Linking bacterial and eukaryotic microbiota to litter chemistry: Combining next generation sequencing with 13C CPMAS NMR spectroscopy

Microbial succession over decomposing litter is controlled by biotic interactions, dispersal limitation, grazing pressure, and substrate chemical changes. Recent evidence suggests that the changes in litter chemistry and microbiome during decomposition are interdependent. However, most previous studies separately addressed the microbial successional dynamics or the molecular changes of decomposing litter. Here, we combined litter chemical characterization by 13C NMR spectroscopy with next generation sequencing to compare leaf litter chemistry and microbiome dynamics using 30 litter types, either fresh or decomposed for 30 and 180 days. We observed a decrease of cellulose and C/N ratio during decomposition, while lignin content and lignin/N ratio showed the opposite pattern. 13C NMR revealed significant chemical changes as microbial decomposition was proceeding, with a decrease in O-alkyl C and an increase in alkyl C and methoxyl C relative abundances. Overall, bacterial and eukaryotic taxonomical richness increased with litter age. Among Bacteria, Proteobacteria dominated all undecomposed litters but this group was progressively replaced by members of Actinobacteria, Bacteroidetes, and Firmicutes. Nitrogen-fixing genera such as Beijerinckia and Rhizobium occurred both in un- decomposed as well as in aged litters. Among Eukarya, fungi belonging to the Ascomycota phylum were dominant in undecomposed litter with the typical phyllospheric genus Aureobasidium. In aged litters, phyllo- spheric species were replaced by zygomycetes and other ascomycetous and basidiomycetous fungi. Our analysis of decomposing litter highlighted an unprecedented, widespread occurrence of protists belonging to the Amebozoa and Cercozoa. Correlation network analysis showed that microbial communities are non-randomly structured, showing strikingly distinct composition in relation to litter chemistry. Our data demonstrate that the importance of litter chemistry in shaping microbial community structure increased during the decomposition process, being of little importance for freshly fallen leaves.