Gregory M. Mueller

Biodiversity of fungi : inventory and monitoring methods

Fungi and Their Allies Preparation, Preservation, and Use of Fungal Specimens in Herbaria Preservation and Distribution of Fungal Cultures Electronic Information Resources Fungal Biodiversity Patterns Molecular Methods for Discriminating Taxa, Monitoring Species, and Assessing Fungal Diversity Fungi on Living Plant Substrata, Including Fruits Terrestrial and Lignicolous Macrofungi Lichenized Fungi Sequestrate Fungi Microfungi on Wood and Plant Debris Endophytic Fungi Saprobic Soil Fungi Fungi in Stressful Environments Mutualistic Arbuscular Endomycorrhizal Fungi Yeasts Fungicolous Fungi Insect- and Other Arthropod-Associated Fungi Fungal Parasites and Predators of Rotifers, Nematodes, and Other Invertebrates Fungi Associated With Vertebrates Coprophilous Fungi Anaerobic Zoosporic Fungi Associated with Animals Fungi in Freshwater Habitats Marine and Estuarine Mycelial Eumycota and Oomycota Mycetozoans Fungi Associated with Aquatic Animals

An estimate of the lower limit of global fungal diversity

We conservatively estimate that there is a minimum of 712,000 extant fungal species worldwide, but we recognize that the actual species richness is likely much higher. This estimate was calculated from the ratio of fungal species to plant species for various ecologically defined groups of fungi in well-studied regions, along with data on each groups’ level of endemism. These calculations were based on information presented in the detailed treatments of the various fungal groups published in this special issue. Our intention was to establish a lower boundary for the number of fungal species worldwide that can be revised upward as more information becomes available. Establishing a lower boundary for fungal diversity is important as current estimates vary widely, hindering the ability to include fungi in discussions of ecology, biodiversity and conservation. Problems inherent in making these estimates, and the impact that additional data on fungal and plant species diversity will have on these estimates are discussed.

Fungal biodiversity: what do we know? What can we predict?

Although fungi are among the most important organisms in the world, only limited and incomplete information is currently available for most species and current estimates of species numbers for fungi differ significantly. This lack of basic information on taxonomic diversity has significant implications for many aspects of evolutionary biology. While the figure of 1.5 million estimated fungal species is commonly used, critics have questioned the validity of this estimate. Data on biogeographic distributions, levels of endemism, and host specificity must be taken into account when developing estimates of global fungal diversity. This paper introduces a set of papers that attempt to develop a rigorous, minimum estimate of global fungal diversity based on a critical assessment of current species lists and informed predictions of missing data and levels of endemism. As such, these papers represent both a meta-analysis of current data and a gap assessment to indicate where future research efforts should be concentrated.

Global diversity and distribution of macrofungi

Data on macrofungal diversity and distribution patterns were compiled for major geographical regions of the world. Macrofungi are defined here to include ascomycetes and basidiomycetes with large, easily observed spore-bearing structures that form above or below ground. Each coauthor either provided data on a particular taxonomic group of macrofungi or information on the macrofungi of a specific geographic area. We then employed a meta-analysis to investigate species overlaps between areas, levels of endemism, centers of diversity, and estimated percent of species known for each taxonomic group for each geographic area and for the combined macrofungal data set. Thus, the study provides both a meta-analysis of current data and a gap assessment to help identify research needs. In all, 21,679 names of macrofungi were compiled. The percentage of unique names for each region ranged from 37% for temperate Asia to 72% for Australasia. Approximately 35,000 macrofungal species were estimated to be “unknown” by the contributing authors. This would give an estimated total of 56,679 macrofungi. Our compiled species list does not include data from most of S.E. Europe, Africa, western Asia, or tropical eastern Asia. Even so, combining our list of names with the estimates from contributing authors is in line with our calculated estimate of between 53,000 and 110,000 macrofungal species derived using plant/macrofungal species ratio data. The estimates developed in this study are consistent with a hypothesis of high overall fungal species diversity.

A ‘dirty’ business: testing the limitations of terminal restriction fragment length polymorphism (TRFLP) analysis of soil fungi

Terminal restriction fragment length polymorphism (TRFLP) is an increasingly popular method in molecular ecology. However, several key limitations of this method have not been fully examined especially when used to study fungi. We investigated the impact of spore contamination, intracollection ribosomal DNA internal transcribed spacer (ITS) region variation, and conserved restriction enzyme recognition loci on the results produced by TRFLP to characterize soil fungal communities. We find that (i) the potential for nontarget structures such as spores to contribute DNA to target sample extractions is high; (ii) multiple fragments (i.e. ‘extra peaks’) per PCR primer‐restriction enzyme combination can be detected that are caused by restriction enzyme inefficiency and intracollection ribosomal DNA ITS variation; and (iii) restriction enzyme digestion in conserved vs. variable gene regions leads to different characterizations of community diversity. Based on these results, we suggest that studies employing TRFLP need to include information from known, identified fungi from sites within which studies take place and not to rely only on TRFLP profiles as a short cut to fungal community description.

Phylogeography and biogeography of fungi.

The rigorous study of processes shaping geographic distributions of lineages is a relatively new and emerging field in mycology. While it was previously generally believed that most fungi have wide distributions and largely unstructured populations, recent studies have shown that this is not the case. The study of distributions in tandem with molecular approaches to phylogeny has recently made substantial advances to our understanding of the diversity and biogeography of fungi. Comprehensive species inventories have provided a better picture of the actual distribution of these organisms, while robust phylogenies based on molecular characters have provided both data that allow interpretation of current distributions and testable hypotheses regarding the processes responsible for distribution patterns. This commentary provides an introduction to five papers in this issue of Mycological Research that focus on fungal phylogeography. These papers are based on oral contributions given at two symposia at the International Mycological Congress (IMC8) held in Cairns (Australia) in August 2006.

Accumulation of several heavy metals and lanthanides in mushrooms (Agaricales) from the Chicago region

Abstract This study explored the differences in metal uptake in sporocarps of ectomycorrhizae-forming fungi relative to (1) fungal species; (2) collection location; (3) differential metal uptake and variation within single-species, single-area populations; and (4) mobile metal content of soil substrate for the fungi. In addition, this study examined levels of some of the lanthanides in these mushrooms, as lanthanide uptake in higher fungi has not been quantified to date. In 1995 and 1996, sporocarps from three species of ectomycorrhizal fungi (Amanita flavorubescens, Amanita rubescens, and Russula pectinatoides) were collected from Cowles Bog, Indiana Dunes National Lakeshore (near an industrial area) and the Palos forest preserves (near a residential area). Soil was also collected from the Cowles Bog plots; metals were extracted from the soil, either with local Lake Michigan water or with nitric acid. These two extractions were meant to simulate the natural soil equilibrium concentrations of soluble metals and the maximum possible effects of any fungal chelating chemicals, respectively. An inductively coupled plasma mass spectrometer was used to analyze soil extracts and nitric acid digests of whole sporocarps for the target analytes. The metals found at elevated levels in the mushrooms included four of environmental interest (Ag, Cd, Ba, and Pb) and three lanthanides (La, Ce, and Nd). Significant differences in uptake of metals were observed between A. rubescens and R. pectinatoides, while A. rubescens and A. flavorubescens were not significantly different. With regard to location, more cadmium was found in Cowles Bog collections of A. rubescens, while Palos forest A. rubescens had more of the lanthanides and barium. Significant specimen-to-specimen variation occurred in all populations examined. Correlation analysis between pairs of trace elements within each sporocarp population revealed strong positive correlations between the lanthanides. Sporocarps concentrated more metal than was made available by the lake water extraction of soil and less metal than was made available by the nitric acid extraction of soil.

Mitochondrial DNA polymorphisms in Laccaria bicolor, L. laccata, L. proxima and L. amethystina

The mitochondrial DNA (mtDNA) from 25 Laccaria bicolor , 8 L. laccata , 3 L. proxima and 2 L. amethystina isolates was examinated by restriction fragment length polymorphism (RFLP) analysis. Whole-cell DNA from Laccaria isolates was digested with Bam HI, Bgl II and Hind III and probed in Southern hybridizations with cloned mitochondrial fragments from L. bicolor . Analysis of restriction fragment length polymorphisms indicated that mtDNA fragment patterns can be used as additional taxonomic characters for delimiting Laccaria spp. However, intraspecific variation was very high with almost no similarity observed among the four biological species of L. laccata or between the 2 isolates of L. amethystina . A high level of variability was also detected within L. bicolor and several subgroups, possibly biological species, were discernible among the L. bicolor isolates examined. No major discrepancies were observed between the trends indicated by RFLP results on mtDNA and those on nuclear ribosomal DNA. As most of the Laccaria isolates had unique overall mitochondrial pattern, this variability could be used for isolate typing.

Pooled samples bias fungal community descriptions

We tested the accuracy of molecular analyses for recovering the species richness and structure of pooled fungal communities of known composition. We constructed replicate pools of 2–20 species and analysed these pools by two separate pooling‐DNA extraction procedures and three different molecular analyses (Automated Ribosomal Intergenic Spacer Analysis (ARISA), terminal restriction fragment length polymorphism (T‐RFLP) and clone library‐sequencing). None of the methods correctly described the known communities. Only clone library‐sequencing with high sequencing per pool (∼100 clones) recovered reasonable estimates of richness. Frequency data were skewed with all procedures and analyses. These results indicate that the error introduced by pooling samples is significant and problematic for ecological studies of fungal communities.

Beyond dead trees: integrating the scientific process in the Biodiversity Data Journal

Driven by changes to policies of governments and funding agencies, Open Access to content and data is quickly becoming the prevailing model in academic publishing. Open Access benefits scientists with greater dissemination and citation of their work, and provides society as a whole with access to the latest research. Open Access is, however, only one facet of scholarly communication. Core scientific statements or assertions are intertwined and hidden in the scholarly narratives, and the data underlying these statements are often obscured to the point that replication of results is impossible (Nature Editorial 2012). This is in part a result of the way scientific papers are written as narratives, rather than sources of data. An often cited reason for the lack of published data is the absence of a reward mechanism for the individuals involved in creating and managing information (Smith 2009, Costello 2009, Vision 2010, McDade et al. 2011, Duke and Porter 2013). Preparing data for publication is a time consuming activity that few scholars will undertake without recognition from their peers. Data papers are a potential solution to this problem (Chavan and Penev 2011, Chavan and Penev 2013). They allow authors to publish data and receive reward through the traditional citation process. Coupling tools to rapidly and simply generate publications will incentivise this behaviour and create a culture of data curation and sharing within the biodiversity science community. If we are going to incentivise the mass publication of data, we also need mechanisms to ensure quality. Traditional peer review is one of the bottlenecks in standard publication practice (Hauser and Fehr 2007, Fox and Petchey 2010). A common criticism of peer review is the lack of transparency and accountability on the part of the reviewers. To cope with the additional volume of papers created by data publication and to move to a more transparent system, we need to rethink peer review. We need both new methods of reviewing and new tools to automate as much of the review process as possible. This requires a new publishing platform, not just a new journal. An abundance of small isolated datasets does not, however, allow us to address the fundamental problems within the biodiversity science community. These islands of data are only of value if connected and interlinked. The task of interlinking is performed by biodiversity data aggregators like the Global Biodiversity Information Facility (GBIF) and Encylopedia of Life (EOL) which form the backbone of data-driven biodiversity research. By automating the submission of data to these aggregators, we can increase their value to more than the sum of their parts, making small data big. A renewed appreciation of the value of small data will help to reduce the vast amount of research data that exists only on laptops and memory sticks - data that is often lost when people change roles or retire. Works of potentially very limited length can hold intrinsic value to the community, but are almost impossible to publish in traditional journals chasing impact factors. Examples include single species descriptions, local checklists and software descriptions, or ecological surveys and plot data. An infrastructure that allows datasets of any size to be important means we can publish them at any time. There is no need to wait for datasets to reach a critical mass suitable for publication in a traditional journal. Today, we are pleased to announce the official release of the first series of papers published in Biodiversity Data Journal (BDJ). After years of hard work in analyzing, planning and programming the Pensoft Writing Tool (PWT), we now have a publishing platform that addresses the key concerns raised above. This provides the first workflow to support the full life cycle of a manuscript - from writing through submission, community peer-review, publication and dissemination, all within a single online collaborative environment. Shortening distance between “data” and “narrative” publishing Most journals nowadays clearly separate data from narrative (text). Moreover, data publishing through data centres and repositories has almost become a separate sector within the scholarly publishing landscape. BDJ is not a conventional journal, nor is it a conventional “data journal”. It aims to integrate data and text in a single publication by converting several kinds of biodiversity data (e.g., species occurrences, checklists, or data tables) into the text for human-readable use, while simultaneously making data units from the same article harvestable and downloadable. The text itself is marked up and presented in a highly structured and machine readable form. BDJ aims to integrate small data into the text whenever possible. Supplementary data files that underpin graphs, hypotheses and results can also be uploaded on the journal’s website and published with the article. Nonetheless, this is usually not possible for large or complex data, for which we recommend deposition in an established open international repository (for details, see Penev et al. 2011): Large primary biodiversity data sets (e.g., institutional collections of species-occurrence records) should be published with the GBIF Integrated Publishing Toolkit (IPT); small data sets of this kind are imported into the article text through an Excel template, available in PWT. Genomic data should be deposited with INSDC (GenBank/EMBL/DDBJ), either directly or via a partnering repository, e.g. Barcode of Life Data Systems (BOLD). Transcriptomics data should be deposited in Gene Expression Omnibus (GEO) or ArrayExpress. Phylogenetic data should be deposited at TreeBASE, either directly or through the Dryad Data Repository. Biodiversity-related geoscience and environmental data should be deposited in PANGAEA. Morphological images other than those presented in the article should be deposited at Morphbank. Images of a specific kind should be deposited in appropriate repositories if these exist (e.g., Morphosource for MicroCT data). Videos should be uploaded to video sharing sites like YouTube, Vimeo or SciVee and linked back to the article text. Similarly, audio files should go to platforms like FreeSound or SoundCloud, and presentations to Slideshare. In addition, multimedia files can also be uploaded as supplementary files on the journal’s website. 3D and other interactive models can be embedded in the article’s HTML and PDF. Any other large data sets (e.g., ecological observations, environmental data, morphological and other data types) should be deposited in the Dryad Data Repository, either prior to or upon acceptance of the manuscript. Other specialised data repositories can be used if these offer unique identifiers and long-term preservation. All external data used in a BDJ paper must be cited in the reference list, and links to these data (as deposited in external repositories) must be included in a separate data resources section of the article. All datasets, images or multimedia are freely downloadable from the text under the Open Data Commons Attribution License or a Creative Commons CC-Zero waiver / Public Domain Dedication. The article text is available under a Creative Commons (CC-BY) 3.0 license. Primary biodiversity data within an article can be exported in Darwin Core Archive format, which makes them interoperable with biodiversity tools based on the Darwin Core standard. By facilitating open access to the data that underlie every publication, BDJ is setting a new standard in transparency and repeatability in biodiversity science. Perpetual and universal access to primary data stimulates scientific progress by helping authors build upon existing datasets. BDJ’s commitment to supporting automated data aggregation and interlinking is happening alongside multiple advances in biodiversity informatics infrastructure that herald the dawning of an era of collaborative, big-data biodiversity science (Page 2008, Patterson et al. 2010, Thessen and Patterson 2011, Parr et al. 2012).