Karin Glaser

Interannual variation in land-use intensity enhances grassland multidiversity

Significance Land-use intensification is a major threat to biodiversity. So far, however, studies on biodiversity impacts of land-use intensity (LUI) have been limited to a single or few groups of organisms and have not considered temporal variation in LUI. Therefore, we examined total ecosystem biodiversity in grasslands varying in LUI with a newly developed index called multidiversity, which integrates the species richness of 49 different organism groups ranging from bacteria to birds. Multidiversity declined strongly with increasing LUI, but changing LUI across years increased multidiversity, particularly of rarer species. We conclude that encouraging farmers to change the intensity of their land use over time could be an important strategy to maintain high biodiversity in grasslands. Although temporal heterogeneity is a well-accepted driver of biodiversity, effects of interannual variation in land-use intensity (LUI) have not been addressed yet. Additionally, responses to land use can differ greatly among different organisms; therefore, overall effects of land-use on total local biodiversity are hardly known. To test for effects of LUI (quantified as the combined intensity of fertilization, grazing, and mowing) and interannual variation in LUI (SD in LUI across time), we introduce a unique measure of whole-ecosystem biodiversity, multidiversity. This synthesizes individual diversity measures across up to 49 taxonomic groups of plants, animals, fungi, and bacteria from 150 grasslands. Multidiversity declined with increasing LUI among grasslands, particularly for rarer species and aboveground organisms, whereas common species and belowground groups were less sensitive. However, a high level of interannual variation in LUI increased overall multidiversity at low LUI and was even more beneficial for rarer species because it slowed the rate at which the multidiversity of rare species declined with increasing LUI. In more intensively managed grasslands, the diversity of rarer species was, on average, 18% of the maximum diversity across all grasslands when LUI was static over time but increased to 31% of the maximum when LUI changed maximally over time. In addition to decreasing overall LUI, we suggest varying LUI across years as a complementary strategy to promote biodiversity conservation.

Biodiversity of Klebsormidium (Streptophyta) from alpine biological soil crusts (Alps, Tyrol, Austria, and Italy)

Forty Klebsormidium strains isolated from soil crusts of mountain regions (Alps, 600–3,000 m elevation) were analyzed. The molecular phylogeny (internal transcribed spacer rDNA sequences) showed that these strains belong to clades B/C, D, E, and F. Seven main (K. flaccidum, K. elegans, K. crenulatum, K. dissectum, K. nitens, K. subtile, and K. fluitans) and four transitional morphotypes (K. cf. flaccidum, K. cf. nitens, K. cf. subtile, and K. cf. fluitans) were identified. Most strains belong to clade E, which includes isolates that prefer humid conditions. One representative of the xerophytic lineage (clade F) as well as few isolates characteristic of temperate conditions (clades B/C, D) were found. Most strains of clade E were isolated from low/middle elevations (<1,800 m above sea level; a.s.l.) in the pine‐forest zone. Strains of clades B/C, D, and F occurred sporadically at higher elevations (1,548–2,843 m a.s.l.), mostly under xerophytic conditions of alpine meadows. Comparison of the alpine Klebsormidium assemblage with data from other biogeographic regions indicated similarity with soil crusts/biofilms from terrestrial habitats in mixed forest in Western Europe, North America, and Asia, as well as walls of buildings in Western European cities. The alpine assemblage differed substantially from crusts from granite outcrops and sand dunes in Eastern Europe (Ukraine), and fundamentally from soil crusts in South African drylands. Epitypification of the known species K. flaccidum, K. crenulatum, K. subtile, K. nitens, K. dissectum, K. fluitans, K. mucosum, and K. elegans is proposed to establish taxonomic names and type material as an aid for practical studies on these algae, as well as for unambiguous identification of alpine strains. New combination Klebsormidium subtile (Kützing) Mikhailyuk, Glaser, Holzinger et Karsten comb. nov. is made.

Trace Elements Induce Predominance among Methanogenic Activity in Anaerobic Digestion

Trace elements (TE) play an essential role in all organisms due to their functions in enzyme complexes. In anaerobic digesters, control, and supplementation of TEs lead to stable and more efficient methane production processes while TE deficits cause process imbalances. However, the underlying metabolic mechanisms and the adaptation of the affected microbial communities to such deficits are not yet fully understood. Here, we investigated the microbial community dynamics and resulting process changes induced by TE deprivation. Two identical lab-scale continuous stirred tank reactors fed with distiller’s grains and supplemented with TEs (cobalt, molybdenum, nickel, tungsten) and a commercial iron additive were operated in parallel. After 72 weeks of identical operation, the feeding regime of one reactor was changed by omitting TE supplements and reducing the amount of iron additive. Both reactors were operated for further 21 weeks. Various process parameters (biogas production and composition, total solids and volatile solids, TE concentration, volatile fatty acids, total ammonium nitrogen, total organic acids/alkalinity ratio, and pH) and the composition and activity of the microbial communities were monitored over the total experimental time. While the methane yield remained stable, the concentrations of hydrogen sulfide, total ammonia nitrogen, and acetate increased in the TE-depleted reactor compared to the well-supplied control reactor. Methanosarcina and Methanoculleus dominated the methanogenic communities in both reactors. However, the activity ratio of these two genera was shown to depend on TE supplementation explainable by different TE requirements of their energy conservation systems. Methanosarcina dominated the well-supplied anaerobic digester, pointing to acetoclastic methanogenesis as the dominant methanogenic pathway. Under TE deprivation, Methanoculleus and thus hydrogenotrophic methanogenesis was favored although Methanosarcina was not overgrown by Methanoculleus. Multivariate statistics revealed that the decline of nickel, cobalt, molybdenum, tungsten, and manganese most strongly influenced the balance of mcrA transcripts from both genera. Hydrogenotrophic methanogens seem to be favored under nickel- and cobalt-deficient conditions as their metabolism requires less nickel-dependent enzymes and corrinoid cofactors than the acetoclastic and methylotrophic pathways. Thus, TE supply is critical to sustain the activity of the versatile high-performance methanogen Methanosarcina.

The influence of environmental factors on protistan microorganisms in grassland soils along a land-use gradient

In this study, we investigated the effect of land use intensity, soil parameters and vegetation on protistan communities in grassland soils. We performed qualitative (T-RFLP) and quantitative (qPCR) analyses using primers specifically targeting the 18S rRNA gene for all Eukarya and for two common flagellate groups, i.e. the Chrysophyceae and the Kinetoplastea. Both approaches were applied to extracted soil DNA and RNA, in order to distinguish between the potentially active protists (i.e. RNA pool) and the total protistan communities, including potentially inactive and encysted cells (i.e. DNA pool). Several environmental determinants such as site, soil parameters and vegetation had an impact on the T-RFLP community profiles and the abundance of the quantified 18S rRNA genes. Correlating factors often differed between quantitative (qPCR) and qualitative (T-RFLP) approaches. For instance the Chrysophyceae/Eukarya 18S rDNA ratio as determined by qPCR correlated with the C/N ratio, whereas the community composition based on T-RLFP analysis was not affected indicating that both methods taken together provide a more complete picture of the parameters driving protist diversity. Moreover, distinct T-RFs were obtained, which could serve as potential indicators for either active organisms or environmental conditions like water content. While site was the main determinant across all investigated exploratories, land use seemed to be of minor importance for structuring protist communities. The impact of other parameters differed between the target groups, e.g. Kinetoplastea reacted on changes to water content on all sites, whereas Chrysophyceae were only affected in the Schorfheide. Finally, in most cases different responses were observed on RNA- and DNA-level, respectively. Vegetation for instance influenced the two flagellate groups only at the DNA-level across all sites. Future studies should thus include different protistan groups and also distinguish between active and inactive cells, in order to reveal causal shifts in community composition and abundance in agriculturally used systems.

Primer pairs for the specific environmental detection and T-RFLP analysis of the ubiquitous flagellate taxa Chrysophyceae and Kinetoplastea

Bacterivorous protists play a key role in microbial soil food webs, however due to the lack of specific PCR protocols targeting selected protist taxa, knowledge on the diversity and dynamics of these groups is scarce. We developed specific PCR primers in combination with a T-RFLP protocol for the cultivation-independent analysis of two important taxa of bacterivorous flagellates, the Chrysophyceae and Kinetoplastea, in soil samples. Sequence analysis of clone libraries originating from two soils in temperate regions demonstrated the specificity of the respective primer pairs. Clone sequences affiliating to the Chrysophyceae mainly clustered within the clade C2, which has been known so far for its presence mainly in cold climatic regions, whereas Kinetoplastea sequences were mainly related to the Neobodonid clade. Based on an in silico restriction analysis of database sequence entries, suitable restriction enzymes for a T-RFLP approach were selected. This in silico approach revealed the necessity to use a combination of two restriction enzymes for T-RFLP analysis of the Chrysophyceae. Soil T-RFLP profiles reflected all T-RFs of the clone library sequences obtained from the same soils and allowed to distinguish flagellate communities from different sites. We propose to use these primer pairs for PCR detection and rapid fingerprint screening in environmental samples and envisage their use also for quantitative PCR or next generation sequencing approaches.

Assimilation of cellulose-derived carbon by microeukaryotes in oxic and anoxic slurries of an aerated soil.

ABSTRACT Soil microeukaryotes may trophically benefit from plant biopolymers. However, carbon transfer from cellulose into soil microeukaryotes has not been demonstrated so far. Microeukaryotes assimilating cellulose-derived carbon in oxic and anoxic soil slurries were therefore examined by rRNA-based stable-isotope probing. Bacteriovorous flagellates and ciliates and, likely, mixotrophic algae and saprotrophic fungi incorporated carbon from supplemental [U-13C]cellulose under oxic conditions. A previous study using the same soil suggested that cellulolytic Bacteria assimilated 13C of supplemental cellulose. Thus, it can be assumed that ciliates, cercozoa, and chrysophytes assimilated carbon by grazing upon and utilizing metabolic products of Bacteria that hydrolyzed cellulose in the soil slurries.

Habitat-specific composition of morphotypes with low genetic diversity in the green algal genus Klebsormidium (Streptophyta) isolated from biological soil crusts in Central European grasslands and forests

ABSTRACT Terrestrial filamentous green algae of the widely distributed, cosmopolitan genus Klebsormidium (Klebsormidiophyceae, Streptophyta) are typical components of biological soil crusts (BSCs). These communities occur in all climatic zones and on all continents, where soil moisture is limited or where there has been disturbance. BSCs form water-stable aggregates that have important ecological roles in primary production, nitrogen fixation, nutrient cycling, water retention and stabilization of soils. Although available data on Klebsormidium are limited, its functional importance in BSCs is regarded as high. Therefore, in the present study Klebsormidium strains were isolated from BSCs sampled from various grassland and forest plots of different land use intensities in Central Europe, as provided by the Biodiversity Exploratories, and its intraspecific genetic diversity was evaluated. Previous phylogenetic analyses revealed a relationship between sequence similarity and habitat preference with a higher genetic diversity than expected from a morphological classification. We isolated and sequenced 75 Klebsormidium strains. The molecular phylogeny based on the ITS regions showed that all strains belong to either the previously described clade B/C or clade E. This classification was supported by morphological characteristics: strains assigned to clade B/C were identified as Klebsormidium cf. flaccidum or Klebsormidium cf. dissectum, and strains from clade E as K. nitens or Klebsormidium cf. subtile. Within one clade the strains showed low sequence divergences. These minor differences were independent of the sampling region and land use intensity. Interestingly, most of the strains assigned to clade E were isolated from forest sites, whereas strains from clade B/C occurred equally in grassland and forest sites. Therefore, it is reasonable to assume that habitat with its microenvironmental conditions, and not biogeography, controls genetic diversity in Klebsormidium.

New barcoded primers for efficient retrieval of cercozoan sequences in high-throughput environmental diversity surveys, with emphasis on worldwide biological soil crusts

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 hypervariable 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 (c. 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 grassland 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 operational taxonomic units (OTUs) at 97% similarity threshold from 26 samples, with c. 50,000 sequences per site, and only 8% of noncercozoan sequences. We could report a further increase in the diversity of Cercozoa, as 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.

New Taxa of Streptophyte Algae (Streptophyta) from Terrestrial Habitats Revealed Using an Integrative Approach

Two new genera (Streptosarcina and Streptofilum) and three new species (Streptosarcina arenaria, S. costaricana and Streptofilum capillatum) of streptophyte algae were detected in cultures isolated from terrestrial habitats of Europe and Central America and described using an integrative approach. Additionally, a strain isolated from soil in North America was identified as Hormidiella parvula and proposed as an epitype of this species. The molecular phylogeny based on 18S rRNA and rbcL genes, secondary structure of ITS-2, as well as the morphology of vegetative and reproductive stages, cell ultrastructure, ecology and distribution of the investigated strains were assessed. The new genus Streptosarcina forms a sister lineage to the genus Hormidiella (Klebsormidiophyceae). Streptosarcina is characterized by packet-like (sarcinoid) and filamentous thalli with true branching and a cell organization typical for Klebsormidiophyceae. Streptofilum forms a separate lineage within Streptophyta. This genus represents an easily disintegrating filamentous alga which exhibits a cell coverage of unique structure: layers of submicroscopic scales of piliform shape covering the plasmalemma and exfoliate inside the mucilage envelope surrounding cells. The implications of the discovery of the new taxa for understanding evolutionary tendencies in the Streptophyta, a group of great evolutionary interest, are discussed.

Polyols and UV-sunscreens in the Prasiola-clade (Trebouxiophyceae, Chlorophyta) as metabolites for stress response and chemotaxonomy

In many regions of the world, aeroterrestrial green algae of the Trebouxiophyceae (Chlorophyta) represent very abundant soil microorganisms, and hence their taxonomy is crucial to investigate their physiological performance and ecological importance. Due to a lack in morphological features, taxonomic and phylogenetic studies of Trebouxiophycean algae can be a challenging task. Since chemotaxonomic markers could be a great assistance in this regard, 22 strains of aeroterrestrial Trebouxiophyceae were chemically screened for their polyol‐patterns as well as for mycosporine‐like amino acids (MAAs) in their aqueous extracts using RP‐HPLC and LC‐MS. d‐sorbitol was exclusively detected in members of the Prasiolaceae family. The novel MAA prasiolin and a related compound (“prasiolin‐like”) were present in all investigated members of the Prasiola‐clade, but missing in all other tested Trebouxiophyceae. While prasiolin could only be detected in field material directly after extraction, the “prasiolin‐like” compound present in the other algae was fully converted into prasiolin after 24 h. These findings suggest d‐sorbitol and prasiolin‐like compounds are suitable chemotaxonomic markers for the Prasiolaceae and Prasiola‐clade, respectively. Additional UV‐exposure experiments with selected strains show that MAA formation and accumulation can be induced, supporting their role as UV‐sunscreen.