Recent developments in the taxonomic affiliation and ... - Springer Link

Appl Microbiol Biotechnol (2011) 90:41–57 DOI 10.1007/s00253-011-3143-4

MINI-REVIEW

Recent developments in the taxonomic affiliation and phylogenetic positioning of fungi: impact in applied microbiology and environmental biotechnology Kerstin Voigt & Paul M. Kirk

Received: 15 November 2010 / Revised: 19 January 2011 / Accepted: 19 January 2011 / Published online: 20 February 2011 # Springer-Verlag 2011

Abstract The goal of modern taxonomy is to understand the relationships of living organisms in terms of evolutionary descent. Thereby, the relationships between living organisms are understood in terms of nested clades—every time a speciation event takes place, two new clades are produced. Life comprises three domains of living organisms, these are the Bacteria, the Archaea and the Eukaryota. Within the eukaryotic domain, the fungi form a monophyletic group of the eukaryotic crown group and are thus high up in the evolutionary hierarchy of life. Fungus-like organisms possess certain morphological features of fungi, such as the hyphal organization of the Oomycota or the spores and reproductive structures inside a fructification of plasmodiophorids (Plasmodiophoromycota) and slime moulds (Mycetozoa). The first group are algae which secondarily lost their plastids during evolution and contain cellulose in their cell walls. Both osmotrophic phyla, the Oomycota and the Plasmidiophoromycota belong to the Chromista and Rhizaria, K. Voigt (*) School of Biology and Pharmacy, Institute of Microbiology, Department of Microbiology and Molecular Biology, Jena Microbial Resource Collection, University of Jena, Neugasse 25, 07743 Jena, Germany e-mail: [email protected] K. Voigt Department of Molecular and Applied Microbiology, Leibniz-Institute for Natural Product Research and Infection Biology (Hans Knöll Institute), Beutenbergstrasse 11a, 07745 Jena, Germany P. M. Kirk CABI UK Centre, Bakeham Lane, TW20 9TY Egham, Surrey, UK

respectively, whereas the last group, the cellular and plasmodial slime moulds (Mycetozoa) are phagotrophic amoeboid protists belonging to the Amoebozoa. These fungus-like organisms are not considered further in this review. The Fungi sensu stricto comprise a heterogenous, often inconspicuous group of microorganisms which (1) are primarily heterotrophic with an (2) osmotrophic style of nutrition containing (3) chitin and its derivatives in the cell wall. This review discusses species concepts and current strategies in fungal taxonomy, phylogenetic affiliations of miscellaneous fungus-like groups like the microsporidia, perspectives of fungal nomenclature, and their impact on natural product research. Keywords Fungal nomenclature . Systematics . Phylogenomics . Evolution . Aquatic and terrestrial fungi . Fungal barcoding

Preliminary consideration: biochemistry meets phylogeny and taxonomy Advances in phylogenetics have opened the way for DNA sequences to be incorporated into taxonomy, which challenges the historical classification based on morphology and other traits. To the same extent as phylogenetic studies have helped to reshape the classification of the kingdom Fungi, they influence our understanding of the search for natural products by providing an evolutionary context. From the existence of a particular natural compound in a species, it can be assumed that this compound may be produced from more species in the same genus or phylogenetic clade. Consequently, phylogenetically driven concepts enhance the speed of the identification and the localization of “hot spots” for fungal resources of natural compounds. Therefore, a new

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term is introduced in this review, the natural resource unit (NRU), which defines a taxonomic group which has been derived from a monophyletic phylogenetic clade and produces a specific natural product as a synapomorphic property of that particular clade. If we acclaim that phylogenetic classifications based on modern taxonomic research identify NRUs as a biochemical equivalents to OTUs (operational taxonomic units; Graur and Li 2000), we may also accept that OTUs which identify NRUs need not have scientific names. Moreover, the new taxonomy could contribute to the search for novel natural products by implementing new fungal taxa in naturally organismic alliances rather than in single organisms. Thus, taxonomy enters a new era with the traditional criteria falling behind, of which more is outlined in the following sections.

Introduction: a historical outline of fungal systematics versus taxonomy Taxonomy deals specifically with the identification, description and naming (nomenclature) of organisms and represents a necessary adjunct to the organization of information on biological diversity, while scientific classification is focused on placing the organisms within hierarchical groups showing their relationships to other organisms (Schuh and Brower 2009). Systematics uses taxonomy and classification as primary tool in understanding the evolution of organisms by studying their diversification and relationships through time in planet Earth's biosphere. Traditionally, the kingdom Fungi comprise four phyla, the Ascomycota and Basidiomycota (representing the derived or higher fungi) and the Chytridiomycota and Zygomycota (representing the basal or lower fungi). The classification of the fungi has changed tremendously over the past three decades (Hawksworth et al. 1983, 1995; Kirk et al. 2001, 2008); for example, the introduction of the Glomeromycota as a new phylum for the arbuscular mycorrhizal fungi (Schüssler et al. 2001) and exclusion of the oomycetes, plasmodiophorids and slime moulds from the fungal into the chromistan and protist kingdoms (Adl et al. 2005). The use of genes have largely accelerated the research on fungal phylogenetics influencing and revolutionizing the systematics of the fungi. A six-locus phylogeny incorporating the whole kingdom Fungi confirmed the justification of the Dikarya (comprising the Ascomycota and Basidiomycota) and provided phylogenetic evidence for the paraphyletic evolution of the basal lineages of fungi (James et al. 2006a). The Blastocladiomycota and the Neocallimastigomycota were separated from the Chytridiomycota sensu stricto (James et al. 2006b; Hibbett et al. 2007, respectively), and the Zygomycota were disintegrated as a distinct phylum, being replaced by the subphyla

Appl Microbiol Biotechnol (2011) 90:41–57

Mucoromycotina, Kickxellomycotina, Entomophthoromycotina, and Zoopagomycotina (Hibbett et al. 2007). Recently, the Mortierellales were separated from the Mucoromycotina and ascribed to a distinct subphylum, the Mortierellomycotina (Hoffmann et al. 2011). A phylogenetic tree visualizing the evolution of the fungi is shown in Fig. 1. Recently, the core group of fungi was fused with the nucleariids to form the Holomycota based on phylogenomic (Liu et al. 2009) or the Nucletmycea as an equivalent name for this grouping based on five-locus phylogenies (Brown et al. 2009). From a traditional taxonomic point of view, the number of fungal species described so far ranges between 72,000 and 120,000 (Hawksworth and Rossman 1997; Hawksworth 2001), which is less than 10% of an estimated 1.5 million extant fungal species (Hawksworth 1991, 2001). If environmental samples based on metagenomics data are taken into account, the estimated number of fungal species may be as high as 3.5 million (O'Brien et al. 2005). To survey progress in species recognition, names of new species (i.e., excluding infraspecific taxa, new names and new combinations) of the Ascomycota, Basidiomycota, and Glomeromycota were surveyed by Hibbett et al. (2009). The overall rate of new species description has been fairly poor during the past 10 years with an average of only 223 species per year, mostly Ascomycota. The gap between the number of species described and the estimated total number of fungi impedes our understanding of fungal diversity and ecology because traditionally emphasis has been given to the macromycetes, forming spore-bearing structures which are visible to the naked eye and, particularly, the plant pathogenic fungi. From an ecological point of view, the discovery of fungal species in the anthropogenic zones were prioritized during the past. Nevertheless, more than 90% of the fungi remain unrecognized and unidentified, the majority of which may be found in undisturbed areas or in associations with plants, insects, animals, or as lichen-forming fungi, particularly in the tropics (Hawksworth 2001) or in the deep sea (Burgaud et al. 2009). In addition, hidden species (cryptic species), previously considered to belong to species already described, are discovered, for example in the basidiomycotan generus Armillaria (Pegler 2000), the ascomycotan genera Trichoderma (Gams and Bissett 1998), Letharia (Kroken and Taylor 2001), and Fusarium (e.g., Baayen et al. 2000; O'Donnell 2000; O'Donnell et al. 2000) and in the zygomycetous genus Absidia (Hoffmann et al. 2009; Alastruey-Izquierdo et al. 2010; Gherbawy and Voigt 2010).

Identification and delimitation of fungi: a survey Classical approaches to identify fungi range from comparisons with the fossil record (Hawksworth et al. 1995), the use of


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Fig. 1 The evolution of the fungi and allied fungi-like microorganisms conducted by a Bayesian inference analysis based on unambiguously aligned amino acid sequences of a total of 1,262 aligned amino acid characters comprising actin, beta tubulin, and translation elongation factor 1alpha (323, 438, 500, respectively) from 80 taxa (modified after Gherbawy and Voigt 2010) using 5 million generations in MrBayes v3.1.2 (Huelsenbeck and Ronquist 2001). The tree is rooted to the outgroup taxon Thermotoga maritima representing the bacterial domain and using the FTsZ homolog of the eukaryotic

tubulin (Erickson 1997; Löwe and Amos 1998; McKean et al. 2001), the MreB (TM1544) homolog of the eukaryotic actin (van den Ent et al. 2001), and the prokaryotic translation elongation factor Tu which is functionally homologue to the eukaryotic EF-1alpha (Gonen et al. 1994). Bootstrap proportions are given above the branches for supporting clade stability. Classification was performed in accordance to Adl et al. (2005), Cavalier-Smith (2004), Cavalier-Smith and Chao (2003), Steenkamp and Baldauf (2004), and Kirk et al. (2008)

growth physiological (Pitt 1979) and biochemical markers (Bridge 1985; Paterson and Bridge 1994), the composition of the cell wall (Bartnicki-Garcia 1970, 1987) and isoenzyme patterns (Kohn 1992; Maxson and Maxson 1990), the existence of pigments (Besl and Bresinsky 1997), secondary metabolite profiles (Frisvad and Filtenborg 1990) to observations on the ultrastructure (Kimbrough 1994; James et al. 2006b). All these parameters are used in comparison with macromorphological and micromorphological characters to obtain criteria, which are taxon-specific (Fig. 2; Voigt 1995). Closely related fungi can differ in their pathological effects, toxicogenic products, beneficial attributes, and ecological niches (Roe et al. 2010). The nucleic acidbased generation of polymorphisms like PCR-RFLP, RAPD, microsatellite PCR and AFLP, or their combination with ELISA strategies became the most prominent and popular method during the 1990s and at the edge of the new millennium. Since the pioneering work of Hebert et al.

(2003) and the establishment of the master international Consortium for the Barcoding of Life (CBOL; www. barcodeoflife.org) in 2004, DNA barcoding using nucleotide sequences of specific marker genes redeemed DNAfingerprint identification and revolutionized the molecular identification of the fungi (Gherbawy and Voigt 2010).

Taxonomy and the multidimensional concept of species During the course of species identification, an overwhelming number of about 15 species concepts were established (for reviews, see: Ruse 1969; Sbordoni 1993; Mayden 1997). Five major concepts of species are currently discussed in fungal taxonomy research (Table 1): the biological (BSC), the morphological (MSC), the evolutionary (ESC), the phylogenetic (PSC), and the genealogical concordance species concept. Due to the lack of recognition data, the ESC (Simpson 1951; Wiley 1978) is not helpful for

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Fig. 2 Schematic illustration of methods used for species and below-species identification of fungi (modified after Voigt (1995))

identifying species, while the MSC, BSC, and PSC do specify criteria for recognizing species (as reviewed by Taylor et al. 2000). Therefore, the term “species concept” is reserved for the theoretical taxon, and the term “species recognition” is used for the operational taxon of the species concepts commonly discussed (Taylor et al. 2000). The species definitions of the major species recognition concepts are outlined in Table 1. More species concepts are reviewed by Ruse (1969). The dominant and most traditional fungal species concept is that of a morphological species based on species-specific morphological characters which often were extended to physiological characters, e.g., growth at different temperatures or water activities (Pitt 1979), the presence of secondary metabolites (Frisvad and Filtenborg 1990) or pigments (Besl and Bresinsky 1997), which interferes with the phenetic species concept (Ruse 1969). The biological species is a set of actually or potentially interbreeding populations which reproduce naturally and produce fertile offspring based on shared reproductive systems, including mating behavior as proposed by Paterson (1978) for the recognition concept. In fungi, mating behavior is, especially in the basal groups, often tedious to apply. For example, species in the mucoralean genus Absidia, the mating partners form abortive zygospores as a result of interbreeding between individuals designated to different species, genera, and families (Hoffmann and Voigt 2009; Alastruey-Izquierdo et al. 2010). A phylogenetic species is defined by a derived character which arose monophyletically and is shared by the members of the species, a synapomorphy (Rosen 1978). The drawback of the PSC is in the subjective nature as to where to place the limit of the species among the individuals grouping. This subjectivity of determining limits of a species is avoided by relying on the concordance of more than one gene genealogy as proposed by Avise and Ball (1990). Where

the different gene trees are concordant, they have the same tree topology due to fixation of formerly polymorphic loci following genetic isolation, as labelled by Taylor et al. (2000), the Genealogical Concordance Phylogenetic Species Recognition.

Phylogeny meets taxonomy: databases are keys to a modern fungal biology Phylogeny has largely influenced concepts of species and their recognition. Modern DNA sequenced-based species identification has been widely utilized in connection with large-scale phylogenetic studies such as the Assembling the Tree of Life (AToL: www.phylo.org/atol) and the Tree of Life web project (ToLweb: www.tolweb.org/tree) which are implemented by CIPRES (Cyberinfrastructure for Phylogenetic Research: www.phylo.org). Concurrently, DNA barcoding became an emerging gold-standard for species recognition and delimitation, with the pioneering work of Hebert et al. (2003) leading to the establishment of the master international Consortium for the Barcoding of Life (CBOL: www.barcodeoflife.org) in 2004 followed by a call of the European Commission for a network in “Taxonomy for Biodiversity and Ecosystem Research” issued the same year inspiring the collective agreement for the European Distributed Institute of Taxonomy (EDIT: www.e-taxonomy.eu) which started on the first of March 2006. Projects such as the Barcode of Life Initiative (BOL: www.barcodeoflife.org) and the International Barcode of Life (iBOL: www.ibol.org) followed by the European Consortium for the Barcode of Life (ECBoL: www.ecbol.org) and many other more specialized barcoding campaigns and centers serve as organizational platforms for scientists and working groups

Taylor et al. 2000 based on the Genealogical Concordance Species Concept (Avise and Ball 1990)

Phylogenetic species recognition (PSR) Genealogical Concordance Phylogenetic Species Recognition (GCPSR) 4

5

Morphological species recognition Evolutionary species concept (ESC) 2 3

Cracraft 1983

Mayr 1940

a group “…of actually or potentially interbreeding natural populations which are reproductively isolated from other such groups” A population or group of population which differs morphologically from other populations “a single lineage of ancestor-descendent populations which maintains its identity from other such lineages and which has its own evolutionary tendencies and historical fate” “…the smallest diagnosable cluster of individual organisms within which there is a pattern of ancestry and descent” a concordant branch appearing in different single gene trees Biological species recognition 1

Reviewed by Ruse (1969) Wiley 1978

Reference A species is… No. Species concept

Table 1 Species concepts at a glance


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interested in taxonomic work. Improvement of the taxonomic facilities is mediated by consortia like the Consortium of European Taxonomic Facilities (CETAF: www. cetaf.org) or the Global Biodiversity Information Facility (GBIF: www.gbif.org) due to conservation and digitization of taxonomic collections and associated information and development of information services for scientific, commercial, and public use and improved access to those. From the molecular data management's point of view, the data are hosted and managed by Barcode of Life Data Systems (BOLD: www.boldsystems.org) and the International Nucleotide Sequence Database Collaboration (INSDC: www.insdc.org), which consists of a joint effort to collect and disseminate databases containing DNA and RNA sequences involving three computerized databases: GenBank (www.ncbi.nlm.nih.gov/genbank), European Molecular Biology Laboratory (EMBL: www.embl.de), and the DNA Data Bank of Japan (DDBJ: www.ddbj.nig. ac.jp). The barrier between phylogenetic reconstruction species' recognition has been relaxed which largely influenced the view on fungal taxonomy. In mycology, projects like the Assembling the Fungal Tree of Life (AFToL: www.aftol.org) and its preceding parental project Deep Hypha (Blackwell et al. 2006) largely inspired the All Fungi DNA barcoding project (Rossmann 2007) in the search for genes which are suitable for phylogenetic reconstruction and identification. The suitability of molecular barcode markers, of which more than 16 are discussed to date, largely varies among the different fungal groups (as reviewed by Begerow et al. 2010; Roe et al. 2010; Schmitt et al. 2009; Gherbawy and Voigt 2010). The Fungal Barcoding project (www.fungalbarcoding.org) has adopted a set of six loci by the members of the international on-line community for DNA barcoding professionals across the globe (connect.BarcodeofLife. net), who provide comparative data sets for a study proposing an official barcode as accepted by CBOL, to be submitted by May/June 2011. The nuclear ribosomal DNA cluster encoding the small (18S) and large (28S) subunit ribosomal RNA which is interrupted by the internal transcribed (ITS) spacer 1 and 2 including the 5.8S rDNA may concurrently serve for identification above and below species-level, respectively. Thus, 18S and 28S is useful for the reconstruction of long-term speciation events above species ranks, the rapidly evolving ITS1 and 2 region facilitates the recognition of species and subspecies (White et al. 1990). For most fungi, the ITS has become the default marker for species identification. However, the loci representing single elements in the nuclear rDNA cluster are highly repetitive and often evolve in a nonconcerted manner resulting in aberrant copies of the same locus. ITS sequence heterogeneities were reported in a number of fungal species (O'Donnell and Cigelnik 1997;

46

Okabe et al. 2001; Wang and Yao 2005; Simon and Weiss 2008; Woo et al. 2010). When present, such sequence heterogeneity will pose difficulties for fungal identification as a diverging population of amplicons will be amplified in initial PCR assays followed by the occurrence of double or multiple nucleotide peaks in the sequence traces. Proteincoding genes have the major advantage that they are single or have small numbers of copies easing the identification of orthologous marker genes which can be used for phylogenetic and taxonomic purposes (Voigt et al. 1999; Fig. 1). Generally, the exonic sequences of slowly evolving genes encoding highly conserved proteins like translation elongation factor-1alpha, actin, and tubulin are suitable for above genus level identification, while their introns are useful and can be used species and below species-level identification (Hoffmann et al. 2009; Alastruey-Izquierdo et al. 2010). However, their moderately repetitive nature, which is especially pronounced in the basal lineages of the fungi, leads to ambiguous designations to species used as references and hampers the unequivocal identification of species (Gherbawy and Voigt 2010). Thus, the search for novel marker genes which are single-copy genes and provide strong signals for species designation independently from their phylogenetic affiliation is still ongoing. The single-copy genes Mcm7 (MS456) and Tsr1 (MS277) are good candidate genes for species identification across a wide range of derived fungi (Schmitt et al. 2009), but their suitability for species recognition within the basal fungi, the chytridiomycetes, and zygomycetes sensu lato, still needs to be tested. Apart from the advantages which phylogenetic approaches possess over those based on morphology and reproductive behavior (see above), patterns of species limits are deviating for each locus if applied in single analyses demonstrating the limitations of relying on a single locus for identifying and inferring species limits among closely related fungal species (Roe et al. 2010). Therefore, the importance of the application of multi-locus species delimitation and identification has led to the wide acceptance of the Genealogical Concordance Phylogenetic Species Recognition (GCPSR) in fungal taxonomy (Taylor et al. 2000).


health serve as important models for biomedical research and provide a wide range of evolutionary comparisons at key branch points in the billion years of fungal evolution. Since the release of the first completed fungal genome, which is the genome of the ascomycetous yeast Saccharomyces cerevisiae, since October 1996, a flood of genome projects were initiated (Fig. 3), encompassing a total of 272 fungal genomes (as of 15th of November, 2010): 17 complete, 127 assembly, 128 in progress. Out of a total of 17 completed genome projects, 14 are ascomycetes, 1 basidiomycete, and 2 are microsporidia (Table 2). Among the 14 ascomycete genomes, two correspond to the S. cerevisiae strain S288c. This fungus was the first whose genome was fully sequenced by the Wellcome Trust Sanger Institute and re-sequenced by the Saccharomyces Genome Database in 2010. It is not surprising that due to biotechnological relevance and minute genome sizes, the majority of the genome-sequenced ascomycetes are ascomycetous yeasts (Saccharomycotina) where sizes of the haploid genomes range between 8.74 Mb (Ashbya gossypii) and 20.5 Mb (Yarrowia lipolytica). Only two filamentous ascomycetes (Pezizomycotina), Aspergillus nidulans and Magnaporthe grisea (30 and 40 Mb, respectively), were completed in terms of a fully sequenced genome (Table 2). Genome projects targeting ascomycetes dominate the past and the present (Fig. 3). Out of a total of 127 fungal genomes in assembly status, the overwhelming majority (103) are ascomycetes with just 16 basidiomycetes, four basal fungi (Rhizopus oryzae, Allomyces macrogynus, Batrachochytrium dendrobatidis, Spizellomyces punctatus) and four microsporidia (Enterocytozoon bieneusi, Nematocida parisii ERTm1, Nosema ceranae, and Octosporea bayeri). Out of a total of 128 fungal genomes which are in progress, 76 are ascomycetes, 44 basidiomycetes, five from the basal lineages of fungi (Mortierella alpina, Mortierella verticillata, Mucor circinelloides f. lusitanicus, Batrachochy-

Current state of phylogenetic strategies in a genomic era: the rise of phylogenomics At the junction between the past and the new millennium, the fungal and genomics communities have been working together to define a Fungal Genome Initiative which is an effort to jumpstart research on the fungal kingdom by prioritizing a set of fungi for genome sequencing (Birren et al. 2002). Fungi that present either biotechnological importance or serious threats to human

Fig. 3 Diagram illustrating the ratio between basal and derived lineages of fungi and their fully sequenced genomes as of 15th of November, 2010

Aspergillus nidulans Candida dubliniensis Candida glabrata

Debaryomyces hansenii

Kluyveromyces lactis

Lachancea thermotolerans

Magnaporthe oryzae Pichia pastoris

Saccharomyces cerevisiae Saccharomyces cerevisiae Schizosaccharomyces pombe Yarrowia lipolytica

Zygosaccharomyces rouxii

Basidiomycota Cryptococcus neoformans var. neoformans Microsporidia Encephalitozoon cuniculi E. intestinalis

2 3 4

5

6

7

8 9

10 11 12 13

14

II 15

III 16 17

Ascomycota Ashbya gossypii

I 1

No. Species

214684

559307

559292 5592922 284812 284591

242507 644223

559295

284590

284592

227321 42374 284593

284811

NCBI TaxID

GB-M1 284813 ATCC50506 876142

JEC21

559307

S288c S228c 972 hCLIB122

70-15 GS115

NRRL Y1140 CBS 6340

CBS767

ATCC 10895 FGSC A4 CD36 CBS138

Strain

2.5 2.22

19.05

9.76

12.07 12.08 14 20.5

40 9.4

10.4

10.69

12.22

30 16 12.28

8.74

Genome size [Mb]

11 11

14

7

16 16 3 6

7 4

8

6

7

8 13

7

No. of chromosomes

11/24/2001 08/16/2010

01/07/2005

06/05/2009

10/25/1996 02/03/2010 02/21/2002 07/02/2004

01/30/2006 05/25/2009

06/05/2009

07/02/2004

07/02/2004

09/24/2009 02/16/2009 07/02/2004

03/06/2004

Genoscope, Evry cedex, France (see Katinka et al. 2001) University of British Columbia, Canada (see Corradi et al. 2010)

TIGR Cryptococcus neoformans Database

Eurofungbase (Eurofung) Wellcome Trust Sanger Institute Génolevures : ‘Genomic Exploration of the Hemiascomycete Yeasts’ Génolevures : ‘Genomic Exploration of the Hemiascomycete Yeasts’ Génolevures : ‘Genomic Exploration of the Hemiascomycete Yeasts’ Génolevures : ‘Genomic Exploration of the Hemiascomycete Yeasts’ North Carolina State University Molecular Miomedical Research, VIB, University of Gent, Belgium Wellcome Trust Sanger Institute Saccharomyces Genome Database S. pombe European Sequencing Consortium (EUPOM) Génolevures : ‘Genomic Exploration of the Hemiascomycete Yeasts’ Génolevures : ‘Genomic Exploration of the Hemiascomycete Yeasts’

Zoological Institute, University Basel, Switzerland

Release date [month/day/ Center/Consortium year]

Table 2 Completed fungal genome sequencing projects (as of 15th of November 2010; source: National Center of Biotechnology Information at www.ncbi.nlm.nih.gov/genomes/leuks.cgi)

Appl Microbiol Biotechnol (2011) 90:41–57 47

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tridium dendrobatidis, Necallimastix sp.), and three microsporidia (Antonospora locustae, Encephalitozoon hellem, and Nosema bombycis). The preference for the ascomycetes in recent genome projects is still persistent and the reality emphasizing the gap between derived and basal fungal genome projects should be addressed because it falls woefully short of expectation. Out of a total of 43 additional genome projects, which were posted between the 15th of November 2010 and the 19th of January 2011 at GenBank, 33, 7, 1, and 1, new genome projects were announced for the ascomycetes, the basidiomycetes, the zygomycetes (Phycomyces blakesleeanus) and the microsporidia (Nematocida parissi strain ERTm3), respectively.

The definition of fungi revisited: are microsporidia fungi? Since their first recognition as pathogens in silkworms by Nägeli (1857), microsporidia have been identified as infectious agents of many contagious diseases in vertebrates (Canning et al. 1986) and invertebrates (Morris and Adams 2002; Wittner and Weiss 1999). Of 143 genera and over 1,200 species described (Wittner and Weiss 1999), insects are frequently found to be their host (as reviewed by Chen et al. 2009). The taxonomic position of the microsporidia is uncertain and has been actively discussed for more than two decades (Patterson and Zölffel 1991; Patterson and Larsen 1991; Cavalier-Smith and Chao 2003; Chen et al. 2009). Microsporidia are heterotrophic, non-flagellate, unicellular protists which are obligate intracellular parasites lacking, like other metazoan parasites, for example diplomonads and parabasalids, mitochondria and peroxisomes (Hashimoto et al. 1998). Mitochondria are hypothesized to be secondarily lost due to the mitochondrial origin of a 70kDa heat shock protein gene present in their nuclear genomes (Hirt et al. 1997; Peyretaillade et al. 1998b) and subsequently by the presence of over a dozen mitochondrion-derived protein-coding genes in the genome of the microsporidian species Encephalitozoon cuniculi (Katinka et al. 2001). Contrarily to other amitochondriate eukaryotes, which generally involve the substitution of pyruvate dehydrogenase by pyruvate-ferredoxin oxidoreductase, they possess several units of a pyruvate dehydrogenase complex which may have been derived from a mitochondrial source (Fast and Keeling 2001). Because of their strictly host-dependent life cycle, the genome of the microsporidia is highly reduced as shown by an unusual chromosomal dispersion of ribosomal genes and a LSU rRNA reduced to the universal core (Peyretaillade et al. 1998a). Genome sizes range between 2.3 Mb in Encephalitozoon intestinalis (Corradi et al. 2010), which is associated with gastrointestinal diseases in humans, and


24 Mb in O. bayeri, a pathogen of Daphnia magna (Corradi et al. 2009). Most (9 out of 12) of the microsporidia genome sized so far possess reduced genomes below 11 Mb (see Méténier and Vivarès 2001 for review). For comparison, the size of the S. cerevisiae genome is about 12 Mb, which is itself considered small for a eukaryote. So what makes the microsporidian genome small? There are two main ways in which a genome can be reduced: either it can reduce the number of genes it encodes via concurrent conservation of gene order, or it can compress them into a smaller space by loss or extreme reduction of intra- and intergenic non-coding DNA and repetitive elements such as microsatellites, active or relict transposable elements (as reviewed by Keeling and Slamovits 2004). Also, the coding potential of the genomes of the microsporidia genome sequenced so far, E. cuniculi (Katinka et al. 2001) and E. intestinalis (Corradi et al. 2010), is very low, with less than 2,000 protein-encoding genes showing a substantial reduction by gene loss which is not random and represents loss of complete metabolic or regulatory pathways. As the parasite becomes more hostdependent, some metabolic functions are provided by the host cell, so genes involved in such functions may become redundant and lost after inactivation by mutation. Thus, the adaptation to a parasitic lifestyle typically precipitates a number of profound changes to various levels of biological organization. These changes can lead to a seemingly paradoxical mixture of characteristics; on one hand, parasites may evolve extremely complex and sophisticated mechanisms to invade their host and evade its defenses, while on the other hand, they may also appear more “simple” by dispensing with characteristics they no longer need as they increasingly depend on host metabolism for nutrients and energy (see for review: Keeling and Fast 2002; Keeling and Slamovits 2004). The complexity of microsporidian cells rests in their unique and highly adapted infection machinery. Microsporidia form distinctive spores that contain a coiled filament which everts to inject the infectious organisms into the host cell (Larsson 1999). This introverted polar filament represents a synapomorphy for the microsporidia (Patterson 1999). Because of its analogy to a cnidocyte, the stinging cell that fires a toxin projectile, the diagnostic feature of a cnidarian, microsporidia were considered as cnidarians during the past (Siddall et al. 1995). Functionally the stinging sell of the cnidarians is analogous to the coiled filament of the microsporidia. The mechanism is based on the increase of osmotic pressure during germination of the spore which accumulates intrasporal sugars until its rigid wall ruptures at its thinnest point at the apex (Undeen and Van der Meer 1999). The posterior vacuole swells, forcing the polar filament to rapidly eject from the rupture, simultaneously everting itself to form a tube. The endoplasmatic contents of the spore are forced through the tube at an


extremely rapid speed, and if it has penetrated another cell during its rapid ejection, the parasite is injected into the cytoplasm of this potential host (Keohane and Weiss 1999). The coiled filament of the microsporidia is also functionally analogous to the gun cell of the oomycete Haptoglossa which also relies on basal vacuole expansion, followed by a rapid inversion of the projectile inside out. The oomycetes were long considered to be fungi due to similarities in life style and morphology, characters which do not indicate fungal origin. Oomycetes are non-photosynthetic protists belonging to the stramenopiles (Chromista; for review, see Cavalier-Smith 2004). The basal oomycete Haptoglossa mirabilis is characterized by the formation of a gun cell, which is activated by mechanical breaking of a line of weakness dislodging the plug in the bore (Barron 1987). The tubular system inside the cell everts, like the finger of a glove, from the fixed end at the muzzle. Upon triggering, the basal vacuole takes in water and expands rapidly pushing the protoplasmic contents of the cell up the everted tube. The apical portion of the everted tube, containing reserve wall material, expands to accomodate the protoplasmic contents of the cell and everts the projectile at rapid speed through the host cuticle. The swollen apical portion containing the protoplasmic contents of the gun cell breaks away from the everted tube to form the infective sporidium inside the host animal. Haptoglossa targets nematodes and rotifers. Its intrageneric diversity is high due to existence of two distinct types of gun cells (see Glockling and Beakes 2002 for comparison of different Haptoglossa species at various life cycle stages). However, projectiles which are functionally extrusomes are quite common among protists. They are associated with the membrane and contain material that can be ejected from the cell in order to form either a capsule/cyst or a pointed projectile that serves for protection or predation like the trichocysts of the ciliate Paramecium (Cavalier-Smith 2004). The projectile-filaments probably appear to be proximately convergent (which is defined by the phenotypic convergence of subcellular to multicellular analogues; Leander 2008) in all three kingdoms. However, the coiled filament of the microsporidia is usually surrounded by massive arrays of ribosomes, which are aberrant from the eukaryotic type of ribosomes. These ribosomes are 70S organelles and not of the typical (80S) eukaryotic type (Fast et al. 1999; Delbac et al. 2001; Bacchi et al. 2002). In addition, the 5.8S and 28S rRNAs are fused, as they are in bacteria (Fast et al. 1999). In fact, there is no spacer sequence between the two in the rDNA, and the two are transcribed as a single molecule, again, as in bacteria (Peyretaillade et al. 1998a). It was thought for some time that these ribosomes were a link with prokaryotic ribosomes, which does not appear to be the case (Peyretaillade et al. 1998a; Delbac et al. 2001). The rRNA sequences are

49

far more closely aligned with eukaryotic rRNAs in both structure and sequence (Peyretaillade et al. 1998a). The distribution of rDNA sequences in the genome shows no pattern. In some microsporidia, all rDNA sequences are located on a single chromosome, while in others, the rDNA sequences appear to be randomly distributed (Peyretaillade et al. 1998a). However, like prokaryotic ribosomes, microsporidian ribosomes contain a large component of small polyamines. Presumably, as in prokaryotes, these molecules bind to the rRNA. Taken these facts all together and changing the point of view towards the radiation of the eukaryotes from the prokaryotes, the projectile-mediated mechanism, which is common in microsporidia, oomycetes and cnidarians, may represent an ancient property of protists derived from a common ancestor of the eukaryotic crown group, the fungi, plants and animals, respectively. As the gun cell of Haptoglossa is a plesiomorphic character of basal oomycetes, the coiled filament of the microsporidia may be a plesiomorphic character of ancient eukaryotes at the fungus-animal transition zone. As remnant of the predatory and phagotrophic lifestyle, it may have been lost during the course of evolution towards osmotrophy in derived lineages. Nevertheless, the phylogenetic position of the microsporidia is still the subject of a lively debate. They are argued by some to be the most primitive eukaryotes as revealed by rDNA (Vossbrinck et al. 1987), single protein phylogenies (Kamaishi et al. 1996) and their combination (Tanabe et al. 2002), and by others to be allied to the fungi based on multi-protein and multi-locus phylogenies (Keeling et al. 2000; Gill and Fast 2006). Moreover, alpha- and betatubulin gene phylogenies suggested a zygomycete origin of the microsporidia (Keeling 2003), whereas an eight-locus genealogies place the microsporidia as a sister group to a combined ascomycete and basidiomycete clade (Gill and Fast 2006). The hypothesis that microsporidia may be true fungi that descended from a zygomycete ancestor, suggesting that microsporidia may possess an extant sexual cycle was supported by the presence of a variety of syntenic gene clusters governing sexual reproduction and many other traits (Lee et al. 2008, for review of the evolution of sex in fungi, see Lee et al. 2010). Contrarily, sexual reproduction and horizontal gene transfer generates genetic diversity in microsporidia (Lee et al. 2009), which may cause a falsified picture of genome evolution in the microsporidia due to an accelerated rate of genomic rearrangements, such as translocations, insertions, and deletions. Also, shared ancestry or convergent evolution may cause a plesiomorphic ancestry of the sex gene cluster implying that its gene order (synteny) and the total number of syntenic gene clusters in general are not significantly more similar between microsporidia and zygomycetes than between microsporidia and any other fungal taxon or even humans (Koestler and Ebersberger 2011). That would

50

mean that the microsporidia evolved apart from the fungi towards the ancestral moiety of protists at the common root between animals and fungi. Evidence for that scenario is given by a phylogenomic analysis of Ebersberger et al. (2009a), placing the microsporidia basal to the fungi and the presence of two amino acid deletion conserved in all fungal EF-1alpha proteins, which is absent in microsporidia (Tanabe et al. 2002). The contradicting and conflicting phylogenetic interpretations, when different marker genes harboring diverging phylogenetic signals were used, are summarized in Fig. 4, highlighting the need for a reappraisal of the taxonomic affiliation and a careful reconsideration of the phylogenetic placement of the microsporidia. Microsporidia form spores which sometimes bear an apparent resemblance to fungi. At the morpho-chemical level, the endosporous chitin is in the form of alpha-chitin, the same cross-linked type thought to be a synapomophy of arthropods (Bohne et al. 2000). Given the close ecological relationship between arthropods and microsporidia, this seems unlikely to be a coincidence. The close link between chitin synthesis and development of a symbiotic protist population was shown by Lewis and Forschler (2010) who tested five different chitin synthase inhibitors resulting in a decrease of parabasalian protists in the intestine of subterranean termites. Like all fungi except the chytrids, microsporidia lack flagella or any other “9+2” structure (Fast and Keeling 2001). To sum it up, arguments against the fungal origin of microsporidia preponderate (Table 3). It is noteworthy that the density of both of the completely sequenced Encephalitozoon genomes (E. cuniculi and E. intestinalis) is similar to that of the nucleomorph genome of the marine cryptomonad alga Guillardia theta (Douglas et al. 2001) and may represent a maximum gene density for nuclear genomes (Keeling and Slamovits 2004). Fig. 4 Summary of changes in the taxonomic position of microsporidia during the past three decades


Trends, prospects, perspectives for the future Due to improvements in next-generation sequencing equipment, enabling large amounts of data to be delivered at unprecedented speed, multi-locus phylogenies will be displaced by the application of phylogenomic approaches on a genome-wide scale in order to detect speciation events at any arbitrary taxonomic level; concurrent with the development of novel biological applications (Schuster 2008). Yet, published multi-locus phylogenies are often without compelling statistical clade stability supports, making the use of genome-level data necessary in order to resolve the fungal phylogeny with confidence as shown by Liu et al. (2009) on 118 nuclear and 13 mitochondrial loci. Genome-level datasets will question the current scheme of fungal classification as demonstrated for the Glomeromycota as well as the Blastocladiomycota challenging their evolution as distinct phyla (Liu et al. 2009). Traditional phylogenetics will be displaced by phylogenomics which takes the genomic architecture and the evolution of whole genomes into account. The widespread use of genomic and expressed sequence tag (EST) data in molecular systematics and molecular taxonomic studies is often hindered by the lack of efficient and reliable approaches for automated ortholog predictions. Existing methods either depend on a known species tree or cannot cope with redundancy in EST data. A novel approach, HaMStR, combines a profile Hidden Markov Model search with subsequent BLAST search to extend existing ortholog cluster with sequences from additional taxa in order to mine EST and genomic data for the presence of orthologs to a curated set of genes (Ebersberger et al. 2009b). This approach has been applied to further resolve the arthropod and the fungal tree of life (Meusemann et al. 2010; Ebersberger et al. 2009a). The application of a data set comprising 128 genes and 146 taxa

Translation elongation factor-1alpha Phylogenetic reconstruction

Peroxisomes

Mitochondria

rDNA cluster

Ribosomes

Genome reduction and compaction

alpha- and beta tubulin

Present in the endospore layer of the spore Like all terrestrial fungi lacking flagella or any other “9+2” structure

Alpha-chitin Motility organs

Infection

Pro (unique for fungi)

Type of comparative criterium

Table 3 The fungal origin of the microsporidia: pros and cons

Bohne et al. (2000) Fast and Keeling (2001)

Reference

RNA polymerase II

rDNA

2 amino acid deletion unique for the microsporidia but absent in the core fungi

Keeling and Slamovits (2004) Hirt et al. (1999)

Tanabe et al. (2002)

Patterson (1999) Barron (1987) Glockling and Beakes (2002) Extraordinarily high Keeling and Slamovits (2004) Douglas et al. (2001) 70S organelles with polyamines, aberrant from the typical 80 S eukaryotic type Bacchi et al. (2002) Fast et al. (1999) Delbac et al. (2001) Peyretaillade et al. (1998a) Lack of ITS between 5.8S and 28S rDNA leading to fusion of the 5.8S and 28S rRNA as in Delbac et al. (2001) bacteria Peyretaillade et al. (1998a) Lacking Patterson and Zölffel (1991) Hirt et al. (1997) Peyretaillade et al. (1998b) Hashimoto et al. (1998) Lacking ditto

By coiled introverted filaments in spores, layered system of sacs (polaroplast) involved in discharge of infectious cells into hosts

Synapomorphy of arthropods

Con (unique for microsporidia but shared with non-fungal groups)

Appl Microbiol Biotechnol (2011) 90:41–57 51

52

in supermatrix and 161 taxa in supertree analyses resulted in a stable backbone for the fungal tree topology especially in the deep branches. The combined evidence from the trees support the deep-level stability of the fungal groups and advances our understanding towards a comprehensive natural system for the classification of the fungi, which indicates the classification of the basal fungi, especially their relationship with the microsporidia. Together with the prediction of gene clusters, phylogenomic analyses provide insights into the evolution of whole fungal genome, facilitating the phylogeny-based prediction of gene clusters responsible for natural products and serving as a guide to direct further sequencing initiatives. For example, the exploration and inclusion of more in-depth surveys of large genome-sized microsporidia other than O. bayeri with an estimated genome size of 24 MB (Corradi et al. 2009), may shed light on the organization and the evolutionary history of microsporidia. Out of a total of 18 genome projects from basal lineages of fungi and allied taxa, nine are of microsporidia and were given the highest priority to date (for review, see the genome projects listed at www.ncbi. nlm.nih.gov/genomes/leuks.cgi as of 15th November, 2010). More genome projects of the basal fungi are needed in order to resolve the root of the fungal clade and their phylogenetic relationship with allied groups.

The organization of taxonomic information in the -omics era The rules by which fungi are named are defined by the International Code of Botanical Nomenclature (ICBN; McNeill et al. 2006). Sequence-based taxon description will trigger the need for a modernization of these rules. That change from the traditional morphology-based system requires an engaged conversation within the taxonomic community, which is organized by different commissions. The International Commission on the Taxonomy of Fungi (ICTF: www.taxonomy.org) is a Commission of the Mycology Division of the International Union of Microbiological Societies (IUMS: www.iums.org) and also the International Mycological Association (IMA: www.ima-mycology.org). The mission of the ICTF is to promote scientific work on fungal taxonomy, especially those species of economic importance, to encourage the dispersal of its results and to develop good taxonomic practice, both by facilitating the development of high scientific standards among practising taxonomists, and by providing information to people who need to access or interpret taxonomic information. All major taxonomic studies are on-line database-driven or databasemediated. Global initiatives to promote the study of fungal biodiversity and to deliver content are MycoBank (www. MycoBank.org; Crous et al. 2004) and the Fungal Planet


(www.fungalplanet.org). An international project to index all scientific names for the fungi is Index Fungorum (www. indexfungorum.org), the global nomenclator for fungi and microorganisms traditionally treated as fungi such as the slime moulds, plasmodiophorids and the oomycetes. In contrast to the International Plant Names Index (IPNI: www.ipni.org), Index Fungorum also indicates the taxonomic status of a name by links to Species Fungorum (www. speciesfungorum.org), highlighting these links to the correct name in green; other names are in blue (and currently lack a taxonomic opinion) or are in red (where the name usages is misapplied to a different species). Future perspectives may lie in the development and implementation of a single global nomenclator (as part of the GNA; see below) for all life on earth, including prokaryotes and eukaryotes, making separate nomenclatural databases for the different organism groups superfluous. The encyclopedia of life (EoL: www.eol.org) provides an on-line accessible platform, LifeDesk, for scientific communities working on taxonomy and nomenclature of the overall organisms of Earth; a similar application is the scratchpad from the EDIT project (www.e-taxonomy.eu). Also, there is an effort underway to coordinate multiple interests in developing common solutions for creating a complete and integrated framework for all biodiversity data names which is the Global Names Architecture (GNA: gbif. org/informatics/name-services/global-names-architecture), the development of which is mediated by GBIF and EoL (Patterson et al. 2010).

The impact of fungal nomenclature on natural product research Fungi produce an impressive variety of low molecular weight bioactive compounds endowed with a multitude of biological activities. The increasing number of fungal genome sequences clearly demonstrates that their biosynthetic potential is far from being fully exploited. Thus, mining the full-genome sequences of fungi leads to a highspeed discovery of genes and gene clusters which are potentially involved in the production of secondary metabolites. The conditions under which a given gene cluster is naturally expressed are largely unknown. Therefore, the potential for natural product synthesis under non-laboratory conditions may be worthwhile. Just recently, evidence has been provided for a trans-domain interaction-mediated induction of otherwise silent biosynthesis genes (Schroeckh et al. 2009). It has been shown that an intimate physical contact with actinobacteria triggers the biosynthesis of archetypal polyketides in the ascomycete Emericella nidulans. The current strategies that have been successfully applied during the last years to activate these silent gene clusters in filamentous fungi, especially in the genus


Aspergillus, are summarized by Brakhage and Schroeckh (2010). Bacteria were often reported to be endosymbionts in fungi (Bianciotto et al. 2000; Bertaux et al. 2003; Partida-Martinez and Hertweck 2005; Hoffman and Arnold 2010), and it is speculated that this symbiosis enables the fungal host to adapt to changed environmental conditions and to occupy ecological niches other than that originally occupied. For example, the intracellular symbiotic bacterium from the genus Burkholderia contributes to the pathogenesis of the zygomycete Rhizopus microsporus in plants by the production of the mycotoxin rhizoxin, an antimitotic macrocyclic polyketide metabolite (Partida-Martinez and Hertweck 2005). During the course of evolution biosynthesis genes may either been horizontally transferred or vertically inherited from ancestral fungi. Most arguments favor the horizontal gene transfer hypothesis as shown for the betalactam biosynthesis genes and their regulatory network (Guthke et al. 2007; Sproete et al. 2008; Brakhage et al. 2009). All these molecular mechanisms will in future largely influence our understanding of taxonomy and its importance in natural product research. Species boundaries may need to be re-defined in the light of genome evolution and microbial interactions. The search for new fungal taxa and taxon groups with biotechnological potential and their implementation in naturally occurring organismic alliances will continue to play a significant role for the elucidation of cryptic or novel natural products. Acknowledgement This work was supported by grants of the Deutsche Forschungsgemeinschaft to KV (DFG VO772/7-1, DFG VO772/9-1). The authors express their gratitude to Gordon W. Beakes (University of Newcastle, Newcastle upon Tyne, UK) and Gareth W. Griffith (University of Wales, Aberystwyth, UK) for inspiring discussions about microsporidia and their fungal alliance during the IMC9 in Edinburgh. We also thank Kerstin Hoffmann for assistance in the Bayesian analysis shown in Fig. 1. The authors declare that they have no conflict of interest.

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