Biocrusts are associations of various prokaryotic and eukaryotic microorganisms in the top millimeters of soil, which can be found in every climate zone on Earth. They stabilize soils and introduce carbon and nitrogen into this compartment. The worldwide occurrence of biocrusts was proven by numerous studies in Europe, Africa, Asia and North America, leaving South America understudied. Using an integrative approach, which combines morphological and molecular characters (small subunit rRNA and ITS region), we examined the diversity of key biocrust photosynthetic organisms at four sites along the latitudinal climate gradient in Chile. The most northern study site was located in the Atacama Desert (arid climate), followed by open shrubland (semiarid climate), a dry forest region (Mediterranean climate) and a mixed broad leaved-coniferous forest (temperate climate) in the south. The lowest species richness was recorded in the desert (18 species), whereas the highest species richness was observed in the Mediterranean zone (40 species). Desert biocrusts were composed exclusively of single-celled Chlorophyta algae, followed by cyanobacteria. Chlorophyta, Streptophyta and cyanobacteria dominated semiarid biocrusts, whereas Mediterranean and temperate Chilean biocrusts were composed mostly of Chlorophyta, Streptophyta and Ochrophyta. Our investigation of Chilean biocrust suggests high biodiversity of South American biocrust phototrophs.

We describe the performance of a new metabarcoding approach to investigate the environmental diversity of a prominent group of widespread unicellular organisms, the Cercozoa. Cercozoa is an immensely large group of protists and although it may dominate in soil and aquatic ecosystems, its environmental diversity remains undersampled. We designed PCR primers targeting the hyper-variable region V4 of the small subunit ribosomal RNA (SSU or 18S) gene, which is the recommended barcode marker for Cercozoa. The length of the amplified fragment (ca. 350 bp) is suitable for Illumina MiSeq, the most cost-effective platform for molecular environmental surveys. We provide barcoded primers, an economical alternative to multiple libraries for multiplex sequencing of over a hundred samples. In silico , our primers matched 68% of the cercozoan sequences of the reference database and performed better than previously proposed new generation sequencing primers. In mountain grasslands soils and in biological soil crusts from a variety of climatic regions, we were able to detect cercozoan sequences encompassing nearly the whole range of the phylum. We obtained 901 OTUs at 97% similarity threshold from 26 samples, with ca. 50,000 sequences per site, and only 8% of non-cercozoan sequences. We could contribute to a further increase of the diversity of Cercozoa, since only 43% of the OTUs were 97-100% similar to any known sequence. Our study thus provides an advanced tool for cercozoan metabarcoding and to investigate their diversity and distribution in the environment.