1. 3- NON-RIBOSOMAL GENE RECONSTRUCTION  Core / auxiliary / strain specific genes  Housekeeping genes and accordance with global reconstruction  MLSA  Alignment (aminoacid / nucleotide  depends on the level of resolution)  Filtering alignments  Number of genes for a stable topology  Horizontal gene transfer  Tetranucleotide signatures

2. Housekeeping genes  alternative phylogenies Core genes with phylogenetic signal Auxiliary genes, not present in all populations with low phylogenetic signal Specific genes of a single strain without phylogenetic signal Lan and Reeves. 2000 TRENDS Microbiol 8: 396-401

3. Characteristics of a molecule as molecular clock  Universally present  only 34 orthologous universal genes (Huynen & Bork, PNAS, 1998. 95:5849-5856)  group specific (i.e. phylum, family, genus…) genes with phylogenetic signal can be used  functional constancy  sufficient sequence conservation for reconstruction purposes  sufficient sequence complexity for a good phylogenetic signal Ludwig and Schleifer. 2005 Microbial phylogeny and evolution (Sapp) 70-98. (Oxford University Press) Markers supporting global phylogenies  RNAr 16S  RNAr 23S  EF-Tu (some phyla are paraphyletic e.g. Actinobacteria y Streptomyces)  RNA polimerase rpoB (some phyla are paraphyletic e.g. Epsilonproteobacteria y resto Proteobacteria)  Heat Shock Hsp60 (Bacteria: GroEL, Archaea: Tf-55; some may be paraphyletic)  Aminoacyl tRNA sintetases Markers that do not support global phylogenies  ATPases  DNA girases  Hsp70  RecA Housekeeping genes  not all give the same resolution

4. PHYLOGENY BASED ON NON-RIBOSOMAL GENES: MLSA Stackebrandt et al., 2002, Int J Syst Evol Microbiol. 52:846-849 Gevers et al., 2005, Nature Rev. Microbiol. 3:733-739 MLSA (multilocus sequence analysis):  5-10 full/partial sequences  house keeping genes  primer design difficulties  biases in the selection of genes  time consuming  ↓↓ number for stable topology Amplify and sequence 5-10 housekeeping genes for each strain Concatenate gene sequences Reconstruct the phylogeny genAgenBgenCgenDgenEgenF Str. 1 Str. 2 Str. 3 Str. 4

5. The alignments of proteins of genes are less clear than rRNA Protein sequences vs. rRNA sequences  Codifying DNA harbors information in triplets (codons)  Degenerated code allows silent mutations (not much evolutionarily constraints)  For deep phylogenies, amino acid alignments give better resolution.  DNA phylogenies should only be done with close relative sequences  Generally shorter sequences (300-1000 residues) than rRNA

6. Removing hypervariable positions  reducing phylogenetic noise http://molevol.cmima.csic.es/castresana/Gblocks.html

7. Of all 22 analyzed genes:  57 % Bacteroidetes  27 % Chlorobi  18 % Chlorobi- Bacteroidetes One cannot rely on single gene reconstructions that may produce inconsistent results Single genes may lead to different topologies

8. The amount of genes in the concatenate influence the stability of the tree random selection among the 22 genes  checking branching robustness The bootstrap values improve with the increase of amount of genes in the analysis Below 8 genes one can obtain unstable topologies 12 genes gave the threshold for reliability For taxonomic purposes, 16S rRNA gene sequence analysis is the most parsimonious approach Sória-Carrasco et al., 2008, System Appl Microbiol. 30:171-179

9. MLSA: phylogenetic reconstructions MULTIPLE SEQUENCE ALIGNMENTS  sometimes have better resolution than the 16S rRNA gene  16S rRNA gene can have very low resolution Jiménez et al., 2013, System Appl Microbiol, 36: 383- 391

10. MULTIPLE SEQUENCE ALIGNMENTS (LARGE SETS)  r-MLST (ribosomal protein concatenates)  SPECL (single copy marker genes)  r-MLST (ribosomal protein concatenates)  http://pubmlst.org/rmlst/ http://pubmlst.org/rmlst/  Jolley et al., 2012, Microbiology 158:1005-15  53 ribosomal protein genes (rps genes)  SPECL  (http://vm-lux.embl.de/~mende/specI/)http://vm-lux.embl.de/~mende/specI/  Mende et al., Nat Methods, in revision  on 40 universal, single copy marker genes  Optimized cutoffs (96.5% nucleotide identity) MONOPHYLY: phylogenetic reconstructions (MLSA)

11. Loosing identity due to HGT Kunin et al. 2005. Genome Res. 15:954-959 Phylogenetic incongruences: HGT makes fuzzy the assignment of identities Masive HGT in the microbial world No tree of life is possible TWO SCHOOLS Phylogenetic incongruences: Can be explained by ► gene duplication (paralogy) and deletion (hidden paralogy) ► false orthology assignation ► alignments artifacts Orthology should be carefully checked Soria-Carrasco & Castresana, 2008. Mol. Biol. Evol. 25: 2319-2329 Kurland. 2005. Bioessays 27:741-747

12. pyrE aroA Some times no other explanation (either true or lack of information) Some times a loss of phylogenetic signal

13. Tetranucleotide variation: 4 4 = 256 TETRA:  Genomes have an oligonucleotide usage (not yet understood, related to codon usage)  Similar genomes might have similar usage  ALIGNMENT FREE PARAMETER  may be useful in deciding whether a group of strains deserve a species status Genome Signatures  G+C content ►dinucleotide ► not much informative  Codon usage ► trinucleotide ► more informative  Tetranucleotides (penta-, hexa-…) ►more information but more computing effort

14. Contigs can be ordered by means of their tetranucleotide similarity Teeling et al., 2004 Environ Microbiol. 6:938-947 High regression may indicate similar genome genetic codification Probably fragments of the same organism