (LoI550) was analyzed (2) for both lakes. PCR was realised with several pairs of primers for 16S rRNA genes (N359F, B23S, 781R (1) and rpoC1 gene (3F, 4R, 7F, 8R [K.Waleron, unpubl. data]). Whole Genome Amplification by Multiple Displacement Amplification (MDA) was realised using the REPLI-g kit( Qiagen) (Fig.3). Fig 2. Scheme of the rpo operon. Fig 3.Schematic representation of REPLI-g amplification Fig 1. Lakes BK1 and BK2. Filamentous cyanobacteria Unicellular cyanobacteria Conclusions Fig 5. LoI550 graphics for BK1 (left) and BK2 (right) Fig 4. PCR amplification of fossil DNA from Progress Lake, using different primers. • -MDA has been used as effective method for amplification of samples where fossil DNA was in so low quantities that it could not be detected by direct PCR. • Synechococcus has been frequently detected in the cores, maybe due to a higher resistence to degradation of this group. This observation was already made in the LAQUAN project (www.laquan.ugent.be) • In Lake BK1, the highest diversity is found in the upper layers, corresponding to a peak in the LoI550. Higher productivity in BK2, indicated by LoI550 values (Fig.5), is likely related to high nutrient concentrations following lake isolation. • In Lake BK2, filamentous cyanobacteria belonging to the Nostoc group, were present c. 4000 years BP, suggesting ecological differences with Lake BK1. Filamentous & unicellular cyanobacteria References: (1) Taton, A., S. Grubisic, E. Brambilla, R. De Wit, and A. Wilmotte. 2003. Cyanobacterial diversity in natural and artificial microbial mats of Lake Fryxell (McMurdo dry valleys, Antarctica): A morphological and molecular approach. Applied and Environmental Microbiology 69:5157-5169. (2)Birks, H. J. B., V. J. Jones, and N. L. Rose. 2004. Recent environmental change and atmospheric contamination on Svalbard as recorded in lake sediments - an introduction. Journal of Paleolimnology 31:403-410. Analysis -Size of amplicon is determinant for detection and amplification of fossil DNA, as shown in a preliminary study in Lake Progress (Fig. 4), where primers N359F-B23S (ca 2000 bp) allowed to detect DNA until 9 cm depth, whereas smaller fragments (ca 450 bp), with primers N359F-781R, were detected until 30 cm depth. -In 16S-based DGGE, heterotrophic bacteria that are present in the cores, are well amplified because of better DNA quality. The rpoC1 gene only appears in cyanobacteria and plastids, but not in eubacteria (Fig.2), so we could expect more specific amplifications with this marker. Specific primers for rpoC1 gave amplicons also until 30 cm depth. -In Lake BK1, we found a compartmented diversity, with the sequences in the 6 first layers related to Anabena and Nostoc and clones found elsewhere in Antarctica, that did not appear in the rest of the core. Unicellular Synechococcus sequences were detected in almost all layers. At least 4 sequences were less than 96% related to existing sequences in Genbank. The oldest layer from which cyanobacterial sequences could be retrieved was ca 4000 years old, with a biodiversity related to freshwater Synechoccocus and a clone found in the brackish waters of the Baltic Sea.