37628 - Molecular Phylogenesis Laboratory

Learning outcomes

At the end of the course the student will have detailed knowledge about the main laboratory techniques for the study of molecular phylogeny at different taxonomic levels. In particular, the student will be able to utilize the following: total DNA isolation, electrophoresis, PCR amplification, cloning, genomic restriction, sequencing, bioinformatic analyses of molecular data.

Course contents

Definition of taxonomy, systematics and phylogeny. Taxonomic characters, homology, analogy. Molecular phylogenetics: advantages & disadvantages.

Principles of molecular evolution. Mutation: classification of mutations, point mutations, segmental mutations. Standing Genetic Variation: gene diversity, nucleotide diversity, structural variation. Changes in Allele Frequencies: Selection (co-dominance, dominance and recessiveness, overdominance and underdominance); Random Genetic Drift; Census Population Size and Effective Population Size; Coalescent theory. Gene/Allele Substitution: fixation probability; fixation time; rate of gene substitution; mutational meltdown; nearly-neutral mutations. Fisher's Theorem and the fitness landscape. Linkage Disequilibrium: hitchhiking and selective sweep, hard sweeps and soft sweeps, molecular signatures of selective sweeps, background selection. Epistasis. The distribution of fitness effects.

Patterns of molecular evolution. Rates of nucleotide substitution: rates of substitution in protein-coding sequences, rates of substitution in noncoding regions. Causes of variation in substitution rates: functional constraints, synonymous vs nonsynonymous rates, variation among different gene regions, variation among genes, relaxation of selection, patterns of amino acid replacement. Nonrandom usage of synonymous codons, translational efficiency and translational accuracy. Causes of variation in substitution rates among evolutionary lineages: the DNA repair hypothesis, the generation-time hypothesis, the metabolic rate hypothesis, the varying-selection hypothesis; living fossils.

Choice of molecular markers, homoplasy. From the coalescence theory to phylogenetics: the "species tree - gene tree" problem. Next Generation Sequencing in phylogenetics (phylogenomics).

Methods for studying molecular data. Alignment of protein-coding and non-coding sequences (progressive alignment and iterative approaches); structural alignments. Concatenated datasets; phylogenomics matrices. Observed and expected divergence. Multiple substitutions and substitution models for nucleotides and amino acids. Among-site variation and proportion of invariants. Phylogenetic reconstructions: algorithmic approach (distance-based; UPGMA, Neighbor-Joining) and tree search methods (character-based). Optimality criteria: Maximum Parsimony and Maximum Likelihood trees. Nodal support: resampling (bootstrap, jackknife) and character-based methods (Bremer support). Bayesian Inference and posterior probability. Tree search using the Markov chain Monte Carlo (MCMC) method. Strict, relaxed and local molecular clock. Chronograms and tree calibration using fossil records, biogeography and secondary calibrations. Validation, sensitivity analyses and biases in phylogenetics: nucleotide compositional bias, signal saturation, long branch attraction, incomplete lineage sorting.

General principles of biological wet-lab techniques/protocols.

Practice in molecular biology lab: total DNA isolation, PCR amplification, cloning (insert ligation into plasmid vector; bacterial competent cells transformation), PCR amplification of recombinant bacterial colonies; sequencing.

Practice in data analysis lab: analysis and editing of DNA automatic sequencer chromatograms; NCBI Genbank database, Blast search; multiple sequence alignment (ClustalW, Muscle); genetic distances and choice of the best substitution model. Phylogenetic reconstruction: Neighbor-Joining, Maximum parsimony, Maximum Likelihood and Bayesian inference. Nodal support using bootstrap method. Calibration and chronograms using bayesian methods.

Readings/Bibliography

Suggested textbooks are:
Dan Graur. Molecular and Genome Evolution. 2016, Sinauer Associates.
Naruya Saitou. Introduction to Evolutionary Genomics. 2013, Springer.
Philippe Lemey (ed.) . Phylogenetic Handbook. 2009, Cambridge University Press.
Barry G. Hall. Phylogenetic Trees Made Easy: A How-To Manual. 2011, Sinauer Associates.

Moreover, further study material will be provided by the teacher.

Teaching methods

.ppt presentation; molecular biology laboratory, informatic laboratory.

Assessment methods

The exam at the end of the course aims to assess the achievement of the following learning objectives:

Deep knowledge about theories and processes of molecular evolution and their study

Deep knowledge about wet-lab techniques/protocols for the analysis of molecular markers for phylogenetic studies at different taxonomic levels

Deep knowledge about data analysis methods for molecular phylogenetics

Ability to carry out a proper interpretation of obtained phylogenetic inferences.

The assessment will take place through a written examination on main course topics and an oral discussion focused on specific topics.

Teaching tools

.ppt presentation; molecular biology laboratory, informatic laboratory.

Office hours

See the website of Andrea Luchetti

See the website of Fausto Tinti

See the website of Fabrizio Ghiselli