In-depth Phylogenomic Analysis of Arbuscular Mycorrhizal Fungi Based on a Comprehensive Set of de novo Genome Assemblies

Abstract

Morphological characters and nuclear ribosomal DNA (rDNA) phylogenies have so far been the basis of the current classifications of arbuscular mycorrhizal (AM) fungi. Improved understanding of the evolutionary history of AM fungi requires extensive ortholog sampling and analyses of genome and transcriptome data from a wide range of taxa. To circumvent the need for axenic culturing of AM fungi we gathered and combined genomic data from single nuclei to generate de novo genome assemblies covering seven families of AM fungi. We successfully sequenced the genomes of 15 AM fungal species for which genome data was not previously available. Comparative analysis of the previously published Rhizophagus irregularis DAOM197198 assembly confirm that our novel workflow generates genome assemblies suitable for phylogenomic analysis. Predicted genes of our assemblies, together with published protein sequences of AM fungi and their sister clades, were used for phylogenomic analyses. We evaluated the phylogenetic placement of Glomeromycota in relation to its sister phyla (Mucoromycota and Mortierellomycota), and found no support to reject a polytomy. Finally, we explored the phylogenetic relationships within Glomeromycota. Our results support family level classification from previous phylogenetic studies, and the polyphyly of the order Glomerales with Claroideoglomeraceae as the sister group to Glomeraceae and Diversisporales.

Keywords: Glomeromycota; genomics; phylogenetic; single nuclei sequencing; topology.

Figure 1

Best maximum likelihood tree inferred with RAxML from a concatenated alignment of 371 single copy orthologs shared among >50% of the taxa. The same topology was recovered using IQ-TREE and Bayesian inference. All nodes have a bootstrap support value of 100 in both analyses, and posterior probabilities of 1. Mucoromycota was used as outgroup. Stars following the taxon name mark newly sequenced strains from this study. Current taxonomic assignment based on Redecker et al. (2013) is color coded, at the levels of family and order. Strain identifers are included in the taxa label when more than one node has the same species name. See expanded tree in Supplementary Figure 7.

Figure 2

Evaluation of support among individual gene trees for alternative hypotheses of the relationships within Glomeromycota based on three datasets. (A) Glomeromycota dataset with single copy orthologs (SCOs) that are present in >50% of the taxa (27 taxa/1737 SCOs). (B) Glomeromycota dataset including SCOs that are present in all the taxa (27 taxa/31 SCOs). (C) Glomeromycota dataset with a selection of 15 taxa (see methods and Supplementary Table 4) including SCOs that are present in all taxa. Bar graphs represent the gene tree quartet frequencies for three possible branching orders within Glomeromycota. T1 corresponds to the ASTRAL topology, T2 and T3 correspond to alternative topologies in ASTRAL. Dashed horizontal lines marked the expectation of a hard polytomy. The topologies inferred with the concatenation-based method (Maximum Likelihood) are marked with an asterisk (^*). Local posterior probabilities are indicated only when below 1.0.

Figure 3

IQ-TREE network analysis visualized in SplitsTree5 with maximum dimension splits filter of 2, using the dataset containing all Glomeromycota taxa, and 1,737 SCOs shared among >50% of the taxa. See Supplementary Figure 13 for expanded network with full branch lengths.