Phylogenetic reconstruction reveals cryptic species diversity within the fungal order Sebacinales Brian R. Murphy, Trevor R. Hodkinson School of Natural Sciences, Trinity College Dublin Dublin, Ireland. murphb16@tcd.ie Bryn T.M. Dentinger, Laura M. Suz Jodrell Laboratory, Royal Botanic Gardens Kew, Richmond, Surrey, TW9 3AB England Abstract— Sebacinales are a large, potentially useful and ecologically diverse order of fungi that requires in-depth DNA sequencing for barcoding and phylogenetics, along with accurate morphological data. For identification using sequences it is essential to have data from well classified and studied reference collections so that genotype and phenotype can be compared. DNA from macroscopic fungarium sebacinalean fungi was extracted and characterized by DNA sequencing and phylogenetic analyses. Nuclear ribosomal ITS regions were successfully amplified and sequenced for 15 Sebacinales species. Likelihood of DNA amplification was negatively correlated with increasing age of the specimens. Phylogenetic analysis of these sequences combined with 187 sequences obtained from GenBank revealed significant cryptic diversity within Sebacinales. The type genus Sebacina was not recovered as monophyletic. These results suggest there may be a need for reclassification of some Sebacinales and that alternative approaches such as next generation sequencing may be required to efficiently obtain DNA sequences from older fungarium specimens. The reference ITS barcode sequences generated from this study will provide an invaluable tool for researchers working on applied and basic aspects of these fungi to infer ecological and evolutionary patterns. Keywords- Cryptic diversity; DNA barcoding; Fungarium specimens; Sebacinales; Phylogeny I. INTRODUCTION Sebacinales is a fungal order with extremely versatile and diverse mycorrhizal associations, including ectomycorrhizae, ericoid and orchid mycorrhizae [1,2,3,4,5,6,7,8]. Sebacinales are almost universally present as symptomless endophytes [9] and have been found in bryophytes, pteridophytes and all families of herbaceous angiosperms studied to date [9]. Some sebacinalean endophytes, e.g. Piriformospora indica and Sebacina vermifera show great promise as biocontrol and biofertilising agents in crop plants [9,10,11,12,13]. Due to their ubiquity and ancient symbiotic history, Sebacinales are a largely underappreciated universal hidden force in plant ecosystems [10,14]. One of the major obstacles to advancing our understanding of Sebacinales is a vastly incomplete reference database for what now appears to be a large number of cryptic taxa. This insufficiency makes it difficult for ecological surveys to identify and quantify sebacinalean taxa, and incomplete taxon sampling for phylogenetic studies renders our inference of evolutionary patterns unreliable. A modern taxonomic treatment of the group based on molecular phylogenetic analysis to aid in diagnosis and description of novel diversity is needed. Therefore, we aimed to improve the available set of reference sequences by generating Internal Transcribed Spacer (ITS) barcode sequences from fully identified, vouchered sebacinalean fungi from the extensive fungarium collections at the Royal Botanic Gardens, Kew (K), and place them in a phylogenetic context to enhance the capacity of researchers focusing on applied and basic aspects of these important fungi to infer ecological and evolutionary patterns. Fungi are instrumental in ecosystem functioning and nutrient cycling, and are commercially important as pests, fermenters, decomposers and pathogens. Some species of Sebacinales have great biocontrol and biofertilisation potential in agriculture, but an inability to identify biologically important species hampers efforts to conserve, study, contain or utilize them. II. MATERIALS AND METHODS Approximately 500 specimens of Sebacinales are recorded in the fungarium at K. The choice of specimens to sample was based on a trade-off between specimen age and comprehensive sampling of all sebacinalean species held in K, including holotypes and isotypes. Although we always selected the most recently collected material when it was available, we did also attempt to sequence DNA from specimens that date back to the 19 th century. The K specimens were generally in good condition (i.e. no obvious mold growth or completely disintegrating cells). Candidate K samples were examined using macro and microscopic characters to confirm identities as far as possible, resulting in the choice of thirty-six species of Sebacinales, representing 7 genera, for DNA extraction. Fungal tissues were sampled in duplicate for internal replication of DNA extractions to confirm sequencing results. DNA was extracted using an enzymatic digestion-glass fiber filtration protocol in 96-well plate format adapted for use with a vacuum-manifold as described in [15]. Full- and partial-length nuclear ribosomal internal transcribed spacer (ITS) of DNA regions were amplified using direct and nested PCR with the forward primers ITS1F [16], ITS8F [15], NSSeb1 and ITS3Seb [9]), and the reverse primers ITS2, ITS4 [17] and ITS6R [15], following the cycling conditions in [15]. The extracted DNA sequences were combined in a sequence matrix with 242 identified Sebacinales ITS sequences from GenBank. The dataset was cleaned up by removing sequences which were not sufficiently identified at the species level, leaving 187 sequences in the data matrix. The matrix was realigned using SECTION Natural science - biology GLOBAL VIRTUAL C O N F E R E N C E The 2nd Global Virtual Conference http://www.gv-conference.com Global Virtual Conference April, 7. - 11. 2014 - 473 -