Molecular Systematics EEB 5350 Spring 2019
2 Credits- half-semester module, 19 March-29 April 2016
Lectures: M & W 11:00-12:15 Bio-Pharm 3rd floor conference room.
M 2:30-4:30; Th 2:00-4:00 (Each lab session starts in 3rd floor conference room then moves to BioPharm 325).
Chris Simon, Biopharm 305D, 6-4640, <firstname.lastname@example.org>
Graduate Assistant: Katie Taylor, TLS 479, Katie.email@example.com, 6-5479
Readings: will be posted as PDF’s.
Optional reference books: 1) Paul Lewis's unpublished text; 2) The Phylogenetic Handbook (eds. Philippe Lemey, Marco Salemi, and Anne-Mieke Vandamme, 2010); 3) Inferring Phylogenies (Felsenstein 2004, Sinauer); 4) Molecular Evolution: A phylogenetic Approach (Page & Holmes 1998, Blackwell); 5) Molecular Systematics, 2nd ed. (Hillis, Moritz & Mable, eds. 1996, Sinauer) especially Chapter 11 by Swofford et al. on Phylogenetic Inference.
Lecture Goals: The course will focus on the basics of molecular systematics theory and practice from the point of view of the data. We will explore the ways in which an understanding of processes of evolution of molecular data can help in the construction of evolutionary trees. Lectures will examine some of the most serious problems in evolutionary tree construction: nucleotide bias, alignment, homoplasy, among-site rate variation, taxon sampling, long branches, big trees, heterogeneous rates of evolution among branches, covarion shifts.
Laboratory Goals: Labs will cover basic techniques in molecular systematics from DNA extraction to sequencing, alignment and cloning. This lab will be of interest to both experienced and novice molecular systematists because we will try newly developed kits/techniques and compare them to older ones and we will pursue a class project.
1) For each topic a bibliography will be provided including one focal paper for which the PDF will be posted. Each student will need to turn in a one-page summary of the importance of each focal paper (1 or occasionally 2 papers per week).
2) The week prior to the start of classes you will be given a checklist discussing practical considerations, organization and data checks for molecular systematics. In certain sections you are asked to answer questions and explain how these procedures are modified in your lab.
3) There will be a short "secondary structure alignment assignment" during the semester.
4) Each student will keep a laboratory notebook and hand-in data collected during the course in the form of an alignment and a nexus data file. Various exercises will be performed in laboratory and some will be finished outside of class. These are detailed in the laboratory syllabus.
5) For each Lab, one student will present a 10-15 minute Powerpoint presentation relating to techniques used in that day’s lab. Ursula will be available to advise you, but use web searches and try to do as much as possible on your own. These Powerpoint presentations will be posted on the class website so that in the future when you teach a molecular systematics class, they can be used as a starting point to revise and develop lectures of your own.
Final Exam: The final exam will be a take home test in which each student critiques the first draft of a paper submitted to Systematic Biology (submitted in the past but making comments as if it were submitted today). Each student will also compare the submitted version to the published version. The answer key will be the actual review containing reviewers, associate editors, and editor’s comments (with permission of authors, reviewers and editors) and a list of critical points that need to be considered by the authors.
Final Due Dates: Sunday 1st May: Lab project and notebook due. Take Home FINAL EXAM handed out. Sunday 6th May: Take home final due.
|Lecture 1. An introduction to looking at your data: How molecules evolve. Data checks
| Read Simon et al. 1994. 651-670 (up to the section that starts on the bottom of the second column). Too large to post, will be emailed to you. How Molecules Evolve & Model Choice Bibliography:
||LAB: Data checks at every step. Mechanics of Lab; Qiagen kit extractions. Qiagen kit extraction protocol:
|Wednesday Mar 21
||Lecture 2. How molecules evolve, continued.
| Read Sullivan and Swofford 2001 for Monday March 26th. Among Site Rate Variation Readings:
||Mini-presentation: DNA extraction (Katie) |
LAB: Qiagen extractions continued and plant extractions. CTAB plant extraction protocol:
| Lecture 3. ASRV, models of evolution, and the history of molecular systematics. Calculating the probability of substitution for sites, Fitch and Margoliash invariant sites models & negative binominal models,Weighting stems and loops.
||Mini-presentation: Primer Design ( Katie ) |
LAB: Explanation of class Tettigades project and Making gels, running extractions on gels, DNA extraction quantification, Troubleshooting and improving “universal” primers for COI.
|Wednesday Mar 28
|| Lecture 4. Correlated changes- should consider stems vs loops; How much to down weight and how to partition when weighting is problematic; Different methods for calculating & accommodating ASRV; For probability of substitution, using a tree is more effective than an alignment; The interaction of tree shape and ASRV; The two components of evolutionary trees; (equal weights aka evenly weighted; misnomer “unweighted” parsimony); Effects of Ignoring ASRV
||Read for Monday April 2nd, Bull et al. 1993. Classic paper from the Hillis Lab on partitioning and combing data, Bull et al. 1993.
||Mini-presentation:The polymerase chain reaction ( Zoe ) |
LAB: Setting-up PCR reactions. PCR protocol:
| Lecture 5. History of “combining data”, As many kinds of data as possible, non-specificity hypothesis, To combine or not to combine? That is the question. Lack of agreement among character subsets, Random error vs systematic error, Assumptions of combined analysis, Bull et al. vs. Chippindale & Wiens; ASRV &ALRV, Homothermia
|| Read and Summarize for Class by Monday April 9th Pagel, M. and A. Meade. 2004. Read and Summarize for Class on Wednesday, April 11th Kainer, D. and R. Lanfear. 2015. Combining Data, Partitioning, Species Trees readings , Pagel and Meade 2004 , Kanier and Landfear 2015.
|| Mini-presentation: Different methods for cleaning PCR products for sequencing reactions ( Tanner ) (Tanner's presentation is lost, but here is a presentation on the same topic from a prior year of the class, which might be a useful reference ) |
LAB: Running PCR products on gels, purifying PCR products with ExoSAP-IT, and setting-up sequencing reactions. PCR clean up protocol and Cycle sequencing protocol:
|Wednesday Apr 4
||Lecture 6. Tests for combining data; testing whether the same tree underlies each data partition. Partitioning; Choosing among models for pre-assigned partitions; Automated partition assignment and partition simplification; Model averaging and mixture models
||Mini-presentation: Numts ( Johnny ) |
LAB: Cleaning and putting samples on the ABI; Looking at sequences using Geneious. Sephadex cleaning protocol and loading ABI machine protocol:
|Lecture 7. What is a long branch?; The meaning of “basal”; Node density artifacts; Felsenstein 1978- when will parsimony be positively misleading?; Penny & Hendy 1989- long branch attraction; Huelsenbeck & Hillis simulations to explore tree space. Accuracy of different phylogenetic methods; Swofford et al. 2001. Bias in Phylogeny estimation due to long branches: Parsimony vs. likelihood in tree space; Remaining uncommitted
||Covarion, Heterotachy, Nucleotide Bias Readings
Read and summarize for Class (Due Monday, April 16) Gruenheit, Nicole, Peter J. Lockhart, Mike Steel, and William Martin. 2008.
|Mini-presentation: How Big Dye works, chromatograms, and troubleshooting(Diler )|
LAB: Viewing and interpreting sequencing results, setting up long range PCR. Long range PCR protocol
|Wednesday Apr 11
||Lecture 8. ALRV: heterotachy, covarion models;Among Lineage rate variation: Covarion evolution: codon models
||Mini-presentation: : Depositing sequences in GenBank ( Tanner ) ppt: submission protocol: example feature table: |
LAB: Running long rage PCR gel , cleaning long range PRC product, setting up 2nd short PCR, Make reagents for bead cleanup protocol. Protocol for making bead cleanup mix and testing:
|Lecture 9. Heterotachous evolution continued, Covarion Models, The Case for Stationary Genes, Mixture of Branch Lengths for building trees and studying selection. Covarion Mixture Models.
||Mini-presentation: Ancient DNA & Museum DNA protocols ( Zoe ) |
LAB: Running short rage PCR gel , cleaning PRC product, setting up sequencing reaction, test bead clean up method
|Wednesday Apr 18
||Lecture 10: Problems associated with nodal support
||Nodal Support Readings
Read and Summarize for Next week.... Monday 23 April 18.
Salichos L, Stamatakis A, Rokas A. 2014. Novel information theory-based measures for quantifying incongruence among phylogenetic trees. Molecular Biology and Evolution 31:1261-1271. (No need to summarize the derivation, just the introduction and the applications).
|Mini-presentation: RNA: extraction and what it can be used for ( Diler ) |
LAB: Cleaning and putting samples on the ABI, and starting RNA isolation with Trizol. Trizol RNA from tissue protocol:
|Lecture 11) Nodal support continued. Spectral analysis, Internode certainty, SplitsTrees. Misc. topics: Big Trees; more taxa or more sequences.
||LAB: Finish RNA isolation, Compare sequencing results from long range and typical PCR
|Wednesday Apr 25
||Lecture 12: Secondary structure & alignment. Molecular Clocks
|| Secondary structure assignment and templates for Magicicada and conserved motif template .
Hickson et al. 1996 Conserved sequence motifs, alignment, and secondary structure for the third domain of animal 12s rRNA.
Molecular clock readings:
Structure and alignment readings:
Mini-presentation: Gel electrophoresis ( Johnny )
LAB: Katie presents on Next Generation Sequencing and Applications
Sunday April 29th
| Lab notebook due. Take home final handed out.
|Final Exam due, emailed to Katie who will transmit the anonymous papers to Chris along with a list of pseudonyms
Final Exam Files Reviewers instructions: