- Top of page
- Materials and Methods
- Supporting Information
The arbuscular mycorrhizal fungi (AMF; Glomeromycota; Schüßler et al., 2001) form symbioses with most land plants, in almost any terrestrial ecosystem (Smith & Read, 2008). Despite their considerable ecological importance, the biology and ecology of these fungi are still not well understood. This is partly because of their obligate symbiotic, asexual and hidden lifestyle in soil and roots.
Accurate identification is crucial, for example, in AMF community studies, which increasingly rely on phylotaxonomy solely based on molecular genetic data. Most commonly used is the nuclear small subunit (SSU) rRNA gene, hereafter referred to as SSU. Several SSU-targeting PCR primers (e.g. Simon et al., 1992; Helgason et al., 1998; Lee et al., 2008) that amplify fragments of c. 500–800 bp have been widely applied in ecological studies (Öpik et al., 2008; Zhang et al., 2010). However, in SSU data sets, one phylotype (often defined by a 97% sequence similarity level) may represent different species and, conversely, different phylotypes may indeed belong to just one species. This makes the resolution of closely related species impossible (Walker et al., 2007; Gamper et al., 2009) and we therefore eschew terms such as ‘virtual taxa’ (Öpik et al., 2010) and ‘species’ for phylotypes that are uncertain to represent taxa, and thus in fact are taxonomically undefined. A ‘taxon’ in mycology is clearly defined (see Article 1.1 in McNeill et al., 2006) and a more appropriate term is the ‘molecular operational taxonomic unit’ (MOTU). Standardized MOTUs are a goal for the classification of unknown fungal species from environmental samples (Hibbett et al., 2011), but care has to be taken that the units indeed are based on coherent taxonomic levels (Hawksworth et al., 2011). Standardization could also facilitate traditional biodiversity analyses (Magurran, 2004).
The more variable region covering nuclear internal transcribed spacer 1 (ITS1), the 5.8S rRNA gene and ITS2 rDNA (hereafter referred to as the ITS region) has also been used for detecting AMF (Redecker et al., 2000; Renker et al., 2003; Hempel et al., 2007), but is often inadequate for discriminating very closely related species (Stockinger et al., 2010). As a marker with intermediate sequence variability, the nuclear large subunit (LSU) rRNA gene (hereafter referred to as LSU) has proved useful for AMF detection (Gollotte et al., 2004; Pivato et al., 2007), although several PCR primers used do not amplify particular AMF lineages (Krüger et al., 2009). Other markers such as the genes for mitochondrial LSU rRNA (Börstler et al., 2010; Sýkorováet al., 2011), β-tubulin (Msiska & Morton, 2009), RNA polymerase II subunits 1 and 2 (James et al., 2006; Redecker & Raab, 2006), or H+-ATPase (Corradi et al., 2004; Sokolski et al., 2010) have been used, but either do not allow phylogenetic species identification or are as yet only known for very few species.
The nuclear rDNA region sequence data set that has been assembled over the past decade is becoming taxonomically sufficiently broad to permit molecular ecological field studies of AMF communities. However, comparison among studies is often difficult because of inconsistencies, for example regarding the coverage of the different loci. The ITS region is often used to determine fungal species (e.g. Tedersoo et al., 2008) and will be proposed as the official fungal DNA barcode (C. Schoch et al., unpublished). Unfortunately, for AMF most environmental ITS region phylotypes cannot be affiliated to species, or species-level identities are not determinable using only this short and highly variable region (Stockinger et al., 2009). Thus, neither the conserved SSU nor the highly variable ITS region alone reliably resolves closely related AMF, but this is possible using a c. 1.5-kb rDNA fragment (Stockinger et al., 2010), easily amplifiable with AMF-specific primers (Krüger et al., 2009). This SSU-ITS-LSU fragment covers c. 250 bp of the SSU, the complete ITS region and c. 800 bp of the LSU. Shorter fragments, such as the c. 400- or 800-bp reads provided by 454-sequencing, will provide information about species identities when analysed with reference to a ‘phylogenetic backbone’ based on longer sequences, such as the SSU-ITS-LSU fragment (Stockinger et al., 2010).
In this further effort to establish a reference database, we (re-)analysed nuclear rDNA regions that can be specifically and easily amplified by PCR for AMF (Krüger et al., 2009), resolve closely related species to allow DNA barcoding (Stockinger et al., 2009, 2010), and facilitate the application of deep sequencing technologies for in-field detection of AMF.
- Top of page
- Materials and Methods
- Supporting Information
By publishing > 240 further sequences produced over recent years and re-analyses of available sequences, we have established what we consider a phylogenetic basis for a natural systematics of Glomeromycota and a phylotaxonomic reference database for future environmental (deep) sequencing. For some analyses, we used consensus sequences, which are theoretical constructs and in some instances have to be interpreted with care (Lindner & Banik, 2011). However, in our analyses the use of strict SSU consensus sequences (degenerate base symbols represent all variations) anchors taxa by conserved regions and thus reduces the risks of phylogenetic attraction by shared characters at mutationally saturated sites. We analysed nuclear rDNA sequence data of c. 109 described species and c. 27 as yet unnamed AMF cultures (note that these are approximate numbers, because the species determination may not always be correct). More than 50% (120 species) of the currently c. 230 validly described AMF species are covered by sequences deposited in the public databases, but only 81 (c. 35%) are propagated in the culture collections INVAM (http://invam.caf.wvu.edu), BEG (Glomeromycota in vitro collection; http://www.kent.ac.uk/bio/beg), and GINCO (http://emma.agro.ucl.ac.be/ginco-bel), making re-analyses of or improvements to the sequence database difficult.
Need for a solid molecular genetic basis for the systematics of Glomeromycota
SSU analyses (Schüßler et al., 2001) and the six-gene phylogeny of James et al. (2006) indicated a likely sister grouping of the Glomeromycota to Dikarya. By including basal fungal lineages as well as members of Dikarya, we again found the same sister grouping (Fig. 1). However, analyses of the mitochondrial genome of R. irregularis isolate 494 (Lee & Young, 2009) and of nucleus-encoded amino acid sequences (Liu et al., 2009) questioned this relationship and indicated a possible common ancestry of AMF with Mortierellales, although tree topologies in the latter study varied depending on taxon sampling. Therefore, we must await more data from phylogenetically basal AMF to resolve immediate sister relationships to Glomeromycota, which are nonetheless clearly monophyletic and phylogenetically basal terrestrial fungi.
The present data compilation and analyses formed part of the basis for a major taxonomic reclassification in the Glomeromycota (Schüßler & Walker, 2010), and it is expected to be important as a reference for new species descriptions. For example, the sole use of morphology for the description of Ambispora brasiliensis (Goto et al., 2008) placed an Acaulospora species incorrectly at generic, familial and even ordinal level (Krüger et al., 2011). Similar instances of problematic species descriptions only based on morphology were discussed by Morton & Msiska (2010b), who reported an albino mutant of S. heterogama WV859, which would have been considered as a new morphospecies if found in the field. Another example was the description of Glomus irregulare (Błaszkowski et al., 2008), now R. irregularis, which was mainly based on a limited analysis of intraspecific morphological plasticity. Therefore, including an accurate phylogenetic characterization should improve the quality of formal species descriptions whenever possible. Obviously, this is particularly important for species not represented by publicly available isolates.
Phylogenetically basal lineages –Paraglomerales and Archaeosporales
Only relatively few data are available for evolutionarily ancient phylogenetic lineages of Glomeromycota. Presently there are only three recognized or described species in the Paraglomerales and 11 in the Archaeosporales, but this is probably only a small proportion of all existing species. Our study is the first to yield significant branch support for Paraglomerales as the most ancient lineage of the Glomeromycota (Fig. 1). It also supports Intraspora (Sieverding & Oehl, 2006) as congeneric with Archaeospora (Schüßler & Walker, 2010).
There has been considerable nomenclatural change among the Diversisporales recently. Oehl et al. (2008) split the genus Scutellospora into three new families containing six genera (Scutellospora in the Scutellosporaceae; Racocetra and Cetraspora in the Racocetraceae; Dentiscutata, Fuscutata, and Quatunica in the Dentiscutataceae). Except for Racocetra, Morton & Msiska (2010a) rejected all these new taxa. Nevertheless, it has long been indicated that Scutellospora is nonmonophyletic (e.g. Kramadibrata et al., 2000; da Silva et al., 2006). We support the notion of Morton & Msiska (2010a) that a robust taxon sampling and phylogenetic analysis should form the basis of taxonomic changes; the phylogeny presented herein may provide support for at least some of the genera proposed by Oehl et al. (2008), but certainly not for erecting new families in this clade.
The finding of two different D. aurantia clades exemplifies problems in interpretation of data derived from trap cultures seemingly producing spores of one species (often called single species cultures). It seems possible, but cannot be proved, that the trap culture material with the spores of D. aurantia contained more than one species. For the monospecific genus Otospora (Palenzuela et al., 2008), the O. bareae sequences cluster within Diversispora. This could support the view that O. bareae is a morphologically exceptional member of the Diversisporaceae, but it is perhaps more likely to be the result of a contamination. The sequence of the recently described E. nevadensis (Palenzuela et al., 2010) also clusters unexpectedly, in terms of its morphology, among those of Diversispora. A detailed analysis of Diversisporaceae with focus on D. epigaea, often named ‘Glomus versiforme BEG47’, and including biogeographical aspects is given in Schüßler et al. (2011).
Kuklospora sensu Oehl & Sieverd. (Sieverding & Oehl, 2006) was described based solely on spore morphology. The recent transfer of all Kuklospora species to Acaulospora (Kaonongbua et al., 2010) is congruent with our analyses. In our opinion the species Acaulospora laevis and Acaulospora entreriana are morphologically indistinguishable. They could not be separated in analyses when ITS1 and ITS2 were excluded, but additional data are needed to investigate a possible conspecificity. This also holds true for cultures annotated as Acaulospora mellea, Acaulospora delicata and Acaulospora dilatata, which are not well covered by available long sequences.
A decade ago, Schwarzott et al. (2001) were already proposing that Glomus should be split into several families. These subsequently were operationally named as phyloclades Glomus Group A (GlGrA), GlGrB and GlGrC, until it became clear where the generic type of Glomus, G. macrocarpum, belongs phylogenetically (Schüßler & Walker, 2010). Now, the family Glomeraceae represents the former GlGrA, separated into six genera: Glomus (GlGrAc), Funneliformis and Septoglomus (both GlGrAa), Rhizophagus and Sclerocystis (both GlGrAb). Glomus iranicum and G. indicum sequences form a basal clade in this family, and G. bistratum and G. achrum cluster in a basal polytomy in the Glomeraceae. Robust phylogenetic placements of the last four species and of the proposed monospecific genus Simiglomus (Oehl et al., 2011a) will require additional data. The family Claroideoglomeraceae corresponds to the former GlGrB, and the Diversisporaceae to GlGrC.
For Claroideoglomus, Funneliformis and Rhizophagus, detailed analyses have already been conducted by Stockinger et al. (2010), under the previous generic name Glomus. The uncovered inconsistencies discussed in that study are also recognizable from the phylogenetic trees of the present study, but are not further discussed here. Rhizophagus irregularis was defined (Błaszkowski et al., 2008), as G. irregulare, mainly based on perceived morphological differences from G. intraradices in a former sense, which included DAOM197198. The analysis shown in Fig. 5(b) confirms that the organisms interpreted as different, based on morphology, in fact belong to the same species. Glomus irregulare (now R. irregularis) is conspecific with DAOM197198 (and other cultures of ‘G. intraradices’ in the former sense) (Stockinger et al., 2009, 2010; Sokolski et al., 2010). The molecular data suggest that R. clarus and R. manihotis are conspecific, but this possible synonymy requires further morphological work before taxonomic assessment.
Putative errors in public sequence databases
As discussed repeatedly (e.g. Schüßler et al., 2003; Bidartondo et al., 2008), annotation of sequence entries in public databases is often inadequate or incorrect. There are different types of error; some errors are based on wrong identification or undiscovered species synonymy, some on pot culture or laboratory contaminants, and others perhaps on accidental misannotation. For example, a batch of LSU sequences (FJ461790–FJ461888◂) caused problems in our initial analyses because of numerous species falling into unexpected groups, until we realized that many of the contained sequences seem to be either misannotated or derived from contaminants. Sequences from ‘G. trimurales’, originally annotated as Glomus sp., fell among three different orders, in the genera Diversispora, Claroideoglomus and Rhizophagus. Several entries will be updated (J. Morton, pers. comm.). Our own past errors include the annotation of A. cavernata BEG33 as A. scrobiculata, and mixing up two samples, resulting in mistakenly naming sequences of S. spinosissima W3009/Att664-1 as S. nodosa BEG4 and vice versa. Moreover, we doubt our own annotation of a sequence (Y17652) that was recently used in the description of the monospecific genus Viscospora, now containing ‘Viscospora viscosa’ BEG27. Morphologically, affiliation to Claroideoglomus was surprising and the culture used for sequencing later was found to contain a contaminant C. claroideum-like fungus. A revived culture of BEG27 has been established and will be used to clarify this matter. A surprising issue regarded sequences (JF276401–JF276423) from one database submission, which all have identical counterparts in the database, including short 88- or 211-bp sequences. They must be derived from resubmission of already existing sequences. The ‘Glomus’ SSU sequences JF276412,17,18 and JN040742 are not from members of the Glomeromycota.
An example of putative culture misannotation is DAOM212349. The original voucher number refers to both the C. lamellosum holotype (field-collected) and a pot culture from which specimens designated as ‘isotype’ (which cannot be correct, as, by definition, an isotype has to be from the original type collection) were derived (Dalpéet al., 1992). A later in vitro ROC established from this pot culture was given the same number in the GINCO database, but it contains R. irregularis. Therefore, DAOM212349 must represent either an initially mixed culture or an instance of later contamination. Obviously, the same identifier is used for fungi belonging to two distinct genera. Failure to update public database sequences, to correct errors or to implement taxonomic changes can cause confusion and impair accurate interpretation of analyses. To facilitate correct interpretation of AMF sequence data, third-party annotations are currently implemented in the PlutoF (Abarenkov et al., 2010) based fungal reference sequence database, which now also includes curated metadata for mycorrhizal fungi (Tedersoo et al., 2011).
Systematics and molecular phylogenetics influence more scientific disciplines than is often realized (Mayr, 1968). It is important to correct misclassifications of organisms, the functional, genetic, and ecological traits of which are best interpreted on phylogenetic grounds.
Besides providing a solid phylogenetic backbone, the data set presented here covers the future primary DNA barcode for fungi, namely the ITS region, and the 5′ portion of the LSU, which will be used as an extended barcode. The use of long sequences covering conserved as well as variable regions solves problems intrinsic to the use of short sequences. This will also assist detection of species in the field, but the database will have to be broadened with respect to sequence and taxon coverage. The latter relates to described species, but also to environmental MOTUs, for which species-level recognition is feasible through the use of the SSU-ITS-LSU fragment (Stockinger et al., 2010; Krüger et al., 2011). A frequent problem with AMF is a lack of well-characterized biological material from described species, as many of these biotrophic fungi have so far proved impossible or difficult to culture, even in pots together with host plants. This problem could be alleviated by contributing more isolates (single spore cultures) to public culture collections, which, however, seem to be limited by inadequate funding. This is an unfortunate situation, given the fact that AMF are integral components of nearly all terrestrial ecosystems.
To discover more about AMF–plant preferences and the functional roles of AMF, a solid systematic classification is indispensable. This study provides a reference guide for molecular species identification and phylotaxonomy that will be important for future molecular ecological studies, including the application of next-generation sequencing strategies. More sequences with sufficient lengths would, moreover, facilitate improved understanding of the biogeography and evolution of AMF. In addition, research in practical fields, such as biosafety assessments regarding the impact of genetically modified plants on AMF communities or AMF species traceability in field applications, may depend on a solid data baseline.
- Top of page
- Materials and Methods
- Supporting Information
Fig. S1 Maximum likelihood phylogenetic tree based on the nuclear small subunit–internal transcribed spacer–large subunit rDNA (SSU-ITS-LSU) fragment of Glomeraceae, except for Rhizophagus and Sclerocystis.
Fig. S2 Maximum likelihood phylogenetic tree based on nuclear small subunit–internal transcribed spacer–large subunit rDNA (SSU-ITS-LSU) fragment of Claroideoglomeraceae.
Table S1 List of sequence identifiers derived from this and related studies published by the authors, including metadata
Notes S1 Consensus sequences used for Fig. 1.
Notes S2 Consensus sequences used for Fig. 2.
Notes S3 Consensus sequences used for Fig. 3(a).
Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office.