Filling Gaps in Biodiversity Knowledge for Macrofungi: Contributions and Assessment of an Herbarium Collection DNA Barcode Sequencing Project

Despite recent advances spearheaded by molecular approaches and novel technologies, species description and DNA sequence information are significantly lagging for fungi compared to many other groups of organisms. Large scale sequencing of vouchered herbarium material can aid in closing this gap. Here, we describe an effort to obtain broad ITS sequence coverage of the approximately 6000 macrofungal-species-rich herbarium of the Museum of Natural History in Venice, Italy. Our goals were to investigate issues related to large sequencing projects, develop heuristic methods for assessing the overall performance of such a project, and evaluate the prospects of such efforts to reduce the current gap in fungal biodiversity knowledge. The effort generated 1107 sequences submitted to GenBank, including 416 previously unrepresented taxa and 398 sequences exhibiting a best BLAST match to an unidentified environmental sequence. Specimen age and taxon affected sequencing success, and subsequent work on failed specimens showed that an ITS1 mini-barcode greatly increased sequencing success without greatly reducing the discriminating power of the barcode. Similarity comparisons and nonmetric multidimensional scaling ordinations based on pairwise distance matrices proved to be useful heuristic tools for validating the overall accuracy of specimen identifications, flagging potential misidentifications, and identifying taxa in need of additional species-level revision. Comparison of within- and among-species nucleotide variation showed a strong increase in species discriminating power at 1–2% dissimilarity, and identified potential barcoding issues (same sequence for different species and vice-versa). All sequences are linked to a vouchered specimen, and results from this study have already prompted revisions of species-sequence assignments in several taxa.

[1]  A. Agresti.  An introduction to categorical data analysis , 1997 .

[2]  E. Kristiansson,et al.  A software pipeline for processing and identification of fungal ITS sequences , 2009, Source Code for Biology and Medicine.

[3]  R. Henrik Nilsson,et al.  PlutoF—a Web Based Workbench for Ecological and Taxonomic Research, with an Online Implementation for Fungal ITS Sequences , 2010, Evolutionary Bioinformatics Online.

[4]  Thomas D. Bruns,et al.  Fungal Community Ecology: A Hybrid Beast with a Molecular Master , 2008 .

[5]  T. White.  Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics , 1990 .

[6]  D. Stevenson,et al.  A comparison of algorithms for the identification of specimens using DNA barcodes: examples from gymnosperms , 2007, Cladistics : the international journal of the Willi Hennig Society.

[7]  Robin Sen,et al.  UNITE: a database providing web-based methods for the molecular identification of ectomycorrhizal fungi. , 2005, The New phytologist.

[8]  M. Bidartondo,et al.  How to know unknown fungi: the role of a herbarium. , 2009, The New phytologist.

[9]  Bryn T. M. Dentinger,et al.  Comparing COI and ITS as DNA Barcode Markers for Mushrooms and Allies (Agaricomycotina) , 2011, PloS one.

[10]  N. Puillandre,et al.  New taxonomy and old collections: integrating DNA barcoding into the collection curation process , 2012, Molecular ecology resources.

[11]  T. Bruns,et al.  The molecular revolution in ectomycorrhizal ecology: peeking into the black‐box , 2001, Molecular ecology.

[12]  R. Henrik Nilsson,et al.  An open source software package for automated extraction of ITS1 and ITS2 from fungal ITS sequences for use in high-throughput community assays and molecular ecology , 2010 .

[13]  C. Meyer,et al.  The Controversy , 2022 .

[14]  Erik Kristiansson,et al.  An outlook on the fungal internal transcribed spacer sequences in GenBank and the introduction of a web-based tool for the exploration of fungal diversity. , 2009, The New phytologist.

[15]  R. Henrik Nilsson,et al.  Progress in molecular and morphological taxon discovery in Fungi and options for formal classification of environmental sequences , 2011 .

[16]  P. Bridge,et al.  On the unreliability of published DNA sequences. , 2003, The New phytologist.

[17]  J. Tebbs,et al.  An Introduction to Categorical Data Analysis , 2008 .

[18]  G. Ainsworth.  Ainsworth & Bisby's "Dictionary of the Fungi" , 1972 .

[19]  T. White,et al.  Identification of indigenous and introduced symbiotic fungi in ectomycorrhizae by amplification of nuclear and mitochondrial ribosomal DNA , 1991 .

[20]  E. Kristiansson,et al.  An open source chimera checker for the fungal ITS region , 2010, Molecular ecology resources.

[21]  R. FitzJohn,et al.  Using terminal restriction fragment length polymorphism (T-RFLP) to identify mycorrhizal fungi: a methods review , 2007, Mycorrhiza.

[22]  K. Seifert.  Progress towards DNA barcoding of fungi , 2009, Molecular ecology resources.

[23]  M. Blackwell.  The fungi: 1, 2, 3 ... 5.1 million species? , 2011, American journal of botany.

[24]  John L. Spouge,et al.  Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi , 2012, Proceedings of the National Academy of Sciences.

[25]  David L. Hawksworth,et al.  Ainsworth & Bisby's Dictionary of the Fungi , 1972 .

[26]  G. Mueller,et al.  An estimate of the lower limit of global fungal diversity , 2006, Biodiversity and Conservation.

[27]  Rytas Vilgalys,et al.  Fungal Community Analysis by Large-Scale Sequencing of Environmental Samples , 2005, Applied and Environmental Microbiology.

[28]  R. Henrik Nilsson,et al.  Intraspecific ITS Variability in the Kingdom Fungi as Expressed in the International Sequence Databases and Its Implications for Molecular Species Identification , 2008, Evolutionary bioinformatics online.

[29]  E. Larsson,et al.  Fruiting body-guided molecular identification of root-tip mantle mycelia provides strong indications of ectomycorrhizal associations in two species of Sistotrema (Basidiomycota). , 2006, Mycological research.

[30]  S. Haberman.  The Analysis of Residuals in Cross-Classified Tables , 1973 .

[31]  Rob DeSalle,et al.  The unholy trinity: taxonomy, species delimitation and DNA barcoding , 2005, Philosophical Transactions of the Royal Society B: Biological Sciences.

[32]  R. C. Gardner,et al.  Type I Error Rate Comparisons of Post Hoc Procedures for I j Chi-Square Tables , 2000 .

[33]  R. Henrik Nilsson,et al.  Approaching the taxonomic affiliation of unidentified sequences in public databases – an example from the mycorrhizal fungi , 2005, BMC Bioinformatics.

[34]  R. Luecking.  Taxonomy: a discipline on the brink of extinction Are DNA barcode scanners the future of biodiversity research? , 2008 .

[35]  Andy F. S. Taylor,et al.  The UNITE database for molecular identification of fungi--recent updates and future perspectives. , 2010, The New phytologist.

[36]  R. DeSalle.  Species Discovery versus Species Identification in DNA Barcoding Efforts: Response to Rubinoff , 2006, Conservation biology : the journal of the Society for Conservation Biology.

[37]  D. Rizzo,et al.  AFLP and phylogenetic analyses of North American and European populations of Phytophthora ramorum. , 2004, Mycological research.