Comprehensive Metabolic and Taxonomic Reconstruction of an Ancient Microbial Mat From the McMurdo Ice Shelf (Antarctica) by Integrating Genetic, Metaproteomic and Lipid Biomarker Analyses

Abstract

Paleobiological reconstructions based on molecular fossils may be limited by degradation processes causing differential preservation of biomolecules, the distinct taxonomic specificity of each biomolecule type, and analytical biases. Here, we combined the analysis of DNA, proteins and lipid biomarkers using 16S and 18S rRNA gene metabarcoding, metaproteomics and lipid analysis to reconstruct the taxonomic composition and metabolisms of a desiccated microbial mat from the McMurdo Ice Shelf (MIS) (Antarctica) dated ~1,000 years BP. The different lability, taxonomic resolution and analytical bias of each biomolecule type led to a distinct microbial community profile. DNA analysis showed selective preservation of DNA remnants from the most resistant taxa (e.g., spore-formers). In contrast, the proteins profile revealed microorganisms missed by DNA sequencing, such as Cyanobacteria, and showed a microbial composition similar to fresh microbial mats in the MIS. Lipid hydrocarbons also confirmed Cyanobacteria and suggested the presence of mosses or vascular plant remnants from a period in Antarctica when the climate was warmer (e.g., Mid-Miocene or Eocene). The combined analysis of the three biomolecule types also revealed diverse metabolisms that operated in the microbial mat before desiccation: oxygenic and anoxygenic photosynthesis, nitrogen fixation, nitrification, denitrification, sulfur reduction and oxidation, and methanogenesis. Therefore, the joint analysis of DNA, proteins and lipids resulted in a powerful approach that improved taxonomic and metabolic reconstructions overcoming information gaps derived from using individual biomolecules types.

Keywords: Antarctica; DNA metabarcoding; McMurdo Ice Shelf; lipid biomarkers; metaproteomics; microbial mat communities; microbial metabolism.

Figure 1

McMurdo Ice Shelf (MIS) close to the coast of Bratina Island. In (A), geographic location of the MIS in Antarctica (image of Google Earth). In (B), a view of the MIS from the top of Bratina Island showing the debris coated-ice of the surface (“dirty ice”) and numerous seasonal meltwater ponds (some ice-capped). In (C), location of the desiccated microbial mat in a hillside of a mound at the time of collection (indicated with a white arrow). In (D), one of the meltwater ponds closest to the location of the desiccated microbial mat. Active microbial mats are observed in the meltwater pond.

Figure 2

Sketch of the temporal shifts and physical processes occurring in the MIS close to the coast of Bratina Island. Horizontal black arrows in the ice shelf indicate the sense and direction of the ice compression against the coast of Bratina Island that causes the undulation of the ice. Vertical and inclined dashed arrows represent the sense and direction of the debris ascension as a result of the ice basal accretion and surface ablation. Different colors of the meltwate ponds represent a mixture of conditions (e.g., different salinities), and black boxes indicate the hypothetical location of the microbial mat ~1,000 years ago (submerged in a meltwater pond) and at present (desiccated and exposed to atmospheric conditions).

Figure 3

Fluorescence microscopy images of the desiccated microbial mat showing red autofluorescence of chlorophyll-bearing cells. (A) Shows a picture of the dried and laminated microbial mat. Micrographs in (B,C) show remnants of photosynthetic cells with a large amount of cellular and extracellular material scattered outside the cells (arrows). The micrograph in (D) shows a possible colony of Cyanobacteria and release of photosynthetic pigments (arrow), and in (E), a colony of microalgae with apparent entire cells.

Figure 4

Concentration of DNA, proteins, and lipid compounds in the microbial mat (μg·g⁻¹ of dry weight). In (A), concentration of intracellular and surface-bound extracellular DNA and proteins, and total lipids. “Surface-bound” refers to the fraction of extracellular DNA and proteins attached to organic or inorganic compounds. Error bars are the SD of triplicates. In (B), concentration of the three most abundant lipidic families: straight-chain or normal (n-) alkanes, alkanoic acids, and alkanols. Results from an organic-solvent extraction without a sample (blank control) is also represented for the lipid biomarkers.

Figure 5

Composition and relative abundances of bacterial, archaeal, and eukaryotic phyla in the desiccated microbial mat obtained with SSU rRNA genes (left) and metaproteomics (right) analyses. For proteins, the relative abundance was calculated based on a normalized spectral abundance factor (NSAF) annotated per cent. Phyla with relative abundances below 0.5% were comprised within “Other” groups.

Figure 6

Microbial composition and relative abundance of the most abundant orders (>0.5% over the bacterial, eukaryotic or archaeal community) within the dominant phyla in the microbial mat based on DNA and protein analyses. For Eukarya, only Chlorophyta, Ochrophyta, and Haptophyta were represented as some of the main photoautotrophs in fresh and marine ecosystems. Relative abundances (%) of proteins were calculated based on the NSAF relative to each domain (bacteria, archaea, or eukarya). “un” means unclassified.

Figure 7

Partial ion chromatograms of the three polarity fractions of lipids in the ancient microbial mat; non-polar (A), acidic (B), and polar (C). Hydrocarbons in (A) were measured in the m/z = 57 ion, alkanoic acids in (B), in the m/z = 74 ion, and alcohols in (C), in the m/z = 75 ion. In the three panels, numbers over the peaks indicate carbon chain lengths of the straight-chain or normal series (i.e., n-alkanes, n-alkanoic acids, and n-alkanols). In (B), iso-/anteiso- pairs (marked as i/a over the peaks) refer to branched alkanoic acids with a methyl group in positions N-1 or N-2, respectively.

Figure 8

Proportion of metabolic pathways in the desiccated microbial mat based on the functional assignment of the metaproteome to KEGG pathway maps. The relative abundance of KEGG categories was calculated based on the sum of SAFs of the proteins that are annotated in each KEGG category. “Unclassified” are proteins unable to cross-reference with the KEGG database. “Other KEGG categories” are shown in Supplementary Figure 6.

Figure 9

Reconstructive sketch of the biological composition and metabolisms that operated in the ancient microbial mat on the MIS before desiccation. In (A), the temporal shift of the microbial mat from the hypothetical scenario of a fresh and photosynthetically active microbial mat ~1,000 years ago to the present desiccated state. The length and continuity/discontinuity of the arrows indicate an estimate of the degree of preservation of the three biomolecules studied (lipids > proteins > DNA) with an inverse gradient of taxonomic specificity. In (B), reconstruction of the biological composition and metabolisms across a representative cross-section of the Bratina microbial mat. The cross-section shows a vertical stratification based on previous microbial mats descriptions (Vincent et al., 1993b; De los Rios et al., 2004; Prieto-Barajas et al., 2018). The presence of putative metabolic functions in each layer is the result of an integrative approach based on the identification of microbial taxa detected by DNA (purple circles), proteins (blue circles) and lipid biomarkers (grey circles) analyses. The metabolisms may not be exclusively associated with a certain layer. A global reaction for each metabolism is represented as an example. The most abundant taxa in the microbial mat determining the metabolisms are: Nostocales (Nost.), Synechococcales (Synec.), Chlorellales (Chlor.), Chlamydomonadales (Chlam.), Chromulinales (Chromu.), Mamiellales (Mamie.), Isochrysidales (Isochry.), Naviculales (Navi.), Nephroselmidales (Neph.), Oltmannsiellopsidales (Oltm.), Nitrosomonadales (Nitroso.), Rhizobiales (Rhiz.), Actinomycetales (Acti.), Chromatiales (Chroma.), Frankiales (Frank.), Rhodobacterales (Rhodob), Campylobacterales (Campy.), Burkholderiales (Burk.), Rhodospirillales (Rhodos.), Clostridiales (Clost.), Desulfobacterales (Desulfob.), Desulfuromonadales (Desulfur), Methanomicrobiales (Methanom.), and Methanosarcinales (Methanos.) For detailed information regarding the metabolisms inferred by the microbial taxa identity, see Supplementary Text 3.