Green algae in soil: assessing their biodiversity and biogeography with molecular-phylogenetic methods based on cultures

Abstract

Green microalgae (Chlorophyta) dominate soils in the northern temperate climate zone, however, their biodiversity is still poorly understood. Available diversity inventories rely on light microscopy and morphospecies, which are hardly distinguishable without molecular markers. Whereas particular attention was paid to molecular diversity of terrestrial microalgae in extreme regions, temperate climate zones remain almost unexplored. We aim to uncover the phylogenetic diversity of green microlagae isolated from Central European soils and from periodically desiccating freshwater creek biofilms. Such terrestrial and semi-aquatic habitats are inhabited by green microalgae, which are presumably able of long-distance dispersal. Their cosmopolitan distribution is presumed, however, supporting molecular evidence is almost missing. By assembling newly obtained green algal sequences together with accessions from remote geographic regions, we further aim to address a question of biogeography of terrestrial microalgae. Soil samples were taken from grassland and forest plots within the German Biodiversity Exploratories. Freshwater biofilms dominated by green microalgae were sampled in two karstwater creeks in Germany. In total, 280 new monoclonal cultures of green microalgae were examined by molecular phylogenetic methods and by light microscopy. By using ribosomal 18S and ITS2 sequences, we recognized about 100 monophyletic species of green microalgae. The newly obtained sequences were blasted against public databases in order to infer taxonomy, distribution and ecology of the detected species. Further monoclonal cultures and environmental clones originated from additional samplings in Germany, Ecuador, the Arctic and Antarctic. Most Chlorophyta isolated from German soils were highly similar (≥ 99.5% threshold) to cultured relatives already known from Europe, predominantly from soils and further terrestrial substrates such as tree barks and rocks. Considering a lower similarity threshold (≥ 99%), about 90% of our cultures matched environmental clones inferred from various terrestrial and aquatic habitats. Similarly, Chlorophyta detected in creek biofilms partly represent terrestrial species, but some are known also from planktonic communities. In soils, we detected novel species mostly within established lineages. Only one soil isolate, provisionally named Navichloris fusiformis, was recognized as a member of a novel monophyletic lineage, comprising accessions from South and North American deserts. In multiple cases, molecular data supported close relatedness between the European soil isolates and species from tropics and polar regions. We show, that well supported monophyletic clades of Stichococcus exhibit either temperate-polar or temperate-tropical distributions. In contrast, some monophyletic clades of Chlorella-like microalgae were so far evidenced only from polar regions and hot deserts. The long-distance dispersal was finally confirmed for particular species of Stichococcus, Chlorella and Klebsormidum. Forest and grassland soils in Central Europe host species of green microalgae otherwise known from a broad spectrum of terrestrial as well as aquatic habitats. New species and even novel lineages can still be uncovered by using standard culturing techniques. Despite the striking similarity between some European and exotic species, unambiguous molecular evidence of the intraspecific long-distance dispersal is still scant. At the present state of knowledge, our data suggest the existence of biogeography of airborne microalgae.