Teaching Genomics > Inquiry-based Integrated Instructional Units > Phylogenetic analysis of the bony fishes: Morphological and mtDNA sequence comparisons

Phylogenetic analysis of the bony fishes: Morphological and mtDNA sequence comparisons

Erica Crespi
Vassar College
Summary

This three-four week laboratory/lecture module is designed to teach students basic concepts, quandaries, and methods of phylogenetics using interdisciplinary techniques within an inquiry-based curriculum. The overarching question the students are challenged with is, what are the phylogenetic relationships among major groups of bony fishes, a group whose evolutionary relationships remain unresolved. Students generate both molecular and morphological datasets on a group of fishes that were purchased from a local fish market. With the collected data, in addition to previously published molecular sequences obtained from Genbank, students generate phylogenetic hypotheses and are challenged to reconcile differences in the hypotheses resulting from the different datasets.


Learning Goals

Context for Use

This particular module was designed within an intermediate-level Comparative Anatomy course to show that a major use of comparative morphological information is to advance the understanding of evolutionary relationships. However, it can easily fit into an Evolution course or a taxon-specific course (e.g., Ichthyology, Herpetology, Botany); the molecular aspects can fit into a Genetics or Molecular Biology course, or even an introductory-level inquiry-based course. This lab is adaptable to any plant or animal or fungus, depending on the interests of the instructor or the resources available at the institution (e.g., specimen collections). Because fresh tissue was obtained from fish market animals, an animal care and use protocol was not needed; however, if live animals are obtained and frozen for this exercise, an IACUC protocol would have been needed. Because tissues can be frozen and maintained for subsequent courses well into the future, the first IACUC may be all that is needed.

Description and Teaching Materials

The three-week module involves a mix of lecture, wet-lab, and computer laboratory components designed to walk the students through the process addressing a phyolgenetic question (see Schedule). The module starts with a hands-on exercise to demonstrate many basic principles of morphological systematics, supplemented by a series of readings about the basic principles of phylogenetics. The module ends with their phylogenetic analysis of data generated during the module, and a critique of a phylogenetic study they have never seen before.

Introduction: The first exercise of the module involves an activity that asks students to derive characters to describe a set of diverse sea shells (often nails or screws are used to completely detach the students biological biases when deriving characters). This teaches them to be observant and discriminating when describing morphological diversity, and introduces them to the idea of "evolutionary distance," which students calculate from their derived characters (see Shell Activity). This can be done during a lab period or in two lecture periods.

Student Reading: The readings involve two chapters of an evolutionary biology textbook to cover Molecular Evolution and Reconstructing Evolutionary Trees (I used Freeman and Herron, Evolutionary Analysis, Prentice Hall), and a series of primary literature articles that were dispersed throughout the module (see reading list below). These articles were chosen to not only validate and explain methodology and techniques we were using, but also to serve as examples of new and old phylogenetic questions that are addressed in the field.

Guided Discovery-wet lab: Students first conduct laboratory techniques to generate the DNA sequence database because of the extensive amount of time that is needed to do this. Students are presented with a collection of 15 fishes from which students had to extract genomic DNA. This array is chosen to represent a diversity of bony fish taxa, but is also constrained to what is available in the fish market. Students work in pairs, and each group extracts DNA from each fish. After the success of the extraction is verified via gel electrophoresis (conducted by instructor), students conduct PCR on each sample to amplify a fragment of the 28S gene. PCR products are run on a gel and purified (conducted by instructor due to time constraints, but could have been done easily by the students in an additional period). These fragments are sequenced by either sending them to an outside core facility or by using an automated sequencer if available. While students wait for the sequences (if sent out, can take up to 4 days), students conduct the morphological analysis of the same fishes, where students work in groups of 3-4 to derive 20-30 characters that would be useful for phylogenetic analysis.

After each laboratory period, at least one intervening lecture period is needed to go over results, discuss important biological concepts of the technique, and introduce the topics and techniques of the next laboratory. Reading assignments given along the way aid in preparing students for each step of the study and give them the background they need for discussion.

Guided Discovery-computer lab: With the morphological and 28S rRNA sequence datasets constructed, students are brought to a computer laboratory facility to learn how to align sequences and construct phylogenetic trees using two basic algorithms (neighbor joining and parsimony) using the MEGA v.3.1 for Windows program (see full reference below). After this initial lesson on how to use the software, students generate phylogenetic trees based on the matrix of morphological characters they derived, and compare the results to the trees derived from their molecular sequence data. Finally, students are asked to collect sequences from a different gene (Cytb) of bony fishes from Genbank, create a dataset, align sequences, and generate another phylogenetic hypothesis based on this gene sequence. Finally, they were asked to critically assess the merit of the hypotheses generated by each dataset based on the quality of the data, the numbers of phylogenetic characters used, the kinds of characters used, the taxonomic sampling included in the analysis, etc. We spent two periods in the computer lab learning how to manage the data and do the analyses; students were then assigned homework to reinforce the use of the tools outside of class time, from which they had to bring in results to class for discussion the following scheduled period. Phylogenetics Module Schedule (Microsoft Word 67kB Jul20 07)
Shell Activity (Microsoft Word 24kB Jul20 07)
Final Assessment Activity (Microsoft Word 40kB Jul20 07)
Sample list of fishes used (Microsoft Word 45kB Jul20 07)
Sample 28S rRNA sequences obtained by students (Microsoft Word 10kB Jul20 07)

Teaching Notes and Tips

Integrating the lab experience: The module is designed to bring the students along on the journey of answering the assigned phylogenetic question, which brought them to the lecture room, wet laboratory, and the computer laboratory as the data are collected, assessed, and analyzed. Therefore, this module truly integrates lecture and laboratory experiences, and each scheduled meeting time marks the next step of the journey.

Scheduling each step depends on the format of the class. Because we met four times a week (three 50-min periods and 1 four-hour lab), the students were able to see the results of their work almost immediately, and the ability to take the next step in the analysis based specifically on their previous results creates a tremendous sense of excitement and ownership of the project. Although it can fit into any course schedule, this module is easier to conduct if taught during two 2-hour sessions a week.

Logistically, the integration between lecture and laboratory is best done when the lecture/discussion room, wet laboratory, and computer laboratory are in close proximity, e.g., the design of the space is based on integrated use! Unfortunately, when I taught this module, the lecture room was on the other side of the building from the lab (which was not designed to allow for lecturing in that room), and the computer lab was in a different building. This led to many students not remembering where they should be from class to class.

With the availability of kits (e.g., Quigen), the molecular biology materials are well-adapted for student use with a very high rate of success, and instructions could be easily derived from the manufacturers protocols. In the first trial of this module, all students successfully extracted genomic DNA from at least one of their samples, and almost all had PCR products. It was important to have redundancy: if all students do all samples, you are bound to have at least a few quality products from each individual taxon for sequencing. Also, all have the experience of doing the technique.

It would have been nice to have our own DNA sequencer for this assignment, as it would have added an additional component to the module. Because we had to ship our samples to an outside core facility (UC Berkeley, or any large university, will analyze shipped sampled with a PO from your department), we had to wait 3-4 days for the results-although it's nice because they send the data electronically. I showed the students the chromatograms that UC Berkeley sent us so they can see how the sequences were generated-and more importantly- how to assess the quality of each sequence for editing and alignment purposes.

The size of the class matters: I did this module with 17 students in one class-and this was near the upper bound of tractability for the instructor.

All readings are geared toward the context of the module and the course. In this case, all readings fit within the Comparative Anatomy course as taught (e.g., evolutionary relationships among protochordates, cartilaginous fishes, and vertebrates; fish-related morphology and phylogenetics).

Assessment

Student understanding of the concepts and techniques is assessed by:
1. Observation of participation in discussion and laboratory
2. Students had to turn in results from the initial shell exercise, mapping morphological traits on trees to determine homoplasy, alignments, and phylogenetic tree construction during the following class for review and discussion. None of these assignments were formerly graded, but they were assessed and comments were returned to the students.
3. Final Assessment Assignment (see Final Assignment): a three-day, open-book/note assignment they were given to turn in at the end of the module that involved:
a. Short answer questions of basic biological concepts covered in module
b. Writing a Materials and Methods section based on their study
c. Critique of a phylogenetic study they never saw before
d. Using MEGA to construct phylogenetic hypotheses from Cytb gene sequence from Genbank, and comparing it to the trees generated from 28S gene sequence they generated, then assessing why the two datasets generated different hypotheses
*One of the short answer questions directly asks students to describe how they perceived phylogenetics to be an integrative science, and asked which aspects of the module were successful and which should be improved.

References and Resources

Molecular Evolutionary Genetics Analysis (MEGA) (available for Windows, DOS, MAC, Linux)-very easy to use for undergraduates although some downloads contained bugs.
http://www.megasoftware.net/m_con_select.html

Freeman S, and Herron JC. Evolutionary Analysis 2nd Ed. Prentice Hall

Hillis DM. 1987. Molecular vs. morphological approaches to systematics. Ann. Rev. Ecol. Syst. 18:23-42.
*review article that clearly overviews the use of morphology and molecular data in systematics, and outlines pros and cons and assumptions of using each technique, and how to reconcile differences in hypotheses based on different sources of data

Mallatt J, Winchell CJ. 2007. Ribosomal RNA genes and deuterostone phylogeny revisited: More cyclostomes, elasmobranches, reptiles, and a brittle star. Mol. Phylogenet. Evol. 43: 1005-1022.
Zardoya R, and Meyer A. 1996. Evolutionary relationships of the coelacanth, lungfish, and tetrapods based on the 28S ribosomal RNA gene. PNAS 93:5449-5454.
*examples of studies 12S rRNA gene in the phylogenetic resolution of deep-rooted lineages; these were used as we were making a progression in anatomy/evolution from protochordates and chondrichthyes to bony fishes.

Teletchea F, Laudet V, Hanni C. 2006. Phylogeny of the Gadidae (sensu Svetovidov, 1948) based on their morphology and two mitochondrial genes. Mol. Phylogenet. Evol. 38:189-199.
*example of morphology characters in fishes and combination of molecular and morphological data

Tang Q, Liu, H, Mayden R, Xiong B. 2006. Comparison of evolutionary rates in the mitochondrial DNA cytochrome b gene and control region and their implications for phylogeny of the Cobitoidea (Teleostei: Cypriniformes). Mol. Phylogenet. Evol. 39:347-357.
*example of study based on cytb and used for discussion of molecular clocks and phylogenetics

Venkatesh B. 2003. Evolution and diversity of fish genomes. Curr. Opin. Genet. Dev. 13:588-592.
Mank J, Avise JC. 2006. Cladogenetic correlates of genomic expansions in the recent evolution of actinopterygiian fishes. Proc. R. Soc. B. 273:33-38.
*articles to extend genomic-level studies of fish diversity

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