Leaf-associated fungal diversity in acidified streams: insights from combining traditional and molecular approaches Hugues Clivot, 1,2 * Julien Cornut, 1,2,3,4 Eric Chauvet, 3,4 Arnaud Elger, 3,4 Pascal Poupin, 1,2 François Guérold 1,2 and Christophe Pagnout 1,2 1 Laboratoire Interdisciplinaire des Environnements Continentaux (LIEC), Université de Lorraine, UMR 7360, Campus Bridoux rue du Général Delestraint, 57070 Metz, France. 2 LIEC, CNRS, UMR 7360, 57070 Metz, France. 3 Laboratoire d’Ecologie Fonctionnelle et Environnement (EcoLab), INP, UPS, Université de Toulouse, UMR 5245, 118 Route de Narbonne, 31062 Toulouse, France. 4 EcoLab, CNRS, UMR 5245, 31062 Toulouse, France. Summary We combined microscopic and molecular methods to investigate fungal assemblages on alder leaf litter exposed in the benthic and hyporheic zones of five streams across a gradient of increasing acidification for 4 weeks. The results showed that acidification and elevated Al concentrations strongly depressed sporulating aquatic hyphomycetes diversity in both zones of streams, while fungal diversity assessed by denaturing gradient gel electrophoresis (DGGE) appeared unaffected. Clone library analyses revealed that fungal communities on leaves were dominated by members of Ascomycetes and to a lesser extent by Basidiomycetes and Chytridiomycetes. An important contribution of terrestrial fungi was observed in both zones of the most acidified stream and in the hyporheic zone of the reference circumneutral stream. The highest leaf breakdown rate was observed in the circumneutral stream and occurred in the presence of both the highest diversity of sporulating aquatic hyphomycetes and the highest contribution to clone libraries of sequences affiliated with aquatic hypho- mycetes. Both methods underline the major role played by aquatic hyphomycetes in leaf decom- position process. Our findings also bring out new highlights on the identity of leaf-associated fungal communities and their responses to anthropogenic alteration of running water ecosystems. Introduction Fungi play a crucial role in detritus-based lotic ecosys- tems as main microbial mediators of allochthonous leaf litter decomposition (Findlay and Arsuffi, 1989; Baldy et al., 1995; Gulis and Suberkropp, 2003). Aquatic fungi, through their leaf-degrading enzyme capabilities, contrib- ute to microbial leaf processing and improve the nutri- tional quality of leaf litter for further consumption by invertebrate detritivores (Bärlocher and Kendrick, 1981; Arsuffi and Suberkropp, 1988). Aquatic hyphomycetes, a polyphyletic group of fungi, are thought to dominate microbial communities growing on decaying leaves (Bärlocher, 1992; Suberkropp, 1992; Gessner et al., 2007). Both leaf litter breakdown and associated aquatic hyphomycete diversity have been considered as useful indicators of the functional integrity of streams (Gessner and Chauvet, 2002) and as potential bioindicators of anthropogenic stress (Solé et al., 2008). In particular, reduced leaf litter breakdown and concurrent loss of aquatic hyphomycete diversity have been reported in numerous studies, notably under high concentrations of metals such as Al in acidified streams (Baudoin et al., 2008), Cd (Moreirinha et al., 2011), Ag (Pradhan et al., 2011), Zn (Fernandes et al., 2009; Niyogi et al., 2009) or even in the context of multiple (i.e. Cu and Zn) contami- nants (Duarte et al., 2008). These observed decreases in species richness under anthropogenic stress have tradi- tionally been assessed using microscopic methods based on the morphological identification of conidia (asexual spores), which varies between species. Recently developed molecular methods allow investi- gation of fungal communities regardless of their life history stage, including mycelia, which constitute the metabolically active part of fungal biomass on leaves (Bärlocher, 2007; 2010; Krauss et al., 2011). Molecular fingerprinting of microbial communities, namely terminal restriction fragment length polymorphism analysis and denaturing gradient gel electrophoresis (DGGE), first showed that the traditional method (conidial identification) Received 14 May, 2013; revised 31 July, 2013; accepted 3 August, 2013. *For correspondence. E-mail hugues.clivot@univ-lorraine.fr; Tel. (+33) 387378657; Fax (+33) 387378512. Environmental Microbiology (2014) 16(7), 2145–2156 doi:10.1111/1462-2920.12245 © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd