Culture-Dependent and Amplicon Sequencing Approaches Reveal Diversity and Distribution of Black Fungi in Antarctic Cryptoendolithic Communities

Abstract

In the harshest environmental conditions of the Antarctic desert, normally incompatible with active life, microbes are adapted to exploit the cryptoendolithic habitat (i.e., pore spaces of rocks) and represent the predominant life-forms. In the rocky niche, microbes take advantage of the thermal buffering, physical stability, protection against UV radiation, excessive solar radiation, and water retention-of paramount importance in one of the driest environments on Earth. In this work, high-throughput sequencing and culture-dependent approaches have been combined, for the first time, to untangle the diversity and distribution of black fungi in the Antarctic cryptoendolithic microbial communities, hosting some of the most extreme-tolerant microorganisms. Rock samples were collected in a vast area, along an altitudinal gradient and opposite sun exposure-known to influence microbial diversity-with the aim to compare and integrate results gained with the two approaches. Among black fungi, Friedmanniomyces endolithicus was confirmed as the most abundant taxon. Despite the much stronger power of the high-throughput sequencing, several species were not retrieved with DNA sequencing and were detectable by cultivation only. We conclude that both culture-dependent and -independent analyses are needed for a complete overview of black fungi diversity. The reason why some species remain undetectable with molecular methods are speculated upon. The effect of environmental parameters such as sun exposure on relative abundance was clearer if based on the wider biodiversity detected with the molecular approach.

Keywords: Antarctica; black fungi; cryptoendolithic communities; extremophiles; metabarcoding.

Figure 1

Map of the study area showing the location of the sampling sites spanning Northern and Southern Victoria Land (continental Antarctica). Sites correspondences are reported in Table S1. A map of Antarctica is included; the red square indicates the Victoria Land.

Figure 2

(A–C) Colonies grown on malt extract agar, isolated from crushed rocks. Linnaeus Terrace north (A), Mt. Elektra (B), and Finger Mountain (C). (D–F) Examples of black fungal colonies in pure cultures isolated from Antarctic endolithic communities: Recurvomyces mirabilis (D), Friedmanniomyces endolithicus (E), and Extremus antarcticus (F).

Figure 3

(A) The pie chart indicates the relative abundance of eukaryotes isolated from the Antarctic endolithic communities analyzed in this study. (B) Venn diagram reporting the distribution of black fungi retrieved from both culture-dependent and high-throughput sequencing (HTS) approaches. Percentages of both shared and unique operational taxonomic units (OTUs) are shown in parentheses. (C,D) Relative abundances of black fungi isolated (C) and recorded from Illumina amplicon sequencing raw data (D).

Figure 4

Map of distribution of black fungi along the Southern (A) and Northern (B) Victoria Land. Sites correspondences are reported in Table S1. The presence of each black fungal species retrieved in this study is reported: M—metabarcoding; C—culture-dependent approach.

Figure 5

Presence/absence and relative abundance of each black fungal species recorded in this study by using a culture-dependent approach (A) or metabarcoding (B).

Figure 6

(A,B) Venn diagrams showing the distribution of black fungi between northern and southern exposure by using both a culture-dependent approach (A) and metabarcoding (B). Both the percentages of OTUs that were shared and found exclusively in each sun exposure are indicated. (C) Correlation analysis between species richness and altitude, calculated using MICtools v1.1.4; only rock-inhabiting fungi (RIF) that correlate with altitude (p < 0.05) are reported.