## 2 Mycobacteria Diversity and Evolution
### Phylogeny and Evolution of the Mycobacterium Genus  
Within the genus _Mycobacterium_, both the growth rate and pigmentation of colonies are still commonly used phenotypic features to characterize different species, however, nowadays the exploitation of the molecular component overcomes the evaluation of phenotypic traits [4]. The 16S rRNA encoding gene was, for many years, the basis of taxonomic works (Figure 1). Taxonomic and phylogenetic research were mainly focused on two hypervariable sequences known as regions A and B [14]. The 16S rRNA-based phylogeny endorses a division between rapid and slow growers, clustering most species in well-defined phylogenetic groups, however, the moderate variability of 16S rRNA among mycobacteria with high intraspecific identity (ranging from 94 to 100%) limits the distinction of several species [14]. Later on, the identification of a phylogenetic signature related to the short helix 18 in the hypervariable region B was reported, with most slow growers presenting a 12-nucleotide insertion that is not present in rapid growers [14]. Moreover, within the slow growers, two particular situations can be highlighted: (1) members of the _M. terrae_ complex have a 14-nucleotide insertion; and (2) the members of the _M. simiae_ complex present sequence features of short helix 18 that match those of ancestral mycobacteria [14]. The relationships among mycobacterial species and identification algorithms have also been evaluated over the years using the intergenic transcribed spacer region (between the genes encoding the 16S and 23S rRNA), and several housekeeping genes, including the 65-kDa heat shock protein (_hsp65_), DNA gyrse subunit B (_gyrB_), and RNA polymerase \(\beta\)-subunit (_rpoB_) [15; 16]. These approaches include gene amplification by PCR, sequencing, restriction fragment length polymorphism analysis, and concatenated sequence analysis [15; 16]. The phylogenetic reconstruction using the concatenated sequences of _lsp65_ and _rpoB_ genes supports the results obtained with 16S rRNA-based works [15; 16]. The entry in the genomics era and the availability of whole-genome sequencing (WGS) platforms and bioinformatics pipelines enabled the revision of the _Mycobacterium_ genus taxonomy. The first two WGS published works, based on a different number of genomes and methodologies, supported the same global conclusions: (1) a clear division between slow and fast growers and an intermediate position occupied by the \(M\). _terrae_ complex; (2) an ancestral position occupied by fast growers; (3) the members of \(M\). _chelone-abscessus_ complex pointed out as the most ancestral cluster [17]. The analyses based on WGS data helped to highlight potential conflicts in the taxonomy, suggested new group assignment, and detected inconsistencies in the labelling of already available genomes [17]. A more recent work conducted by [PERSON] and collaborators (2018) performed comparative genomics analyses with 150 _Mycobacterium_ genomes and gave a new perspective on the _Mycobacterium_ genus. The phylogenonic tree based on core proteins, the high degree of genome relatedness among members of each clade, and the identification of specific molecular signatures, jointly supported the existence of five phylogenetic clades that were named \"Tuberculosis-Simiae\", \"Terrae\", \"Triviale\", \"Fortuitum-Vaccae\", and \"Abscessus-Chelonae\" clades [13]. The Abscessus-Chelonae clade was proposed as the earliest branching lineage within the _Mycobacterium_ genus, as had been anticipated by previous works and supported the notion that slow growers evolved from fast growers [13]. Three of these clades comprise slow-growing species, while the other two clades mostly encompass the fast-growing [13]. This comparative genomics work allowed the identification of 172 molecular markers that are unique to all _Mycobacterium_ species or members of different clades, which in the future can be explored to improve diagnostic algorithms [13].