Amplicon-Metagenomic Analysis of Fungi from Antarctic Terrestrial Habitats

Abstract

In cold environments such as polar regions, microorganisms play important ecological roles, and most of our knowledge about them comes from studies of cultivable microorganisms. Metagenomic technologies are powerful tools that can give a more comprehensive assessment of microbial communities, and the amplification of rDNA followed by next-generation sequencing has given good results in studies aimed particularly at environmental microorganisms. Culture-independent studies of microbiota in terrestrial habitats of Antarctica, which is considered the driest, coldest climate on Earth, are increasing and indicate that micro-diversity is much higher than previously thought. In this work, the microbial diversity of terrestrial habitats including eight islands of the South Shetland Archipelago, two islands on the Antarctic Peninsula and Union Glacier, was studied by amplicon-metagenome analysis. Molecular analysis of the studied localities clustered together the islands of the South Shetland Archipelago, except Greenwich Island, and separated them from the Litchfield and Lagotellerie islands and Union Glacier, which is in agreement with the latitudinal difference among them. Among fungi, 87 genera and 123 species were found, of which species belonging to 37 fungal genera not previously cultivated from Antarctica were detected. Phylogenetic analysis, including the closest BLAST-hit sequences, clustered fungi in 11 classes being the most represented Lecanoromycetes and Eurotiomycetes.

Keywords: Antarctic diatoms; Antarctic fungi; Antarctic microeukaryotes; amplicon-metagenome; cold-terrestrial microeukaryotes.

FIGURE 1

Sampling site locations. Sampling locations used in this work. Detail of principal sampling sites (marked as I-#) at Lagotellerie Island in the Antarctic Peninsula is shown. For details of sampling sites from other locations, see the following references (Carrasco et al., 2012; Barahona et al., 2016; Troncoso et al., 2017).

FIGURE 2

Shared and exclusive OTU of each Locality. Number of OTU in a Locality also present in at least one other Locality (black columns), and those exclusively present in the corresponding Locality (white columns), are shown.

FIGURE 3

Cross-study comparisons among Localities. All localities were compared based on number of shared OTU (left) and geographic distance between them (right). For each case, hierarchical clustering combined with heat-map is shown. Scales are indicated at top of each heat-map and black indicates no BLAST-hits.

FIGURE 4

Cross-study comparisons among sampling Sites. All sampling sites were compared based on number of shared OTU (left) and geographic distance between them (right). For each case, hierarchical clustering combined with heat-map is shown. Scales are indicated at top of each heat-map and black indicates no BLAST-hits.

FIGURE 5

Operational taxonomic unit (OTU) comparisons against NCBI database. (A) Distribution of OTU BLAST-hits according to sources. (B) Distribution of BLAST-hits from species. Species with the most BLAST-hits is indicated on the top of column.

FIGURE 6

Fungal species distribution among Localities. Only results for fungi found in at least 4 Localities are shown.

FIGURE 7

Phylogenetic placement of yeast isolates. Analysis was performed with D1/D2 domain sequences using the maximum likelihood method. Bootstrap values higher than 50% are shown (1,000 replicates). ^∗Sequences obtained in this work. Accession number of sequences obtained from the NCBI database in parentheses.

FIGURE 8

Phylogenetic grouping of the OTU fungal BLAST-hits. Bootstrap values higher than 50 % are shown (500 replicates). (Inner) Coloring according to fungal class: Agaricomycetes (Blue), Dothideomycetes (green), Chytridiomycetes (red), Eurotiomycetes (dark green) Lecanoromycetes (violet), Leotiomycetes (orange), Microbotryomycetes (cyan), Saccharomycetes (magenta) and Tremellomycetes (olive). (Outer) Coloring according to continent of origin: Africa (brown), Asia (red), Europa (magenta), North America (blue), Oceania (yellow), South America (green). ^∗Uncultured fungal sources. OTU obtained in this work are shown in black.

FIGURE 9

Global distribution of closest OTU fungal BLAST-hits. All BLAST-hits (magenta) and BLAST-hits excluding those from uncultured sources (cyan), are represented. Scales indicate number of BLAST hits.