Biodiversity of terrestrial algal communities from soil and air-exposed substrates using a molecular approach Dissertation zur Erlangung des mathematisch-naturwissenschaftlichen Doktorgrades „Doctor rerum naturalium“ der Georg-August-Universität Göttingen im Promotionsprogramm Biologie der Georg-August-University School of Science (GAUSS) vorgelegt von Christine Hallmann aus Bremen Göttingen, 2015 Betreuungsausschuss: Prof. Dr. Thomas Friedl, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Abteilung Experimentelle Phykologie und Sammlung von Algenkulturen, Universität Göttingen PD Dr. Michael Hoppert, Institut für Mikrobiologie und Genetik, Abteilung für Allgemeine Mikrobiologie, Universität Göttingen Mitglieder der Prüfungskommission: Referent: Prof. Dr. Thomas Friedl, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Abteilung Experimentelle Phykologie und Sammlung von Algenkulturen, Universität Göttingen Korreferent: PD Dr. Michael Hoppert, Institut für Mikrobiologie und Genetik, Abteilung für Allgemeine Mikrobiologie, Universität Göttingen weitere Mitglieder der Prüfungskommission: Prof. Dr. Hermann Behling, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Abteilung Palynologie und Klimadynamik, Universität Göttingen Prof. Dr. Erwin Bergmeier, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Abteilung Vegetationsanalyse und Phytodiversität, Universität Göttingen Prof. Dr. Rolf Daniel, Institut für Mikrobiologie und Genetik, Abteilung für Genomische und Angewandte Mikrobiologie, Universität Göttingen Prof. Dr. Joachim Reitner, Geowissenschaftliches Zentrum, Abteilung Geobiologie, Universität Göttingen Tag der mündlichen Prüfung: 24.06.2015 Table of contents General Introduction ......................................................................................................... 1 Results Chapter 1 Molecular diversity of phototrophic biofilms on building stone ................... 6 Chapter 2 Cryptogam covers on sepulchral monuments and re-colonization of a marble surface after cleaning ...................................................................... 39 Chapter 3 Molecular diversity on a marble monument: a case study ........................... 60 Chapter 4 Biodiversity of green phototrophic biofilms on artificial hard surfaces: a case study of aero-terrestrial algae in an urban environment using molecular approaches .................................................................................. 81 Chapter 5 Green algal communities from soil and tree bark in middle European forests: a molecular comparison ............................................................... 112 Chapter 6 Diversity of green algae in grassland and forest soils under different management types - a culture-independent approach ............................... 147 Summary ......................................................................................................................... 192 Danksagung .................................................................................................................... 194 Publications .................................................................................................................... 195 General Introduction General Introduction Eukaryotic microalgae are widespread phototrophic organisms not only in aqueous habitats like freshwater or the marine environment, but are also common and abundant in diverse terrestrial habitats (Hoffmann, 1989; López-Bautista et al., 2007; Karsten et al., 2007; Rindi et al., 2010; Holzinger and Karsten, 2013; Ettl and Gärtner, 2014). Among terrestrial algae, the green algae, along with the prokaryotic cyanobacteria, may be the most diverse group (Hoffmann, 1989; Rindi, 2007; Rindi, 2011). The eukaryotic terrestrial green algae comprise both phyla Chlorophyta and Streptophyta (Lewis and McCourt, 2004; Pröschold and Leliaert, 2007). Terrestrial green algae are particularly diverse in the Chlorophyta where they are distributed on at least three lineages, the classes Trebouxiophyceae, Chlorophyceae and Ulvophyceae (Friedl and Rybalka, 2012; Leliaert et al., 2012). Out of these three classes the Trebouxiophyceae appear to comprise predominantly terrestrial algae as most of its members are known from dry habitats exposed to air (aeroterrestrial algae), soil or lichen symbiosis (Friedl and Rybalka, 2012; Leliaert et al., 2012). With respect to colonization of land the Streptophyta may have been the most successful lineage, because the embryophytes have their origin within the Streptophyta (Karol et al., 2001; Turmel et al., 2007; Wodniok et al., 2011). Common terrestrial algal groups of the Streptophyta are members of the Klebsormidiophyceae (Rindi et al., 2011; Ryšánek et al., 2015). In general, several independent origins of “land plants”, i.e. terrestrial green algae have been discussed (Lewis and Lewis, 2005; Lewis, 2007). Terrestrial algae are obvious at almost all air exposed substrates, e.g. rocks, building stone, monument surfaces (Crispim et al., 2003; Hoppert et al., 2004; Macedo et al., 2009), artificial substrates in urban environment (such as house facades and roof tiles; Karsten et al., 2007), tree barks (Gärtner, 1994; Lüttge and Büdel, 2010; Neustupa and Štifterová, 2013) where green algae often form conspicuous green phototrophic biofilms or crusts, as well as soils. The green algal communities (as well as the cyanobacterial communities) on stone surfaces may be involved in biodeterioration of their substrates growing epi- and endolithically on rocks as well as building material and monument surfaces (Saiz-Jimenez, 1995; Warscheid and Braams, 2000; Crispim et al., 2003; Gaylarde et al., 2003; Hoppert et al., 2005; López-Bautista et al., 2007; Cutler et al., 2013). They also contribute to undesirable coloration observed in urban habitats on various man-made substrates, e.g. building facades and roof tiles (Barberousse et al., 2006; Rindi, 2007; Gladis-Schmacka 1 General Introduction et al., 2014). Soils are the most important non-aqueous habitats for terrestrial algal communities (Zenova et al., 1995). They have beneficial functions in soil, e.g. provide organic matter as food source, perform nitrogen fixation, and interact with higher plants (Metting, 1981; Starks et al., 1981). These green algal communities together with cyanobacteria are among the first organisms colonizing bare soils, deserts or disturbed sites and promote soil stability and constitute water-stable aggregates to prevent erosion (Johansen, 1993; Lewis and Lewis, 2005; Büdel et al., 2014). Traditionally, the composition of terrestrial green algal communities have been assessed by the establishment of enrichment cultures followed by the development of pure unialgal cultures. These isolates have been identified by microscopic features which, however, are often rather spare in distinctive characters which may cause uncertainties in the identification. Only single terrestrial green algal isolates have been identified at the molecular level, i.e. by comparisons of DNA sequence signatures which enable an unambiguous identification and re-identification of algal species. Using traditional culture methods only, there have been considerable uncertainties in the diversity of terrestrial green algal communities due to different identification strategies depending on the authors and taxonomic uncertainties of previously published floristic lists (Hoffmann, 1989; Lukešová and Hoffmann, 1996; Neustupa and Škaloud, 2010). A culture-independent molecular approach provides unambiguous identification of green algal communities e.g. in terrestrial environments. Even if the recovered species may not be exactly named in case of lack of appropriate reference sequences or taxonomic problems, the algal taxa can be unambiguously recovered by their unique molecular signature. This enables not only a more reliable approach to determine the diversity of an algal community, but also permits comparisons among the algal communities of the same and different habitats. 2 General Introduction Aim of the thesis The main objective of this thesis was to test the reliability of a molecular approach for the diversity assessment of terrestrial green algal communities using several exemplar habitats, i.e. open air-exposed-ones as well as soil. It should be further evaluated in as much the green algal species composition can be unambiguously determined so that comparisons of different communities at the same habitat/substrate along a gradient of abiotic parameters or between different habitats and substrates are possible. 18S rRNA gene sequences were used determined from environmental clone libraries. This approach was compared with corresponding analyses based on enrichment cultures or unialgal isolates in selected cases, i.e. chapters 4 and 6. Chapter 1 describes the diversity of air-exposed phototrophic biofilms, including cyanobacteria, on natural building stone. The communities of two wall areas with different expositions towards sun were compared. Chapter 2 and Chapter 3 present the assessment of biofilms consisting green algae and fungi covering stone monuments, their composition and potential involvement in biodeterioration. Additionally, the re-colonization after cleaning and restauration procedures were investigated. The analysis of a green algal biofilm on an exemplar artificial hard substrate in an urban environment is outlined in Chapter 4. In Chapter 5 also a natural air-exposed substrate, different types of tree bark, was studied in the framework of the