Bacterial clades are often ecologically distinct, despite extensive horizontal gene transfer (HGT). How selection works on different parts of bacterial pan-genomes to drive and maintain the emergence of clades is unclear. Focussing on the three largest clades in the diverse and well-studied Bacillus cereus sensu lato group, we identified clade-specific core genes (present in all clade members) and then used clade-specific allelic diversity to identify genes under purifying and diversifying selection. Clade-specific accessory genes (present in a subset of strains within a clade) were characterized as being under selection using presence/absence in specific clades. Gene ontology analyses of genes under selection revealed that different core genes and gene functions were enriched in different clades. Furthermore, some gene functions were enriched only amongst clade-specific core or accessory genomes. Genes under purifying selection were often clade-specific, while genes under diversifying selection showed signs of frequent HGT. These patterns are consistent with different selection pressures acting on both the core and accessory genomes of different clades and can lead to ecological divergence in both cases. Examining variation in allelic diversity allows us to uncover genes under clade-specific selection, allowing ready identification of strains and their ecological niche.

Bacterial phylogenies often contain distinct clusters or “clades”, associated
with different ecological niches despite frequent horizontal gene transfer (HGT)
between clades. An excellent example is the Bacillus cereus (Bc) group, whose
members share common adaptations but are often found in different clades, raising
questions over each clade’s specific ecology and pathogenic potential. Identifying
consistent genetic and phenotypic differences between clades within the Bc group
will determine whether clades truly possess specific ecologies and reduce confusion
regarding the pathogenic potential of the different clades.
The ecology of each Bc clade was explored through identification and
comparison of genes under selection within the core and accessory genomes within
the three largest clades within the Bc group. Each clade had its own distinctive
signature of selection, with Clade 3 showing signatures of adaptation to low
temperatures, supporting the hypotheses of previous studies.
Experimental evolution explored the effect of different thermal niches on the
ability of a Clade 2 B. thuringiensis and Clade 3 B. mycoides strain to adapt to novel
environmental conditions. After 700 generations of adaptation to a novel growth
medium, fitness gains in each strain were found to be highly dependent on
temperature. Single nucleotide polymorphism (SNP) variant analysis of evolved
strains indicated each strain responded differently to each temperature, suggesting
adaptation in these strains was constrained by thermal niche. Inactivation of a
putative thermal niche defining gene, cspA, in B. thuringiensis caused the fitness of
the strain to increase at low temperatures contrary to predictions, with different levels of temperature dependence in variants of the gene from mesophilic and
psychrotolerant strains. However, neither inactivation of the cspA gene or
complementation with the psychrotolerant variant affected the B. thuringiensis
strain’s ability to adapt during a second experimental evolution experiment.
The phenotypic inertia of thermal niche within members of the Bc clades
suggests that thermal niche may form a basis for the maintenance of distinct clades
within the Bc group. The reason for thermal niche constraint is unclear, but further
study can provide insight into how clades with distinct ecologies are maintained over
time.

The Bacillus cereus group encompasses beneficial and harmful species in diverse niches and has a much debated taxonomy. Investigating whether selection has led to ecological divergence between phylogenetic clades can help understand the basis of speciation, and has implications for predicting biological safety across this group. Using three most terrestrial species in this group (B. cereus, Bacillus thuringiensis and Bacillus mycoides) we charactererized ecological specialization in terms of resource use, thermal adaptation and fitness in different environmental conditions and tested whether taxonomic species or phylogenetic clade best explained phenotypic variation. All isolates grew vigorously in protein rich media and insect cadavers, but exploitation of soil or plant derived nutrients was similarly weak for all. For B. thuringiensis and B. mycoides, clade and taxonomic species were important predictors of relative fitness in insect infections. Fully psychrotolerant isolates could outcompete B. thuringiensis in insects at low temperature, although psychrotolerance predicted growth in artificial media better than clade. In contrast to predictions, isolates in the Bacillus anthracis clade had sub-optimal growth at 37°C. The common ecological niche in these terrestrial B. cereus species is the ability to exploit protein rich resources such as cadavers. However, selection has led to different phylogenetic groups developing different strategies for accessing this resource. Thus, clades, as well as traditional taxonomic phenotypes, predict biologically important traits.