A fungal phylogeny based on 42 complete genomes derived from supertree and combined gene analysis

Background  

To date, most fungal phylogenies have been derived from single gene comparisons, or from concatenated alignments of a small number of genes. The increase in fungal genome sequencing presents an opportunity to reconstruct evolutionary events using entire genomes. As a tool for future comparative, phylogenomic and phylogenetic studies, we used both supertrees and concatenated alignments to infer relationships between 42 species of fungi for which complete genome sequences are available.     

PhySIC_IST: cleaning source trees to infer more informative supertrees

Background  

Supertree methods combine phylogenies with overlapping sets of taxa into a larger one. Topological conflicts frequently arise among source trees for methodological or biological reasons, such as long branch attraction, lateral gene transfers, gene duplication/loss or deep gene coalescence. When topological conflicts occur among source trees, liberal methods infer supertrees containing the most frequent alternative, while veto methods infer supertrees not contradicting any source tree, i.e. discard all conflicting resolutions. When the source trees host a significant number of topological conflicts or have a small taxon overlap, supertree methods of both kinds can propose poorly resolved, hence uninformative, supertrees.     

A phylogenomic analysis of the Ascomycota

An automated procedure was developed to extract orthologous sequences from fungal genomes and incorporate them into phylogenomic analyses in a timely and efficient manner. This approach involves parsing an all versus all BLASTP search of 17 proteomes and creating a similarity matrix from e-values, which is then used to cluster proteins into related groups by means of a Markov Clustering algorithm. After performing this analysis at different stringency levels, 854 single copy protein clusters, which were ubiquitously distributed in all 17 proteomes, were identified. These clusters were culled to include only those clusters where all proteins had best hits to and received hits from a protein within the same cluster. The final data set included gapless alignments for 781 clusters of orthologous sequences that were concatenated into one super alignment containing 195,664 amino acid characters. Neighbor-joining distance and maximum likelihood analyses resulted in identical topologies and all except one node received 100% bootstrap support. The node supporting Stagonospora nodorum's position received 83% support or higher; it was also the only taxon differentially resolved in the maximum parsimony analyses. All analyses resolved the two derived subphyla Pezizomycotina and Saccharomycotina, and Schizosaccharomyces pombe as an early diverging lineage of the Ascomycota. Importantly, these analyses resolved the Leotiomycetes as the sister group to the Sordariomycetes, a region of the Ascomycota phylogeny that has remained problematic in molecular phylogenetic studies of more limited character sampling. Additional phylogenetic analyses which included orthologous sequences from an unannotated ascomycotan genome (e.g., Coccidioides immitis) and subsets of orthologs with different characteristics supported this topology. Phylogenetic analyses of the 595 orthologs which included C. immitis resulted in an identical topology to the previous 781 ortholog analysis and correctly placed C. immitis in the Eurotiomycetes. This demonstrated the correct identification of orthologs and the ability to incorporate unannotated genomic data into a common phylogenetic analysis.     

The common ancestral core of vertebrate and fungal telomerase RNAs

Telomerase is a ribonucleoprotein with an intrinsic telomerase RNA (TER) component. Within yeasts, TER is remarkably large and presents little similarity in secondary structure to vertebrate or ciliate TERs. To better understand the evolution of fungal telomerase, we identified 74 TERs from Pezizomycotina and Taphrinomycotina subphyla, sister clades to budding yeasts. We initially identified TER from Neurospora crassa using a novel deep-sequencing–based approach, and homologous TER sequences from available fungal genome databases by computational searches. Remarkably, TERs from these non-yeast fungi have many attributes in common with vertebrate TERs. Comparative phylogenetic analysis of highly conserved regions within Pezizomycotina TERs revealed two core domains nearly identical in secondary structure to the pseudoknot and CR4/5 within vertebrate TERs. We then analyzed N. crassa and Schizosaccharomyces pombe telomerase reconstituted in vitro, and showed that the two RNA core domains in both systems can reconstitute activity in trans as two separate RNA fragments. Furthermore, the primer-extension pulse-chase analysis affirmed that the reconstituted N. crassa telomerase synthesizes TTAGGG repeats with high processivity, a common attribute of vertebrate telomerase. Overall, this study reveals the common ancestral cores of vertebrate and fungal TERs, and provides insights into the molecular evolution of fungal TER structure and function.     

Phylogenomics of <Emphasis Type="Italic">Bartheletia paradoxa</Emphasis> reveals its basal position in Agaricomycotina and that the early evolutionary history of basidiomycetes was rapid and probably not strictly bifurcating

The higher level phylogeny of fungi has been addressed in previous studies, but for those analyses, either taxon sampling or gene sampling was low, or some basal lineages important for the inference of basidiomycete phylogeny were lacking. Here, a phylogenomic analysis based on highly conserved genes and including the enigmatic species Bartheletia paradoxa from Ginkgo biloba is presented. While phylogenetic analyses including also less conserved parts of core eukaryotic genes yielded a basal position for the extremophile genus Wallemia with low support, an exclusion of highly variable parts of these genes suggested Bartheletia paradoxa as the most basal member of the Agaricomycotina, but again with low support. Network analyses suggest a network-like evolution at the base of the Basidiomycota, supported by phylogenies based on single genes and gene clusters with shared topology. When further removing noise by removing poorly resolving genes, strong but not maximum support was obtained for Bartheletia paradoxa being the sister lineage to all other Agaricomycotina. We speculate that the lack of support for the early splits in Agaricomycotina and Basidiomycota can probably be explained by rapid radiation, linked to major evolutionary developments, such as, in the case of Basidiomycota, the advent of basidia in the last common ancestor.     

Building supertrees: an empirical assessment using the grass family (Poaceae)

Large and comprehensive phylogenetic trees are desirable for studying macroevolutionary processes and for classification purposes. Such trees can be obtained in two different ways. Either the widest possible range of taxa can be sampled and used in a phylogenetic analysis to produce a "big tree," or preexisting topologies can be used to create a supertree. Although large multigene analyses are often favored, combinable data are not always available, and supertrees offer a suitable solution. The most commonly used method of supertree reconstruction, matrix representation with parsimony (MRP), is presented here. We used a combined data set for the Poaceae to (1) assess the differences between an approach that uses combined data and one that uses different MRP modifications based on the character partitions and (2) investigate the advantages and disadvantages of these modifications. Baum and Ragan and Purvis modifications gave similar results. Incorporating bootstrap support associated with pre-existing topologies improved Baum and Ragan modification and its similarity with a combined analysis. Finally, we used the supertree reconstruction approach on 55 published phylogenies to build one of most comprehensive phylogenetic trees published for the grass family including 403 taxa and discuss its strengths and weaknesses in relation to other published hypotheses.     

Performance of flip supertree construction with a heuristic algorithm

Supertree methods are used to assemble separate phylogenetic trees with shared taxa into larger trees (supertrees) in an effort to construct more comprehensive phylogenetic hypotheses. In spite of much recent interest in supertrees, there are still few methods for supertree construction. The flip supertree problem is an error correction approach that seeks to find a minimum number of changes (flips) to the matrix representation of the set of input trees to resolve their incompatibilities. A previous flip supertree algorithm was limited to finding exact solutions and was only feasible for small input trees. We developed a heuristic algorithm for the flip supertree problem suitable for much larger input trees. We used a series of 48- and 96-taxon simulations to compare supertrees constructed with the flip supertree heuristic algorithm with supertrees constructed using other approaches, including MinCut (MC), modified MC (MMC), and matrix representation with parsimony (MRP). Flip supertrees are generally far more accurate than supertrees constructed using MC or MMC algorithms and are at least as accurate as supertrees built with MRP. The flip supertree method is therefore a viable alternative to other supertree methods when the number of taxa is large.     

Towards a Supertree of Arthropoda: A Species-Level Supertree of the Spiny,Slipper and Coral Lobsters (Decapoda: Achelata)

While supertrees have been built for many vertebrate groups (notably birds, mammals and dinosaurs), invertebrates have attracted relatively little attention. The paucity of supertrees of arthropods is particularly surprising given their economic and ecological importance, as well as their overwhelming contribution to biodiversity. The absence of comprehensive archives of machine-readable source trees, coupled with the need for software implementing repeatable protocols for managing them, has undoubtedly impeded progress. Here we present a supertree of Achelata (spiny, slipper and coral lobsters) as a proof of concept, constructed using new supertree specific software (the Supertree Toolkit; STK) and following a published protocol. We also introduce a new resource for archiving and managing published source trees. Our supertree of Achelata is synthesised from morphological and molecular source trees, and represents the most complete species-level tree of the group to date. Our findings are consistent with recent taxonomic treatments, confirming the validity of just two families: Palinuridae and Scyllaridae; Synaxidae were resolved within Palinuridae. Monophyletic Silentes and Stridentes lineages are recovered within Palinuridae, and all sub-families within Scyllaridae are found to be monophyletic with the exception of Ibacinae. We demonstrate the feasibility of building larger supertrees of arthropods, with the ultimate objective of building a complete species-level phylogeny for the entire phylum using a divide and conquer strategy.     

Secretomes of medically important fungi reflect morphological and phylogenetic diversity

Secretome represents a main target for understanding the mechanisms of fungal adaptation. In the present study, we focus on the secretomes of fungi associated with infections in humans and other mammals in order to explore relationships between the diverse morphological and phylogenetic groups. Almost all the mammalian pathogenic fungi analyzed have secretome sizes smaller than 1000 proteins and, secreted proteins comprise between 5% and 10% of the total proteome. As expected, the correlation pattern between the secretome size and the total proteome was similar to that described in previous secretome studies of fungi. With regard to the morphological groups, minimum secretome sizes of less than 250 secreted proteins and low values for the fraction of secreted proteins are shown in mammalian pathogenic fungi with reduced proteomes such as microsporidia, atypical fungi and some species of yeasts and yeast-like fungi (Malassezia). On the other hand, filamentous fungi have significantly more secreted proteins and the highest numbers are present in species of filamentous fungi that also are plant or insect pathogens (Fusarium verticilloides, Fusarium oxysporum and Basidiobolus meristosporus). With respect to phylogeny, there are also variations in secretome size across fungal subphyla: Microsporidia, Taphrinomycotina, Ustilagomycotina and Saccharomycotina contain small secretomes; whereas larger secretomes are found in Agaricomycotina, Pezizomycotina, Mucoromycotina and Entomophthoromycotina. Finally, principal component analysis (PCA) was conducted on the complete secretomes. The PCA results revealed that, in general, secretomes of fungi belonging to the same morphological group or subphyla cluster together. In conclusion, our results point out that in medically important fungi there is a relationship between the secretome and the morphological group or phylogenetic classification.     

Adding time-calibrated branch lengths to the Asteraceae supertree

New inference techniques,such as supertrees,have improved the construction of large phylogenies,helping to reveal the tree of life.In addition,these large phylogenies have enhanced the study of other evolutionary questions,such as whether traits have evolved in a neutral or adaptive way,or what factors have influenced diversification.However,supertrees usually lack branch lengths,which are necessary for all these issues to be investigated.Here,divergence times within the largest family of flowering plants,namely the Asteraceae,are reviewed to estimate time-calibrated branch lengths in the supertree of this lineage.An inconsistency between estimated dates of basal branching events and the earliest asteraceous fossil pollen record was detected.In addition,the impact of different methods of branch length assignment on the total number of transitions between states in the reconstruction of sexual system evolution in Asteraceae was investigated.At least for this dataset,different branch length assignation approaches influenced maximum likelihood(ML)reconstructions only and not Bayesian ones.Therefore,the selection of different branch length information is not arbitrary and should be carefully assessed,at least when ML approaches are being used.The reviewed divergence times and the estimated time-calibrated branch lengths provide a useful tool for future phylogenetic comparative and macroevolutionary studies of Asteraceae.     

The evolution of supertrees

Supertrees result from combining many smaller, overlapping phylogenetic trees into a single, more comprehensive tree. As such, supertree construction is probably as old as the field of systematics itself, and remains our only way of visualizing the Tree of Life as a whole. Over the past decade, supertree construction has gained a more formal, objective footing, and has become an area of active theoretical and practical research. Here, I review the history of the supertree approach, focusing mainly on its current implementation. The supertrees of today represent some of the largest, complete phylogenies available for many groups, but are not without their critics. I conclude by arguing that the ever-growing molecular revolution will result in supertree construction taking on a new role and implementation in the future for analyzing large DNA sequence matrices as part of a divide-and-conquer phylogenetic approach.     

Adding time‐calibrated branch lengths to the Asteraceae supertree

Abstract New inference techniques, such as supertrees, have improved the construction of large phylogenies, helping to reveal the tree of life. In addition, these large phylogenies have enhanced the study of other evolutionary questions, such as whether traits have evolved in a neutral or adaptive way, or what factors have influenced diversification. However, supertrees usually lack branch lengths, which are necessary for all these issues to be investigated. Here, divergence times within the largest family of flowering plants, namely the Asteraceae, are reviewed to estimate time‐calibrated branch lengths in the supertree of this lineage. An inconsistency between estimated dates of basal branching events and the earliest asteraceous fossil pollen record was detected. In addition, the impact of different methods of branch length assignment on the total number of transitions between states in the reconstruction of sexual system evolution in Asteraceae was investigated. At least for this dataset, different branch length assignation approaches influenced maximum likelihood (ML) reconstructions only and not Bayesian ones. Therefore, the selection of different branch length information is not arbitrary and should be carefully assessed, at least when ML approaches are being used. The reviewed divergence times and the estimated time‐calibrated branch lengths provide a useful tool for future phylogenetic comparative and macroevolutionary studies of Asteraceae.     

Deduction of probable events of lateral gene transfer through comparison of phylogenetic trees by recursive consolidation and rearrangement

Background  

When organismal phylogenies based on sequences of single marker genes are poorly resolved, a logical approach is to add more markers, on the assumption that weak but congruent phylogenetic signal will be reinforced in such multigene trees. Such approaches are valid only when the several markers indeed have identical phylogenies, an issue which many multigene methods (such as the use of concatenated gene sequences or the assembly of supertrees) do not directly address. Indeed, even when the true history is a mixture of vertical descent for some genes and lateral gene transfer (LGT) for others, such methods produce unique topologies.     

A molecular phylogeny of bryozoans

We present the most comprehensive molecular phylogeny of bryozoans to date. Our concatenated alignment of two nuclear ribosomal and five mitochondrial genes includes 95 taxa and 13,292 nucleotide sites, of which 8297 were included. The number of new sequences generated during this project are for each gene:ssrDNA (32), lsrDNA (22), rrnL (38), rrnS (35), cox1 (37), cox3 (34), and cytb (44). Our multi-gene analysis provides a largely stable topology across the phylum. The major groups were unambiguously resolved as (Phylactolaemata (Cyclostomata (Ctenostomata, Cheilostomata))), with Ctenostomata paraphyletic. Within Phylactolaemata, (Stephanellidae, Lophopodidae) form the earliest divergent clade. Fredericellidae is not resolved as a monophyletic family and forms a clade together with Plumatellidae, Cristatellidae and Pectinatellidae, with the latter two as sister taxa. Hyalinella and Gelatinella nest within the genus Plumatella. Cyclostome taxa fall into three major clades: i. (Favosipora (Plagioecia, Rectangulata)); ii. (Entalophoroecia ((Diplosolen, Cardioecia) (Frondipora, Cancellata))); and iii. (Articulata ((Annectocyma, Heteroporidae) (Tubulipora (Tennysonia, Idmidronea)))), with suborders Tubuliporina and Cerioporina, and family Plagioeciidae each being polyphyletic. Ctenostomata is composed of three paraphyletic clades to the inclusion of Cheilostomata: ((Alcyonidium, Flustrellidra) (Paludicella (Anguinella, Triticella)) (Hislopia (Bowerbankia, Amathia)) Cheilostomata); Flustrellidra nests within the genus Alcyonidium, and Amathia nests within the genus Bowerbankia. Suborders Carnosa and Stolonifera are not monophyletic. Within the cheilostomes, Malacostega is paraphyletic to the inclusion of all other cheilostomes. Conopeum is the most early divergent cheilostome, forming the sister group to ((Malacostega, Scrupariina, Inovicellina) ((Hippothoomorpha, Flustrina) (Lepraliomorpha, Umbonulomorpha))); Flustrina is paraphyletic to the inclusion of the hippothoomorphs; neither Lepraliomorpha nor Umbonulomorpha is monophyletic. Ascophorans are polyphyletic, with hippothoomorphs grouping separately from lepraliomorphs and umbonulomorphs; no cribrimorphs were included in the analysis. Results are discussed in the light of molecular and morphological evidence. Ancestral state reconstruction of larval strategy in Gymnolaemata revealed planktotrophy and lecithotrophy as equally parsimonious solutions for the ancestral condition. More comprehensive taxon sampling is expected to clarify this result. We discuss the extent of non-bryozoan contaminant sequences deposited in GenBank and their impact on the reconstruction of metazoan phylogenies and those of bryozoan interrelationships.     

Increasing data transparency and estimating phylogenetic uncertainty in supertrees: Approaches using nonparametric bootstrapping

The estimation of ever larger phylogenies requires consideration of alternative inference strategies, including divide-and-conquer approaches that decompose the global inference problem to a set of smaller, more manageable component problems. A prominent locus of research in this area is the development of supertree methods, which estimate a composite tree by combining a set of partially overlapping component topologies. Although promising, the use of component tree topologies as the primary data dissociates supertrees from complexities within the underling character data and complicates the evaluation of phylogenetic uncertainty. We address these issues by exploring three approaches that variously incorporate nonparametric bootstrapping into a common supertree estimation algorithm (matrix representation with parsimony, although any algorithm might be used), including bootstrap-weighting, source-tree bootstrapping, and hierarchical bootstrapping. We illustrate these procedures by means of hypothetical and empirical examples. Our preliminary experiments suggest that these methods have the potential to improve the correspondence of supertree estimates to those derived from simultaneous analysis of the combined data and to allow uncertainty in supertree topologies to be quantified. The ability to increase the transparency of supertrees to the underlying character data has several practical implications and sheds new light on an old debate. These methods have been implemented in the freely available program, tREeBOOT.     

Assessment of phylogenomic and orthology approaches for phylogenetic inference

MOTIVATION: Phylogenomics integrates the vast amount of phylogenetic information contained in complete genome sequences, and is rapidly becoming the standard for reliably inferring species phylogenies. There are, however, fundamental differences between the ways in which phylogenomic approaches like gene content, superalignment, superdistance and supertree integrate the phylogenetic information from separate orthologous groups. Furthermore, they all depend on the method by which the orthologous groups are initially determined. Here, we systematically compare these four phylogenomic approaches, in parallel with three approaches for large-scale orthology determination: pairwise orthology, cluster orthology and tree-based orthology. RESULTS: Including various phylogenetic methods, we apply a total of 54 fully automated phylogenomic procedures to the fungi, the eukaryotic clade with the largest number of sequenced genomes, for which we retrieved a golden standard phylogeny from the literature. Phylogenomic trees based on gene content show, relative to the other methods, a bias in the tree topology that parallels convergence in lifestyle among the species compared, indicating convergence in gene content. CONCLUSIONS: Complete genomes are no guarantee for good or even consistent phylogenies. However, the large amounts of data in genomes enable us to carefully select the data most suitable for phylogenomic inference. In terms of performance, the superalignment approach, combined with restrictive orthology, is the most successful in recovering a fungal phylogeny that agrees with current taxonomic views, and allows us to obtain a high-resolution phylogeny. We provide solid support for what has grown to be a common practice in phylogenomics during its advance in recent years. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.     

The Tree versus the Forest: The Fungal Tree of Life and the Topological Diversity within the Yeast Phylome

A recurrent topic in phylogenomics is the combination of various sequence alignments to reconstruct a tree that describes the evolutionary relationships within a group of species. However, such approach has been criticized for not being able to properly represent the topological diversity found among gene trees. To evaluate the representativeness of species trees based on concatenated alignments, we reconstruct several fungal species trees and compare them with the complete collection of phylogenies of genes encoded in the Saccharomyces cerevisiae genome. We found that, despite high levels of among-gene topological variation, the species trees do represent widely supported phylogenetic relationships. Most topological discrepancies between gene and species trees are concentrated in certain conflicting nodes. We propose to map such information on the species tree so that it accounts for the levels of congruence across the genome. We identified the lack of sufficient accuracy of current alignment and phylogenetic methods as an important source for the topological diversity encountered among gene trees. Finally, we discuss the implications of the high levels of topological variation for phylogeny-based orthology prediction strategies.     

Resolving the phylogeny of lizards and snakes (Squamata) with extensive sampling of genes and species

Squamate reptiles (lizards and snakes) are one of the most diverse groups of terrestrial vertebrates. Recent molecular analyses have suggested a very different squamate phylogeny relative to morphological hypotheses, but many aspects remain uncertain from molecular data. Here, we analyse higher-level squamate phylogeny with a molecular dataset of unprecedented size, including 161 squamate species for up to 44 nuclear genes each (33 717 base pairs), using both concatenated and species-tree methods for the first time. Our results strongly resolve most squamate relationships and reveal some surprising results. In contrast to most other recent studies, we find that dibamids and gekkotans are together the sister group to all other squamates. Remarkably, we find that the distinctive scolecophidians (blind snakes) are paraphyletic with respect to other snakes, suggesting that snakes were primitively burrowers and subsequently re-invaded surface habitats. Finally, we find that some clades remain poorly supported, despite our extensive data. Our analyses show that weakly supported clades are associated with relatively short branches for which individual genes often show conflicting relationships. These latter results have important implications for all studies that attempt to resolve phylogenies with large-scale phylogenomic datasets.     

Phylogeny of iguanian lizards inferred from 29 nuclear loci, and a comparison of concatenated and species-tree approaches for an ancient, rapid radiation

Iguanian lizards form a diverse clade whose members have been the focus of many comparative studies of ecology, behavior, and evolution. Despite the importance of phylogeny to such studies, interrelationships among many iguanian clades remain uncertain. Within the Old World clade Acrodonta, Agamidae is sometimes found to be paraphyletic with respect to Chamaeleonidae, and recent molecular studies have produced conflicting results for many major clades. Within the largely New World clade Pleurodonta, relationships among the 12 currently recognized major subclades (mostly ranked as families) have been largely unresolved or poorly supported in previous studies. To clarify iguanian evolutionary history, we first infer phylogenies using concatenated maximum-likelihood (ML) and Bayesian analyses of DNA sequence data from 29 nuclear protein-coding genes for 47 iguanian and 29 outgroup taxa. We then estimate a relaxed-clock Bayesian chronogram for iguanians using BEAST. All three methods produce identical topologies. Within Acrodonta, we find strong support for monophyly of Agamidae with respect to Chamaeleonidae, and for almost all relationships within agamids. Within Pleurodonta, we find strong Bayesian support for almost all relationships, and strong ML support for some interfamilial relationships and for monophyly of almost all families (excepting Polychrotidae). Our phylogenetic results suggest a non-traditional biogeographic scenario in which pleurodonts originated in the Northern Hemisphere and subsequently spread southward into South America. The pleurodont portion of the tree is characterized by several very short, deep branches, raising the possibility of deep coalescences that may confound concatenated analyses. We therefore also use 27 of these genes to implement a coalescent-based species-tree approach for pleurodonts. Although this analysis strongly supports monophyly of the pleurodont families, interfamilial relationships are generally different from those in the concatenated tree, and support is uniformly poor. However, a species-tree analysis using only the seven most variable loci yields higher support and more congruence with the concatenated tree. This suggests that low support in the 27-gene species-tree analysis may be an artifact of the many loci that are uninformative for very short branches. This may be a general problem for the application of species-tree methods to rapid radiations, even with phylogenomic data sets. Finally, we correct the non-monophyly of Polychrotidae by recognizing the pleurodont genus Anolis (sensu lato) as a separate family (Dactyloidae), and we correct the non-monophyly of the agamid genus Physignathus by resurrection of the genus Istiurus for the former Physignathus lesueurii.