Phylogenomic analyses predict sistergroup relationship of nucleariids and Fungi and paraphyly of zygomycetes with significant support

Background Resolving the evolutionary relationships among Fungi remains challenging because of their highly variable evolutionary rates, and lack of a close phylogenetic outgroup. Nucleariida, an enigmatic group of amoeboids, have been proposed to emerge close to the fungal-metazoan divergence and might fulfill this role. Yet, published phylogenies with up to five genes are without compelling statistical support, and genome-level data should be used to resolve this question with confidence. Results Our analyses with nuclear (118 proteins) and mitochondrial (13 proteins) data now robustly associate Nucleariida and Fungi as neighbors, an assemblage that we term 'Holomycota'. With Nucleariida as an outgroup, we revisit unresolved deep fungal relationships. Conclusion Our phylogenomic analysis provides significant support for the paraphyly of the traditional taxon Zygomycota, and contradicts a recent proposal to include Mortierella in a phylum Mucoromycotina. We further question the introduction of separate phyla for Glomeromycota and Blastocladiomycota, whose phylogenetic positions relative to other phyla remain unresolved even with genome-level datasets. Our results motivate broad sampling of additional genome sequences from these phyla.


Background
The investigation of previously little known eukaryotic lineages within and close to the opisthokonts will be key to understanding the origins of Fungi, the evolution of developmental traits in Fungi and Metazoa, and ulti-mately the origin(s) of multicellularity [1][2][3]. In particular, it will help to establish which and how many developmental genes are either shared or specific to these two major eukaryotic groups. In this context, it is essential to determine the precise phylogenetic position of candidate protists that are close to Fungi, Metazoa, or opisthokonts as a whole.
Overall, the phylogenetic position of the 'true' nucleariids remains controversial. In a more recent phylogenetic investigation with four nuclear gene sequences (EF-1α, HSP70, actin and β-tubulin), nucleariids associate confidently with Fungi, but only when selecting two slowevolving chytridiomycetes [18]. When improving the taxon sampling to 18 fungal species, the bootstrap support (BS) value for fungal monophyly drops to 85%, and alternative nucleariid positions are not rejected with the approximately unbiased (AU) test [18,19]. In this context, it seems noteworthy that Nuclearia and fungi other than chytrids are fast-evolving, and that the rate of tubulin evolution varies strongly among species of the latter dataset (correlating to some degree with the independent loss of the flagellar apparatus in non-chytrid fungi and in Nuclearia). Together, these rate differences at the gene and species levels may increase long-branch-attraction (LBA between the two fast-evolving groups) thus causing weaker support for fungal monophyly and the nucleariidfungal sister relationship, or predicting altogether incorrect phylogenetic relationships.
These unresolved questions served as motivation for the current phylogenetic analyses that are based on broad taxon sampling, substantially more nuclear genes (available through expressed sequence tag (EST) or complete genome projects), and comparative analyses of nuclear and mitochondrial gene datasets. To this end, we sequenced several thousand ESTs each from two Nuclearia simplex strains (probably representing separate species based on the high level of sequence divergence between them), and added them to a previous dataset [20] along with new genome data available from Holozoa (C. owc-zarzaki, Amoebidium parasiticum, Sphaeroforma arctica; [5]) and Fungi (Allomyces macrogynus, Batrachochytrium dendrobatidis, and Mortierella verticillata). We then sequenced the mitochondrial genome of one of the two N. simplex strains. Similar to the nuclear genomes of fungi, their mitochondrial genomes also evolve at varying rates thereby introducing a considerable potential for phylogenetic artifacts. However, phylogenetic comparisons between mitochondrial and nuclear data provide valuable, cross-wise indicators of phylogenetic artifacts as the respective evolutionary rates differ between the two genomes. For instance, such comparisons have revealed inconsistencies for the positioning of Schizosaccharomyces species within Taphrinomycotina [21], and of Capsaspora within Holozoa [5,7,8].
If the nucleariids are indeed the closest known relatives of Fungi as claimed [18], this protist group will provide an excellent fungal outgroup that would ultimately facilitate the settling of controversial phylogenetic placement of taxa within Fungi and/or in close neighboring groups. Among the debated issues are the monophyly and appropriate classification of the traditional fungal taxa Chytridiomycota and Zygomycota. Previous analyses based on single or a few genes have been inconsistent in answering these questions, and often lack significant support [22][23][24][25][26][27][28][29][30][31]. For example, the analyses of ribosomal RNA data supports the sister relationship between Glomeromycota and Dikarya (Ascomycota plus Basidiomycota) [29], while analysis of genes encoding the largest and secondlargest subunits of the nuclear RNA polymerase II supports the monophyly of Zygomycota in its traditional definition [25].
Phylogenetic positioning of the extremely fast-evolving Microsporidia (causing strong LBA artifacts in phylogenetic analyses) is another controversial issue of great interest. In some of the most recent analyses, Microsporidia have been placed either close to zygomycetes/Mucorales [32,33], or together with Rozella allomycis [24]. Together with environmental sequences, Rozella species form part of a large, diverse and relatively slowly evolving lineage (designated "Rozellida"). They branch as a sister clade to Fungi [24,34], which raises the additional question whether they should be considered to be true fungi as originally proposed [35]. Testing the above alternative hypotheses on microsporidian affinities by phylogenomic analysis will require much more data from Rozellida (a few genes are known from Rozella allomycis, but largely insufficient for inclusion in our analyses), and from a much wider range of the paraphyletic zygomycetes. Generation of genome-size data will be further critical for applying methods that reduce LBA artifacts such as removal of fast-evolving genes or sequence sites (e.g., [36] and references therein).
Despite these and various other unresolved phylogenetic issues, fungal taxonomy has been substantially redefined in a recent proposal [28]. Chytridiomycota is still treated as a phylum, but now include only Chytridiomycetes and Monoblepharidomycetes. Other traditional chytrid lineages such as Blastocladiomycota and Neocallimastigales have been elevated to phyla based on the analyses of LSU and SSU rRNA [23], although support with these and other molecular markers is inconclusive. In turn, the traditional phylum Zygomycota has been altogether removed from this taxonomy [28], because evolutionary relationships among its members are currently unresolved and suspected to be paraphyletic. Zygomycota are now reassigned into a phylum Glomeromycota plus four subphyla incertae sedis (i.e., uncertain): Mucoromycotina, Kickxellomycotina, Zoopagomycotina and Entomophthoromycotina. To revisit these somewhat contentious issues, we compared results with mitochondrial and nuclear phylogenomic datasets, and further analyzed the effect of extending fungal species sampling, with the two N. simplex strains as the outgroup.

Phylogenomic analysis with the Eukaryotic Dataset supports Nucleariida as sister to Fungi
Phylogenomic analysis of the Eukaryotic Dataset with one of the currently most realistic phylogenetic models (category mixture model (CAT); [37]) confirms the monophyly of major eukaryotic groups including Holozoa, Fungi, Amoebozoa, and Viridiplantae. Further, Amoebidium, Sphaeroforma plus Capsaspora form a monophyletic group, and Nuclearia is without a doubt the closest known sister-group to Fungi (100% BS; Figure 1). Also some higher-order relationships are recovered with significant support, such as opisthokonts and the two recently proposed supergroups JEH (jakobids, Euglenozoa plus Heterolobosea [20]) and CAS (Cercozoa, Alveolata plus Stramenopila [20,38,39]), whereas monophyly of Plantae, Excavata and Chromalveolata is not found. Evidently, the taxon sampling of protists in our dataset is insufficient for (and not aimed at) resolving the phylogenetic relationships among these latter lineages, as it was meant to constitute only a strong and well sampled outgroup to opisthokonts.
Analysis of the Eukaryotic Dataset with maximum likelihood (ML) using RAxML [40] and the commonly used WAG+Γ model generated a similar tree topology ( Figure 1 and additional file 1). Deep opisthokont divergences are predicted consistently and with significant support (BS > 98%), with Nuclearia clearly sister to Fungi (100% BS) and choanoflagellates the closest neighbor of animals. Amoebidium, Sphaeroforma plus Capsaspora form a monophyletic sister group to animals plus choanoflagellates, consistent with a previous analysis [5] but contradicting others [7,8]. The reasons for this incongruence may be related to differences in data and taxon sampling. Our dataset contains 50 eukaryotic species with a close outgroup to Holozoa (i.e., including nucleariids together with fungal representatives), compared with a total of only 30 species in a more extensive previous analysis [7]. In contrast to our analysis using Bayesian inference (BI), ML associates Malawimonadozoa with JEH (77% BS), a tendency noted and discussed previously [20,41], and an issue to be addressed by better taxon sampling in this group (currently, data are available from only two species). Other minor differences between WAG versus CAT model analyses (yet without statistical support in favor of alternatives) are in relationships within Plantae and the placement of Haptophyceae.
We further investigated if the position of Nuclearia next to Fungi might be affected by potential phylogenetic artifacts, such as compositional sequence bias and/or LBA [36,42]. This is suspected because of the highly varying evolutionary rates both within Fungi and in protist outgroups, and the unusual result that better taxon sampling in Fungi reduces phylogenetic support for the Nuclearia position ( [18]; see introduction). To do so, we first eliminated fast-evolving species from the dataset: S. cerevisiae, Blastocystis hominis, Cryptosporidium parvum, Sterkiella histriomuscorum, Diplonema papillatum and Leishmania major. The results from analyses using RAxML were essentially unchanged, both with respect to tree topology and BS values (additional file 2). To counteract sequence bias, we recoded the 20 amino acids into six groups as previously proposed [43]. Again, phylogenetic analysis of this dataset using P4 [44] generated essentially the same tree topology, with some support values decreased due to loss of information by recoding (additional file 3).
Finally, we evaluated the positioning of Nuclearia next to Fungi with the AU and weighted Shimodeira Hasegawa (wSH) likelihood tests [45]. For this, we compared the topology presented in Figure 1 with competing tree topologies in which the two Nuclearia strains were moved as sistergroup to all major eukaryotic lineages, and all possible positions within Opisthokonta. The results of both tests confirm Nuclearia as the closest neighbor group of Fungi, with all alternative topologies rejected at a significance level of p = 0.002 (Table 1). Given the unequivocal support for Nuclearia as the fungal sistergroup, we propose the term 'Holomycota' to refer to the assemblage of Nucleariida plus Fungi.

Mitochondrial phylogeny and genomic features support monophyly of the Holomycota
Phylogenetic analyses of nuclear versus mitochondrial datasets are expected to come to similar conclusions, thus providing independent evidence for the given phyloge-netic relationships. To this end, we sequenced and analyzed the complete mitochondrial DNA (mtDNA) of one of the N. simplex strains (a circular mapping DNA of 74 120 bp; see additional file 4). Note that growth of Nuclearia is complicated (the standard method calls for growth on Petri dishes with a bacterial lawn as food source), and that it is difficult to obtain sufficient cell material for mtDNA purification, explaining why we succeeded for only one of the two Nuclearia species.
The Nuclearia mtDNA contains a high number of introns (21 group I, and one group II), and mitochondrial protein genes appear to be translated with the standard translation code. These features are also widespread in Fungi. In contrast, Holozoa all use a mitochondrial UGA (tryptophan) codon reassignment, and contain no or only a few introns (with the notable exception of Placozoa, an enigmatic group of Metazoa [46]).
Phylogenetic analysis of a dataset with 56 species and 13 of the ubiquitous, most conserved mtDNA-encoded proteins predicts the monophyly of Opisthokonta, Stramenopila, Holozoa and Fungi with confidence, and also recovers Nuclearia as the sister-group of Fungi, albeit with a moderate BS value of 85% (Figure 2). To verify if the limited support for Holomycota is expected (i.e., correlating with the number of available sequence positions in the respective datasets), we performed a variable length Tree of eukaryotes based on Eukaryotic Dataset Figure 1 Tree of eukaryotes based on Eukaryotic Dataset. Trees were inferred with PhyloBayes and rooted following a previous suggestion [69,70]. The values at branches indicate bootstrap support (BS) values (upper value, BI/CAT model; lower value ML/ WAG model). Values below 60% are indicated by a hyphen; when BS values are equal only one is indicated. The posterior probability values using PhyloBayes are 1.0 for all except two branches (0.98 for the branch uniting Viridiplantae and Haptophyceae; 0.90 for the clade indicted by *). The analyses using ML (RAxML, WAG+Gamma; four categories, see additional file 1) support the alternative grouping of Malawimonadozoa and JEH with a BS of 77%. Other minor differences include Plantae relationships and the placement of Haptophyceae, which receive no solid support in both BI and ML analyses.
bootstrap (VLB) analysis. It compares the development of BS values with the number of sequence positions, for the nucleariid/fungal sister relationship. For this, we chose the 29 species shared between the two datasets (for the tree topology of the respective nuclear dataset see additional file 5). The results show that the development of BS values is similar for nuclear and mitochondrial data (Figure 3), and that the available mitochondrial dataset (as well as the above-cited nuclear phylogenies with five genes) is too small to resolve the phylogenetic position of nucleariids with high confidence. A better taxon sampling primarily in nucleariids will be imperative for improved phylogenetic resolution, motivating sequencing projects with new technologies, which are likely to provide mitochondrial as well as nuclear genome sequences -even with the limited amount of cellular material that is available for some taxa (e.g., [47]).

Fungal phylogeny with Nucleariida as outgroup
Analyses of both the nuclear and mitochondrial datasets have been insufficient to assess with confidence, neither zygomycete mono/paraphyly, nor the phylogenetic position of Blastocladiomycota (Blastocladiales) (Figure 1, 2). For instance, a recent mitochondrial multi-gene phylogeny with the first complete Glomus mtDNA sequence groups Glomus and Mortierella, yet lacks significant statistical support [47]. To re-address these questions, we have assembled a large dataset of nuclear-encoded genes from an extended, representative selection of fungal species, plus the two Nuclearia species as outgroup (i.e., the Fungal Dataset). The analyses show overall strong BS for the paraphyly of zygomycetes (Figure 4), i.e., the Entomophthoromycotina represent a significantly supported and completely independent fungal lineage. However, monophyletic Mucoromycotina including Mortierella as recently redefined [28] is not recovered (rendering the taxon Mucoromycotina paraphyletic), neither is the taxon Symbiomycota (Glomeromycota plus Dikarya; [29]). Instead, there is moderate support to group Mucorales plus Dikarya (92% BS in BI) and Glomus as their next neighbor (85% BS in BI). Although the placement of Glomus relative to Mortierella differs between our BI and ML analyses (Figure 4), we assume that the result of the BI analysis with its superior evolutionary model is more reliable. In light of these results, taxonomic reordering based on stable phylogenetic resolution of the traditional zygomycetes will require phylogenomic analyses with a much improved taxon sampling. Currently, nuclear and mitochondrial genome data are available only for single species in the latter two taxa; i.e. Glomus intraradices and M. verticillata.
Rooting of the fungal tree with nucleariids confirms that the traditional chytridiomycetes are also paraphyletic, again assuming that the result of the BI analysis is correct ( Figure 4). Confirmation of this result (justifying an elevation of Blastocladiomycota as a separate phylum; [28]) is highly desirable, as genome-size datasets in Blastocladiomycota are limited to the two moderately distant species Blastocladiella emersonii and A. macrogynus. Similarly, in light of the significant support for a monophyletic Chytridiomycota plus Neocallimastigomycota (100% BS with BI; Figure 4), their division into separate taxonomic higher ranks should be reconsidered, but only after phylogenomic analysis with improved taxon sampling in both groups. Finally, our results motivate genome or EST sequencing in Rozella species (Rozellida), potential relatives of Microsporidia and close neighbors of Fungi. The availability of a largely improved taxon sampling in zygomycetes, chytrids and Rozellida will provide a solid basis for evaluating the proposed placements of Microsporidia -either within or as a sistergroup to Fungi -based on phylogenomic analyses. The results presented here are consistent with previous notions on how Fungi came into being. For example it is thought that the first Fungi probably had branched chytrid-like rhizoids, which developed by enclosure of nucleariid-like filopodia (sometimes branched) into cell walls, during a nutritional shift from phagotrophy to saprotrophy, thus giving rise to fungal hyphae and rhizoids [7]. However, the picture is more complicated as it is widely thought that the ancestral opisthokont also had a single posterior flagellum [48]. This structure was lost during evolution of most but not all fungal lineages (e.g., [9,25,49,50]), with a separate loss in the nucleariid sistergroup. In this sense, nucleariids are unlikely to represent a primitive developmental stage, but rather a secondary reduction resulting in a unicellular, amoeboid life style. Obviously, the clarification of the chain of events leading to the emergence of multicellularity in Fungi is by no means complete. These issues will only become clear with a much broader sampling of genomes from taxa near the animal-fungal divergence and the discovery of additional protist groups that are closely related to Fungi.

Conclusion
Here we demonstrate that phylogenomic analysis with improved evolutionary models and algorithms has a potential for resolving long-standing issues in fungal evolution, by increasing phylogenetic resolution. Yet, while our results support certain aspects of the new taxonomic classification of Fungi they contradict others, suggesting that the introduction of certain higher-level taxa is only preliminary. In particular, the elevation of Neocallimastigales, Blastocladiomycota and Glomeromycota to separate phyla is questionable from a molecular phylogenetics standpoint, and potentially confusing to the larger scientific community. At present, genome analyses continue to suffer from poor sampling in chytrids, zygomycetes and Phylogeny inferred from the mitochondrial dataset Figure 2 Phylogeny inferred from the mitochondrial dataset. For details on figure description, evolutionary models and phylogenetic methods, see legend of Figure 1. Note that as already noted in a previous publication [5], the phylogenetic position of Capsaspora with mitochondrial data differs from that with nuclear data (Figure 1). We attribute this inconsistency to the limited availability of mtDNA sequences from Capsaspora relatives, and a strong LBA artifact introduced by the fast-evolving Bilateria in concert with Trichoplax. Further, the placements of Cryptococcus and Ustilago differ (although without significant support) from those with nuclear data (see Figure 4), although results with the much larger nuclear dataset are more likely to be correct.
close fungal relatives such as nucleariids. This issue will be resolved by the employment of new, increasingly inexpensive genome sequencing technologies. Phylogenomic projects like the current one will help focusing on genome analyses of poorly known phyla and taxa that are key to understanding fungal origins and evolution.

Mitochondrial sequencing and genome annotation
N. simplex (CCAP 1552/2) was grown as described above. The harvested cells were disrupted by addition of SDS plus proteinase K, and mitochondrial DNA was purified following a whole cell lysate protocol [55] and sequenced from a random clone library [56]. For mitochondrial genome assembly we used Phred, Phrap and Consed [57,58]; http://www.phrap.org/. Mitochondrial genes and introns were identified using automated procedures (MFannot, N. Beck and BFL unpublished; RNAweasel, [59]), followed by manual curation of the results.

Dataset construction
A previously published alignment of nuclear-encoded proteins [20] was used for adding the new Nuclearia cDNA sequences generated in our lab, plus extra sequences available from GenBank (a taxonomic broad dataset containing 50 eukaryotes will be referred to as the 'Eukaryotic Dataset'; another one containing 26 fungal species plus the two Nuclearia species as 'Fungal Dataset') using MUST [60] and FORTY (Denis Baurain and HP, unpublished). The number of species has been limited (to allow phylogenomic analyses within reasonable time frames), but only in well-sampled phylogenetic groups of undisputed phylogenetic affinity. Species that were not included are either fast-evolving and/or are incompletely sequenced. Other procedures for dataset construction, in particular the elimination of paralogous proteins, have been described previously [61]. Within opisthokonts, major lineages had to be represented by at least two distant species, and the extremely fast-evolving Microsporidia were excluded, as these are known to introduce phylogenetic artifacts and an overall reduction of phylogenetic resolution (at an extreme leading to misplacement of species; e.g., [62,63]  For a dataset of mitochondrial proteins, 13 ubiquitous genes (cox1, 2, 3, cob, atp6, 9, and nad1, 2, 3, 4, 4L, 5, 6) were selected. Muscle ( [64]), Gblocks ( [65]) and an application developed in-house (mams) were used for automatic protein alignment, removal of ambiguous regions and concatenation. The final dataset contains 56 taxa and 2 710 amino acid positions.

Phylogenetic analysis
Phylogenetic analyses were performed at the amino acid (aa) level using methods that are known to be least sensitive to LBA artifacts ( [36,37,66], and references therein). The concatenated protein datasets were analyzed either by Bayesian inference (BI, PhyloBayes [37]) with the CAT+ Γ model and four discrete gamma categories, or by maximum likelihood (ML, RAxML [40] with the WAG+ Γ model and four discrete categories. BI analyses using the CAT model have been shown to be more reliable than ML, due to the application of a more realistic evolutionary Fungal phylogeny with nuclear data, using Nucleariida as the outgroup Figure 4 Fungal phylogeny with nuclear data, using Nucleariida as the outgroup. For details on figure description, evolutionary models and phylogenetic methods, see legend of Figure 1. Note that the phylogenetic position of Blastocladiomycota is unstable, differing between ML versus BI analyses (we consider the latter to be more reliable).
model. ML analyses were essentially performed to identify differences in topology, pinpointing problematic parts of the tree for which addition of new data would be in order (i.e., preferentially genome sequences from slowly-evolving species, and those that are expected to break long internal branches at questionable tree topologies).
In case of BI and the Eukaryotic Dataset (values for the Fungal Dataset in brackets), chains were run for 3000 (1000) cycles, and the first 1500 (500) cycles were removed as burn-in corresponding to approximately 1,200,000 (400,000) generations. Convergence was controlled by running three independent chains, resulting in identical topologies. The reliability of internal branches for both, ML and BI analyses was evaluated based on 100 bootstrap replicates. For BI, we inferred a consensus tree from the posterior tree topologies of replicates.
Likelihood tests of competing tree topologies were also performed. The site-wise likelihood values were estimated using Tree-Puzzle [67] with the WAG+ Γ model, and pvalues for each topology were calculated with CONSEL [45].

Variable Length Bootstrap analysis
We compared the performance of nuclear and mitochondrial datasets in phylogenetic inference by Variable Length Bootstrap (VLB) analysis [68]. Sequences of 29 common species were taken from the eukaryotic (24,439 aa positions) and mitochondrial (2,710 aa positions) datasets. From these, two respective series of sub-datasets were constructed by randomly choosing 400, 600, 800, 1 000 ... sequence positions. Phylogenetic inferences were then performed using RAxML with the WAG+Γ model and four discrete categories, after which the BS values for the grouping of nucleariids and Fungi were recorded.