Background: Molecular phylogenetics and phylogenomics have greatly revised and enriched the fungal systematics in the last two decades. Most of the analyses have been performed by comparing single or multiple orthologous gene regions. Sequence alignment has always been an essential element in tree construction. These alignment-based methods (to be called the standard methods hereafter) need independent verification in order to put the fungal Tree of Life (TOL) on a secure footing. The ever-increasing number of sequenced fungal genomes and the recent success of our newly proposed alignment-free composition vector tree (CVTree, see Methods) approach have made the verification feasible. Results: In all, 82 fungal genomes covering 5 phyla were obtained from the relevant genome sequencing centers. An unscaled phylogenetic tree with 3 outgroup species was constructed by using the CVTree method. Overall, the resultant phylogeny infers all major groups in accordance with standard methods. Furthermore, the CVTree provides information on the placement of several currently unsettled groups. Within the sub-phylum Pezizomycotina, our phylogeny places the Dothideomycetes and Eurotiomycetes as sister taxa. Within the Sordariomycetes, it infers that Magnaporthe grisea and the Plectosphaerellaceae are closely related to the Sordariales and Hypocreales, respectively. Within the Eurotiales, it supports that Aspergillus nidulans is the early-branching species among the 8 aspergilli. Within the Onygenales, it groups Histoplasma and Paracoccidioides together, supporting that the Ajellomycetaceae is a distinct clade from Onygenaceae. Within the sub-phylum Saccharomycotina, the CVTree clearly resolves two clades: (1) species that translate CTG as serine instead of leucine (the CTG clade) and (2) species that have undergone whole-genome duplication (the WGD clade). It places Candida glabrata at the base of the WGD clade. Conclusion: Using different input data and methodology, the CVTree approach is a good complement to the standard methods. The remarkable consistency between them has brought about more confidence to the current understanding of the fungal branch of TOL.

A faithful phylogeny and an objective taxonomy for prokaryotes should agree with each other and ultimately follow the genome data. With the number of sequenced genomes reaching tens of thousands, both tree inference and detailed comparison with taxonomy are great challenges. We now provide one solution in the latest Release 3.0 of the alignment-free and whole-genome-based web server CVTree3. The server resides in a cluster of 64 cores and is equipped with an interactive, collapsible, and expandable tree display. It is capable of comparing the tree branching order with prokaryotic classification at all taxonomic ranks from domains down to species and strains. CVTree3 allows for inquiry by taxon names and trial on lineage modifications. In addition, it reports a summary of monophyletic and non-monophyletic taxa at all ranks as well as produces print-quality subtree figures. After giving an overview of retrospective verification of the CVTree approach, the power of the new server is described for the mega-classification of prokaryotes and determination of taxonomic placement of some newly-sequenced genomes. A few discrepancies between CVTree and 16S rRNA analyses are also summarized with regard to possible taxonomic revisions. CVTree3 is freely accessible to all users at http://tlife.fudan.edu.cn/cvtree3/ without login requirements.

Shigella species and Escherichia coli are closely related organisms. Early phenotyping experiments and several recent molecular studies put Shigella within the species E. coli. However, the whole-genome-based, alignment-free and parameter-free CVTree approach shows convincingly that four established Shigella species, Shigella boydii, Shigella sonnei, Shigella felxneri and Shigella dysenteriae, are distinct from E. coli strains, and form sister species to E. coli within the genus Escherichia. In view of the overall success and high resolution power of the CVTree approach, this result should be taken seriously. We hope that the present report may promote further in-depth study of the Shigella-E. coli relationship.

Composition vector trees (CVTrees) are inferred from whole-genome data by an alignment-free and parameter-free method. The agreement of these trees with the corresponding taxonomy provides an objective justification of the inferred phylogeny. In this work, we show the stability and self-consistency of CVTrees by performing bootstrap and jackknife re-sampling tests adapted to this alignment-free approach. Our ultimate goal is to advocate the viewpoint that time-consuming statistical re-sampling tests can be avoided at all in using this alignment-free approach. Agreement with taxonomy should be taken as a major criterion to estimate prokaryotic phylogenetic trees.

A tripartite comparison of Archaea phylogeny and taxonomy at and above the rank order is reported: (1) the whole-genome-based and alignment-free CVTree using 179 genomes; (2) the 16S rRNA analysis exemplified by the All-Species Living Tree with 366 archaeal sequences; and (3) the Second Edition of Bergey’s Manual of Systematic Bacteriology complemented by some current literature. A high degree of agreement is reached at these ranks. From the newly proposed archaeal phyla, Korarchaeota, Thaumarchaeota, Nanoarchaeota and Aigarchaeota, to the recent suggestion to divide the class Halobacteria into three orders, all gain substantial support from CVTree. In addition, the CVTree helped to determine the taxonomic position of some newly sequenced genomes without proper lineage information. A few discrepancies between the CVTree and the 16S rRNA approaches call for further investigation.