Carlos J Pardo-De la Hoz , Diane L Haughland , Darcie Thauvette , Sydney Toni , Spencer Goyette , William White , Ian D Medeiros , Luc Cornet , Petr Dvořák , Diego Garfias-Gallegos , Jolanta Miadlikowska , Nicolas Magain , François Lutzoni
Species are a fundamental unit of biodiversity. Yet, the existence of clear species boundaries among bacteria has long been a subject of debate. Here, we studied species boundaries in the context of the phylogenetic history of Nostoc, a widespread genus of photoautotrophic and nitrogen-fixing cyanobacteria that includes many lineages that form symbiotic associations with plants (e.g. cycads and bryophytes) and fungi (e.g. cyanolichens). We found that the evolution of Nostoc was characterized by eight rapid radiations, many of which were associated with major events in the evolution of plants. In addition, incomplete lineage sorting associated with these rapid radiations outweighed reticulations during Nostoc evolution. We then show that the pattern of diversification of Nostoc shapes the distribution of average nucleotide identities (ANIs) into a complex mosaic, wherein some closely related clades are clearly isolated from each other by gaps in genomic similarity, while others form a continuum where genomic species boundaries are expected. Nevertheless, recently diverged Nostoc lineages often form cohesive clades that are maintained by within-clade gene flow. Boundaries to homologous recombination between these cohesive clades persist even when the potential for gene flow is high, i.e. when closely related clades of Nostoc co-occur or are locally found in symbiotic associations with the same lichen-forming fungal species. Our results demonstrate that rapid radiations are major contributors to the complex speciation history of Nostoc. This underscores the need to consider evolutionary information beyond thresholds of genomic similarity to delimit biologically meaningful units of biodiversity for bacteria.
https://doi.org/10.1093/molbev/msaf244

Carlos J Pardo-De la Hoz , Nicolas Magain , Bryan Piatkowski , Luc Cornet , Manuela Dal Forno , Ignazio Carbone , Jolanta Miadlikowska , François Lutzoni
Prokaryotic genomes are often considered to be mosaics of genes that do not necessarily share the same evolutionary history due to widespread horizontal gene transfers (HGTs). Consequently, representing evolutionary relationships of prokaryotes as bifurcating trees has long been controversial. However, studies reporting conflicts among gene trees derived from phylogenomic data sets have shown that these conflicts can be the result of artifacts or evolutionary processes other than HGT, such as incomplete lineage sorting, low phylogenetic signal, and systematic errors due to substitution model misspecification. Here, we present the results of an extensive exploration of phylogenetic conflicts in the cyanobacterial order Nostocales, for which previous studies have inferred strongly supported conflicting relationships when using different concatenated phylogenomic data sets. We found that most of these conflicts are concentrated in deep clusters of short internodes of the Nostocales phylogeny, where the great majority of individual genes have low resolving power. We then inferred phylogenetic networks to detect HGT events while also accounting for incomplete lineage sorting. Our results indicate that most conflicts among gene trees are likely due to incomplete lineage sorting linked to an ancient rapid radiation, rather than to HGTs. Moreover, the short internodes of this radiation fit the expectations of the anomaly zone, i.e., a region of the tree parameter space where a species tree is discordant with its most likely gene tree. We demonstrated that concatenation of different sets of loci can recover up to 17 distinct and well-supported relationships within the putative anomaly zone of Nostocales, corresponding to the observed conflicts among well-supported trees based on concatenated data sets from previous studies. Our findings highlight the important role of rapid radiations as a potential cause of strongly conflicting phylogenetic relationships when using phylogenomic data sets of bacteria. We propose that polytomies may be the most appropriate phylogenetic representation of these rapid radiations that are part of anomaly zones, especially when all possible genomic markers have been considered to infer these phylogenies.
https://doi.org/10.1093/sysbio/syad008

Luc Cornet, Nicolas Magain, Denis Baurain, François Lutzoni
Understanding the evolutionary history of symbiotic Cyanobacteria at a fine scale is essential to unveil patterns of associations with their hosts and factors driving their spatiotemporal interactions. As for bacteria in general, Horizontal Gene Transfers (HGT) are expected to be rampant throughout their evolution, which justified the use of single-locus phylogenies in macroevolutionary studies of these photoautotrophic bacteria. Genomic approaches have greatly increased the amount of molecular data available, but the selection of orthologous, congruent genes that are more likely to reflect bacterial macroevolutionary histories remains problematic. In this study, we developed a synteny-based approach and searched for Collinear Orthologous Regions (COR), under the assumption that genes that are present in the same order and orientation across a wide monophyletic clade are less likely to have undergone HGT. We searched sixteen reference Nostocales genomes and identified 99 genes, part of 28 COR comprising three to eight genes each. We then developed a bioinformatic pipeline, designed to minimize inter-genome contamination and processed twelve Nostoc-associated lichen metagenomes. This reduced our original dataset to 90 genes representing 25 COR, which were used to infer phylogenetic relationships within Nostocales and among lichenized Cyanobacteria. This dataset was narrowed down further to 71 genes representing 22 COR by selecting only genes part of one (largest) operon per COR. We found a relatively high level of congruence among trees derived from the 90-gene dataset, but congruence was only slightly higher among genes within a COR compared to genes across COR. However, topological congruence was significantly higher among the 71 genes part of one operon per COR. Nostocales phylogenies resulting from concatenation and species tree approaches based on the 90- and 71-gene datasets were highly congruent, but the most highly supported result was obtained when using synteny, collinearity, and operon information (i.e., 71-gene dataset) as gene selection criteria, which outperformed larger datasets with more genes.
https://doi.org/10.1016/j.ympev.2021.107100