Conserved signature indels
Conserved signature inserts and deletions in protein sequences provide an important category of molecular markers for understanding phylogenetic relationships. CSIs, brought about by rare genetic changes, provide useful phylogenetic markers that are generally of defined size and they are flanked on both sides by conserved regions to ensure their reliability. While indels can be arbitrary inserts or deletions, CSIs are defined as only those protein indels that are present within conserved regions of the protein.
The CSIs that are restricted to a particular clade or group of species, generally provide good phylogenetic markers of common evolutionary descent. Due to the rarity and highly specific nature of such changes, it is less likely that they could arise independently by either convergent or parallel evolution and therefore are likely to represent synapomorphy. Other confounding factors such as differences in evolutionary rates at different sites or among different species also generally do not affect the interpretation of a CSI. By determining the presence or absence of CSIs in an out-group species, one can infer whether the ancestral form of the CSI was an insert or deletion and this can be used to develop a rooted phylogenetic relationship among organisms.
CSIs are discovered by looking for shared changes in a phylogenetic tree constructed from protein sequences. Most CSIs that have been identified have been found to have high predictive value upon addition of new sequences, retaining the specificity for the originally identified clades of species. They can be used to identify both known and even previously unknown species belonging to these groups in different environments. Compared to tree branching orders which can vary among methods, specific CSIs make for more concrete circumscriptions that are computationally cheaper to apply.
Types
Group-specific
Group-specific CSIs are commonly shared by different species belonging to a particular taxon but they are not present in other groups. These CSIs were most likely introduced in an ancestor of the group of species before the members of the taxa diverged. They provide molecular means for distinguishing members of a particular taxon from all other organisms.Figure 1 shows an example of 5aa CSI found in all species belonging to the taxon X. This is a distinctive characteristic of this taxon as it is not found in any other species. This signature was likely introduced in a common ancestor of the species from this taxon. Similarly other group-specific signatures could be shared by either A1 and A2 or B1 and B2, etc., or even by X1 and X2 or by X3 and X4, etc. The groups A, B, C, D and X, in this diagram could correspond to various bacterial or Eukaryotic phyla.
Group-specific CSIs have been used in the past to determine the phylogenetic relationship of a number of bacterial phyla and subgroups within it. For example a 3 amino acid insert was uniquely shared by members of the phylum Thermotogota in the essential 50S ribosomal protein L7/L12, within a highly conserved region. This is not present in any other bacteria species and could be used to characterize members of Thermotogota from all other bacteria. Group-specific CSIs were also used to characterize subgroups within Thermotogota.
Multi-group or mainline
Mainline CSIs are those in which a conserved insert or deletion is shared by several major phyla, but absent from other phyla.Figure 2 shows an example of 5aa CSI found in a conserved region that is commonly present in the species belonging to phyla X, Y and Z, but it is absent in other phyla. This signature indicates a specific relationship of taxa X, Y and Z and also A, B and C. Based upon the presence or absence of such an indel, in out-group species, it can be inferred whether the indel is an insert or a deletion, and which of these two groups A, B, C or X, Y, Z is ancestral.
Mainline CSIs have been used in the past to determine the phylogenetic relationship of a number of bacterial phyla. The large CSI of about 150-180 amino acids within a conserved region of Gyrase B, is commonly shared between various Pseudomonadota, Chlamydiota, Planctomycetota and Aquificota species. This CSI is absent in other ancestral bacterial phyla as well as Archaea. Similarly a large CSI of about 100 amino acids in RpoB homologs is present in various species belonging to Pseudomonadota, Bacteroidota, Chlorobiota, Chlamydiota, Planctomycetota, and Aquificota. This CSI is absent in other ancestral bacterial phyla as well as Archaea. In both cases one can infer that the groups lacking the CSI are ancestral.
Evolutionary studies based on CSIs
A key issue in bacterial phylogeny is to understand how different bacterial species are related to each other and their branching order from a common ancestor. Currently most phylogenetic trees are based on 16S rRNA or other genes/proteins. These trees are not always able to resolve key phylogenetic questions with a high degree of certainty. However in recent years the discovery and analyses of conserved indels in many universally distributed proteins have aided in this quest. The genetic events leading to them are postulated to have occurred at important evolutionary branch points and their species distribution patterns provide valuable information regarding the branching order and interrelationships among different bacterial phyla.Thermotogota
Recently the phylogenetic relationship of the group Thermotogota was characterized based on the CSI approach. Previously no biochemical or molecular markers were known that could clearly distinguish the species of this phylum from all other bacteria. More than 60 CSIs that were specific for the entire Thermotogota phylum or its different subgroups were discovered. Of these, 18 CSIs are uniquely present in various Thermotogota species and provide molecular markers for the phylum. Additionally there were many CSIs that were specific for various Thermotogota subgroups. Another 12 CSIs were specific for a clade consisting of various Thermotogota species except Tt. Lettingae. While 14 CSIs were specific for a clade consisting of the Fervidobacterium and Thermosipho genera and 18 CSIs were specific for the genus Thermosiphon.Lastly 16 CSIs were reported that were shared by either some or all Thermotogota species or some species from other taxa such as Archaea, Aquificota, Bacillota, Pseudomonadota, Deinococcota, Fusobacteriota, Dictyoglomota, Chloroflexota, and eukaryotes. The shared presence of some of these CSIs could be due to lateral gene transfer (LGT) between these groups. However the number of CSIs that are commonly shared with other taxa is much smaller than those that are specific for Thermotogota and they do not exhibit any specific pattern. Hence they have no significant effect on the distinction of Thermotogota.