Phylogenomic analyses predict sistergroup relationship of nucleariids and Fungi and paraphyly of zygomycetes with significant support
- Yu Liu†1, 3,
- Emma T Steenkamp†2,
- Henner Brinkmann1,
- Lise Forget1,
- Hervé Philippe1 and
- B Franz Lang1Email author
© Liu et al; licensee BioMed Central Ltd. 2009
Received: 23 July 2009
Accepted: 25 November 2009
Published: 25 November 2009
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.
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.
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.
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 ultimately the origin(s) of multicellularity [1–3]. 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.
The candidate organisms choanoflagellates, ichthyosporeans and Ministeria have been convincingly shown to be relatives of Metazoa (combined in a taxon termed Holozoa; ) by using molecular phylogenetics with genomic datasets (e.g., [4–8]). Yet, there are remaining questions about the exact phylogenetic positions of Capsaspora [5, 8] and Ministeria  within Holozoa. Another, less well studied group of protists are Nucleariida, a group of heterotrophic amoeboids with radiating filopodia. Nucleariids lack distinctive morphological features that might allow associating them with either animals or fungi. Their mitochondrial cristae are either discoidal-shaped or flattened [9–11]. Indeed, initial phylogenetic analyses based on single genes have been inconsistent in placing them even within opisthokonts. There has been also confusion due to the inclusion within Nucleariida of Capsaspora owczarzaki, a species that is now excluded from this group and shown to be clearly associated with Holozoa [5, 11–17].
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 slow-evolving chytridiomycetes . 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 nucleariid-fungal 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  along with new genome data available from Holozoa (C. owczarzaki, Amoebidium parasiticum, Sphaeroforma arctica; ) 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 , and of Capsaspora within Holozoa [5, 7, 8].
If the nucleariids are indeed the closest known relatives of Fungi as claimed , 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 [22–31]. For example, the analyses of ribosomal RNA data supports the sister relationship between Glomeromycota and Dikarya (Ascomycota plus Basidiomycota) , while analysis of genes encoding the largest and second-largest subunits of the nuclear RNA polymerase II supports the monophyly of Zygomycota in its traditional definition .
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 . 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 . 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.,  and references therein).
Despite these and various other unresolved phylogenetic issues, fungal taxonomy has been substantially redefined in a recent proposal . 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 , although support with these and other molecular markers is inconclusive. In turn, the traditional phylum Zygomycota has been altogether removed from this taxonomy , 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.
Results and Discussion
Phylogenomic analysis with the Eukaryotic Dataset supports Nucleariida as sister to Fungi
Analysis of the Eukaryotic Dataset with maximum likelihood (ML) using RAxML  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  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 . 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 (; 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 . Again, phylogenetic analysis of this dataset using P4  generated essentially the same tree topology, with some support values decreased due to loss of information by recoding (additional file 3).
Comparison of alternative tree topologies with AU and wSH tests.
Best tree (see Figure 1)
Nuclearia sister of Holozoa
Nuclearia sister of Opisthokonta
Nuclearia sister of Asco- + Basidio- + Zygomycetes
Nuclearia sister of Capsaspora + Amoebidium + Sphaeroforma
Nuclearia sister of Metazoa + Monosiga
Nuclearia sister of Allomyces
Nuclearia sister of Spizellomyces
Nuclearia sister of Capsaspora
Nuclearia sister of Amoebidium + Sphaeroforma
Nuclearia sister of Amoebozoa
Nuclearia sister of Opisthokonta + Amoebozoa
Nuclearia sister of Asco- + Basidiomycetes
Nuclearia sister of Monosiga
Nuclearia sister of Metazoa
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 phylogenetic 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 ).
Fungal phylogeny with Nucleariida as outgroup
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; ) 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 . However, the picture is more complicated as it is widely thought that the ancestral opisthokont also had a single posterior flagellum . 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.
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 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.
Construction of cDNA libraries and EST sequencing
Two N. simplex (CCAP 1552/2 and 1552/4) cDNA libraries were constructed following recently published protocols . Cells were grown in liquid standing cultures in WCL medium http://megasun.bch.umontreal.ca/People/lang/FMGP/methods/wcl.html supplemented with 0.5 × Cerophyll, with E. coli cells as food, which were pre-grown on LB medium in Petri-dishes as food. Plasmids were purified using the QIAprep 96 Turbo Miniprep Kit (Qiagen), sequencing reactions were performed with the ABI Prism BigDye™ terminator version 3.0/3.1 (Perkin-Elmer, Wellesley, MA, USA) and sequenced on an MJ BaseStation (MJ Research, USA). Trace files were imported into the TBestDB database http://tbestdb.bcm.umontreal.ca/searches/login.php for automated processing, including assembly as well as automated gene annotation by AutoFact [53, 54].
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  and sequenced from a random clone library . 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, ), followed by manual curation of the results.
A previously published alignment of nuclear-encoded proteins  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  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 . 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]). Sampling within the protist outgroup of the Eukaryotic Dataset is also not comprehensive (Stramenopila, Alveolata, and Euglenozoa) and limited to slow-evolving representatives of major eukaryotic lineages. The final Eukaryotic Dataset contains 118 proteins (24 439 amino acid positions) and the Fungal Dataset 150 proteins (40 925 amino acid positions). Proteins included in the nuclear datasets are listed in additional file 6.
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 (), Gblocks () 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 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 ) with the CAT+ Γ model and four discrete gamma categories, or by maximum likelihood (ML, RAxML  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 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  with the WAG+ Γ model, and p-values for each topology were calculated with CONSEL .
Variable Length Bootstrap analysis
We compared the performance of nuclear and mitochondrial datasets in phylogenetic inference by Variable Length Bootstrap (VLB) analysis . 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.
We thank Mary L. Berbee (University of British Columbia, Canada) and Rytas Vilgalys (Duke University) for providing Mortierella and Batrachochytrium cDNA libraries for sequencing, the NHGRI/Broad Institute for access to several new genome sequences (Allomyces, Mortierella, Spizellomyces and Capsaspora) and the Canadian Research Chair Program (BFL, HP), the Canadian Institute of Health Research (BFL) and the 'Bourses d'Excellence biT' (CIHR; YL) for salary and interaction support.
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