Conserved Signature Indels (Csis) Are an Important Category of Shared Derived Molecular Markers

Defending Darwin: Do Microbial Genomes Support or Challenge the Darwinian Mode of Evolution?

Radhey S. Gupta McMaster University, Canada © by author

ECCMID 2013, ESCMID Online Lecture LibraryBerlin, April 28, 2013

Current State of Microbiology

16S rRNA tree • 16S rRNA trees provide the current framework for understanding microbiology. • About 30 main groups/phyla within Bacteria have been identified. These groups are also seen in most other phylogenetic trees. • Interrelationships among different groups are not resolved. • Most higher taxa are distinguished only in phylogenetic (or statistical) terms. In most cases, no biochemical or molecular markers are known that are specific for these groups. © by author Ludwig and Klenk, Bergey’s Manual of Systematic Bacteriology (2005)

Some Important Issues in Microbiology Requiring Understanding

• ESCMIDAbility to distinguish different Online groups of microorganisms Lecture in more definitive Library molecular terms • Development of a reliable classification system reflecting their evolutionary relationships. • Understanding what unique molecular, biochemical or other properties distinguish different groups of microbes from each other. Are Genome Sequences helpful in Understanding Microbial Evolution?

Widely held view

Doolittle, W.F. (1999) Science 284,2124 Koonin and Wolf (2012) Front. Cell. Infect. Microbiol. 284,2124 © by author Prokaryotic genomes have acquired extensive information from sources other than through vertical descent. Only a vague outline of the main domains/phyla (STOL) can be inferred. ESCMID OnlineA treeLecture-like evolutionary relationship Library (i.e. Darwinian mode of evolution) is very weakly supported or not at all supported.

Are any of the Critical Issues Related to Microbial Phylogeny/Taxonomy better understood? Merhej and Raoult (2012) Front. Cell. Infect. Microbiol. 2: 113 Main Approaches Leading to These Inferences

• Phylogenetic tree constructions - incongruity in the observed trees. • Analyses of closest Blast hits

Both of these approaches are inaccurate and inferences based upon them are affected by numerous variables.

“All methods of phylogenetic tree inference are imperfect. They perform particularly badly when dealing with lineages that evolve at different rates and … where different positions change at vastly different rates. Unfortunately, these are real- © bynd author world conditions.” (Carl Woese, In “The Prokaryotes”, 2 ed.)

Results of Blast analyses are greatly influenced by relative representation of different taxa in the dataset. Their interpretation requires an understanding of the interrelationshipsESCMID among different Online taxa, which is lackingLecture at present. Library Main Arguments Against Darwin’s Theory of Evolution

• The genes from exogenous sources (LGTs, viruses, gene gain or loss, etc.) are the major sources of genetic variation; • The changes occurring by these means do not constitute infinitesimal changes. • Evolutionary relationships indicated by such genes do not support nor can they be depicted, by a tree-like branching pattern.

• The main focus of Darwin’s theory was on macroscopic characteristics and not on© Genome by Evolutionauthor – these two are very different things. • Exogenous information, in most cases, in the short term, only ESCMIDaffects genome evolution; Online In terms ofLecture species evolution, Library most of these changes generally have very little or no effect. Darwin’s Views on Determining Evolutionary Relationships Among Organisms

Genetic basis of heredity, or different mechanisms by which genetic variation is produced, was not known to Darwin……..

“We can understand, the very important distinction between real affinities and analogical (convergent evolution) or adaptive resemblances (changes produced due to external factors) …The analogical or adaptive characters, although of the utmost importance to the welfare of the being, are almost valueless to the systematist.” (Charles Darwin, The Origin of Species, Chapter XIII)

Darwin also clearly specified the characters© by that author are most useful for genealogical studies.

“The characters which show true affinity between any two or more species, are those which have been inherited from a common parent” (i.e.ESCMID Shared Derived Characters Online or Synapomorphies Lecture) Library “…. we have to discover the lines of descent by the most permanent characters, however slight their vital importance may be.” (Charles Darwin, The Origin of Species ,Chapter XIII) Genome Sequences Provide the Necessary Resource for Discovery of Shared Derived Molecular Markers

Two kinds of shared derived >3000 prokaryotic Molecular Markers that are genomes completely specific for different groups of sequenced organisms.

Conserved Signature Conserved Signature ESCMID Onlineindels (CSIs) Lecture in ProteinsLibrary (CSPs) specific genes/proteins for Different lineages Conserved Signature Indels (CSIs) are an Important category of Shared Derived Molecular Markers

www.bacterialphylogeny.com

• Useful CSIs are flanked on both© sides by with authorconserved regions. • Present at different phylogenetic depths, enabling demarcation of clades of various taxonomic ranks. • Based upon their presence or absence, a rooted evolutionary relationship can be ESCMIDdetermined independent Online of phylogenetic trees.Lecture Library • CSIs are also useful for identifying cases of LGTs. Conserved Signature Proteins (CSPs)

The genes-proteins (CSPs) that are uniquely found in different lineages provide another important class of molecular markers for evolutionary studies. © by author ESCMID Online Lecture Library Prokaryotic Phyla that can be Clearly Distinguished Based upon Multiple Unique CSIs and CSPs

Actinobacteria Chlorobi Thermotogae

Chlamydiae Aquificae Synergistetes

Proteobacteria (Alpha, Deinococcus- Firmicutes Beta, Gamma and Thermus Epsilon) Betaproteobacteria Bacteroidetes Cyanobacteria © by author Epsilonproteobacteria Chloroflexi Crenarchaeota

GammaproteobacteriaESCMID Online Spirochaetes Lecture Archaea Library Some Examples of the Applications of CSIs and CSPs for Determining Evolutionary Relationships Among Prokaryotic Phyla © by author ESCMID Online Lecture Library Molecular Signatures for the Chlamydiae

Intracellular parasites; Important pathogens of humans and animals; Evolutionary relationships and taxonomy not resolved based upon 16S rRNA and other gene/protein sequences.

Are CSIs and CSPs useful in resolving these relationships?

Simkania negevensis Protochlamydia amoebophila

>50 CSPs Parachlamydia acanthamoebae and 4 CSIs Waddlia chondrophila Chlamydiales order

69 CSPs + Chlamydophila felis >12 CSIs 20 CSPs + Chlamydophila caviae >5CSIs Chlamydophila psittaci Chlamydophila Chlamydophila abortus © by authorChlamydophila pneumoniae

Chlamydia trachomatis Chlamydia ESCMID100 CSPs + >10CSIs Online LectureChlamydia muridarum Library Chlamydiaceae 20 CSPs + >5CSIs Family

Griffiths, Ventresca and Gupta (2006) BMC Genomics, Jan 25, 7:14; Gupta and Griffiths (2006) Trends Microbiol. 27: 527-535; Gupta et al. (2012) Front. Micobiol. 3: 327 Conserved Indel Specific for the Chlamydiae

Gyrase B

Proteobacteria

MurA EF-P MgtE TrmD Chlamydiae Ctprotease GlmU RpoA Tgt LysRS Other Gram -ve © by author EF-Tu bacteria ESCMID Online Lecture Library Gram-positive bacteria

Griffiths, E. and Gupta, R. S. Microbiology (2002) 148, 2541-2549. Griffiths, Petrich and Gupta, Microbiology (2005) 151, 2647-2657 Molecular Signatures for Actinobacteria

• One of the largest phyla within bacteria; Highly diverse in morphology and physiology. • Harbors many clinically, industrially and environmentally important members. • Recognized solely on the basis of their branching in the 16S rRNA trees.

©A largeby number author of CSIs and CSPs that are specific for either all Actinobacteria or its different taxonomic clades have been identified. ESCMID Online Lecture Library

Gao and Gupta (2005) IJSEM, 55: 240-2412; Gao et al. (2006) Anto. Von Leeuwenhoek 90: 69-91; Gao and Gupta (2012) Microbiol. Mol. Biol. Rev. 76: 66-112 Bergey’s Manual, 2005, Example of a CSI that is Specific for Actinobacteria

Cox1

Proteobacteria

Originally <20 Actino genomes; Other Gram(-) Bacteria 2 aa deletion present in all actinobacteria (>250) except one of the deepest branching species.

Firmicutes >2000 Non-Actino homologs 2-aa deletion : 0/>2000 © by author Many other CSIs in widely Actinobacteria distributed proteins are also ESCMID Online Lecture Libraryspecific for Actinobacteria

Gao and Gupta (2005) IJSEM, 55: 240-2412; Gao and Gupta (2012) Microbiol. Mol. Biol. Rev. 76: 66-112 Signature Proteins that are Specific for Actinobacteria

Gao and Gupta (2006) Anto. von Leeuwenhoek 90: 69-91; Microbiol.Mol. Biol. Rev. (2012) 76:66-112 Phylogenetic Framework and Molecular Signatures for the Main Clades of Actinobacteria

Gao and Gupta (2012) Microbiol. Mol. Biol. Rev. 76: 66-112. The Use of CSIs and CSPs for Detecting Lateral Gene Transfers (LGTs)

Between Actinobacteria and Chlamydiae Chlamydiae branches GlyA Griffiths and Gupta (2006) J. Mol. Evol. 63: 283-296. within Actinobacteria

© by author ESCMID Online Lecture Library “the very same characters are analogical when one class or order is compared with another, but give true affinities when the members of the same class or order are compared one with another” “In comparing one group with a distinct group, we summarily reject analogical or adaptive characters, and yet use these same characters within the limits of the same group”. (Charles Darwin, The Origin of Species ,Chapter XIII) Blast Analyses and Lateral Gene Transfers

Thermotogae are hyperthermophilic bacteria; Analysis of top Blast hits on proteins from their genomes has led to inference that they have undergone extensive LGTs.

Evidence for lateral gene transfer between On the chimeric nature, thermophilic origin, and Archaea and Bacteria from genome sequence phylogenetic placement of the Thermotogales of Thermotoga maritima Zhaxybayeva et al. (2007) PNAS 106: 5865 Nelson et al. (1999) Nature 399: 323-329

Thermotogae specific Thermotogae 24.00% specific 13.60% © by author ESCMID Online Lecture Library Molecular Signatures for Thermotogae

Systematic studies were undertaken to identify CSIs that are specific for the Thermotogae species or shared with other organisms. More than 80% of the identified CSIs correspond to different clades of Thermotogae species.

TreeESCMID based upon concatenated Online Lecture Library sequences for 15 conserved proteins

Gupta and Bhandari (2011) ANTO, 100: 1-34; Bhandari et al. (2012) Front. Cell. Infect. Microbiol. 2; Art. 98; Bhandari and Gupta, R. S. (2013) The Prokaryotes, vol. 4 (in press) Molecular Signatures for Thermotogae

• About 15-20% of the observed CSIs are also present in a limited number of species from other phyla, which may be due to LGTs or convergent evolution. • However, no specific relationship of Thermotogae to Firmicutes or any other bacterial phyla or Archaea could be inferred based upon these CSIs.

Gupta and Bhandari (2011) ANTO, 100: 1-34; Bhandari et al. (2012) Front. Cell. Infect. Microbiol. 2; Art. 98; Bhandari and Gupta, R. S. (2013) The Prokaryotes, vol. 4 (in press) Do These Results Support or Challenge the Darwinian Model of Evolution?

The results obtained are remarkably similar to those Darwin would have predicted.

Vertically inherited Novel genes from different sources

© by author The relationships deduced by different molecular signatures are also strongly supported by phylogenetic analyses; ESCMID“The arrangement of the groups Online within each class, inLecture due subordination and relation Library to the other groups, must be strictly genealogical” “When several characters, let them be ever so trifling, occur together throughout a large group of beings having different habits, we may feel almost sure, on the theory of descent, that these characters have been inherited from a common ancestor. And we know that such correlated or aggregated characters have special value in classification.” (Charles Darwin, The Origin of Species ,Chapter XIII) Summary

• Large numbers of molecular signatures (synapomorphies) have been identified that are specific for different prokaryotic taxa at multiple phylogenetic depths.

• The clades identified by these signatures are also supported by phylogenetic trees. Few signatures supporting other alternate relationships were observed.

• The hierarchical arrangements of the prokaryotic taxa, as indicated by these signatures is exactly as would be predicted/expected based on Darwin’s theory of evolution.

• Although LGTs and other means of gene acquisition are important evolutionarily mechanisms for generating variations, in most cases they do not disguise the strong and© consistent by author signal resulting from vertical descent. • The identified molecular signatures exhibit high degree of predictive ability. Sequence data from new genomes strongly support the specificities of previously identified CSIs and CSPs. These molecular markers also provide ESCMIDpowerful new means Online for diagnostic, systematicLecture and biochemical Library studies. Is Darwin’s Theory Challenged by the Genetic Revolution?

Pro-View Con-View

Microbial genomes are a Genome sequences are composite of information enabling discovery of from different sources. numerous robust molecular markers for different Very weak or no useful prokaryotic taxa leading to signal can be detected to a reliable understanding of support the Darwin’s the microbial evolution. The theory of Evolution. species distribution of these markers is entirely in accordance with the © by author Darwin’s theory. Darwin had the foresight to recognize different sources of variation and their ESCMID Online Lecture Libraryrelative importance for understanding the genealogical relationships. Acknowledgements

My Group at Collaborators: McMaster Dr. Herb Schellhorn Emma Griffiths Shirley Wong McMaster Univ. Beile Gao Vaibhav Bhandari Dr. Haroun Shah Hafiz-Sohail Naushad Health Protection Agency, Nadia Ahmod London, UK. Mobolaji Adeolou Pranay Chandra Ali Shami © by author ESCMID Online Lecture Library