Genomic Analysis of Microfossils in Lake Sediments

Abstract

Botryococcus braunii is a microscopic, colonial green alga that may be found in fresh and brackish waters throughout the globe. B. braunii is unique in that it constitutively synthesises and secretes copious amounts of various long-chain (C23-C40) hydrocarbons, generically termed “botryococcenes”. Botryococcanes, the hydrogenated forms of botryococcenes, comprise 1% of the fossil hydrocarbons found in petroleum deposits and in oil-shales. Microfossils identified as Botryococcus by optical and scanning electron microscopy are also abundant in these strata, but the actual identity and precise relationship between these microfossils and extant Botryococcus species is not known.

In this investigation, the relationship between living Botryococcus algae and microfossils identified as Botryococcus using traditional palaeontological analysis and light-microscopy was investigated by analysis of ancient DNA (aDNA). The material used was identified in sediments from Boswell Lake (British Columbia, Canada), a Holocene lake that had remained undisturbed since the glacial retreat. New flow-cytometry methods were developed to rapidly purify enough of the relevant microfossils, from which aDNA was extracted and sequenced. Pollen grains were purified using the same flow-cytometry method and from the same horizons as the Botryococcus microfossils and used to age the sedimentary horizons by 14C radiocarbon dating. Samples of the purified microfossils were imaged by scanning electron microscopy for comparison with published images of fossils identified as Botryococcus from kerogens. In addition, metaDNA from the relevant horizons was extracted and sequenced by NGS, and a chemical analysis for botryococcene derivatives performed using two-dimensional gas chromatography (2D-GC).

The genomic analyses show that the sub-fossils identified in Boswell Lake are likely to be representatives of B. braunii, race B. The geochemical analysis identified hydrocarbons that migrate as botryococcenes on 2D-GC in the strata whence the sub-fossils were purified. The SEM images indicate that the microfossils purified from Boswell Lake have similar morphologies to those found in kerogens. Taken together, these data strongly support the proposition that petroleum and kerogen deposits are unusually rich in B. braunii and that these algae have a lineage potentially dating 500 million years. The metagenomic analysis enabled similar conclusions to be reached regarding the presence of B. braunii within the sediment, without the need for targeted microfossil purification. While this analysis was less precise due to the under-representation of algal genomes in the public sequence databases, the metagenomics approach employed was particularly well suited to the temporal analysis of prokaryotic microcosms within Lake Boswell, the succession of which could be associated with periods of climatic variation.

The analytical methods described herein are generally applicable to understanding microbial systems over geological periods, and may be used to generate important insights into the cause and effect relationships between microbial populations and environmental perturbation.