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

Erica Crespi
Vassar College
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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 (whole) that were purchased from a local fish market that span many orders of fish within the Actinopterygii (the main group of fishes), but also representat other closely related groups. We also happened to have a frozen gar, bowfin, and eel that were really good outgroups. 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

  • Students will learn the basic morphology of fishes (or any taxonomic group) and will be able to quantitatively derive characters from a diversity of fishes that are useful (and many that are not useful!) for phylogenetic analysis.
  • Students will apply their understanding of basic molecular processes that generate genetic variation underlying evolutionary processes to select gene regions for phylogenetic analysis; students will generate hypotheses about the evolutionary history of organisms based on divergence in DNA sequence data.
  • Students will understand basic concepts in systematics and phylogenetics, including homoplasy (convergent evolution and reversals), molecular clocks, genome diversity and basic assumptions, attributes, and analytical methods (parsimony, genetic! distance matrices, character mapping) used in morphological/molecular systematics.
  • Students will be involved in discussion, wet lab, computer lab, and lecture to experience how integration of different biological fields is needed to answer a single question and gain appreciation for interdisciplinary approaches to science.
  • Students will learn bioinformatics skills using computer-based resources and tools to manipulate and analyze data, including BLAST searches, sequence alignment, and tree-building programs for phylogenetic analysis (MEGA and MacClade).
  • Students will gain an appreciation for the subjectivity of science and become more comfortable with ambiguity in interpretation of results, i.e., there is almost never "one right answer."

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. Students have taken our introductory courses, and have some experience with GenBank when they come in. However, this module or something like it can easily fit into an Evolution course, a taxon-specific course (e.g., Ichthyology, Herpetology, Botany), or a biodiversity course; and parts of it can be omitted depending on the purpose of the course (see Teaching Tips). 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.

It is important to have class sizes at 20 students or less if possible, as there is a lot to keep track of as the module progresses. I have done this course with one lecture-one with one lab section for a class of 19 in one year, and one lecture with two lab sections to accommodate a class of 32 the next time I taught it.

Description and Teaching Materials

This four 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. 28s rRNA fragment ( 81kB Jun21 09)

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:

Making phylogenetic trees based on morphological characters using MacClade software: After the shell activity, students create phylogenetic trees using this software, which allows students to enter a character matrix, and allows them to find the "most parsimonious" phylogenetic hypothesis (tree with least number of "evolutionary steps"). This program also allows you to look at individual traits and see where character states evolved so students can visualize homoplasy (i.e., multiple evolutions), and identify traits that are suitable for phylogenetic analysis (those that show no homoplasy), and those that are not (most of the traits they chose to use). One 50-minute "lecture" period was used to introduce students to the program, and they were allowed to complete their first tree for homework. This program is used again when they create a phylogenetic hypothesis based on morphological characters they derive from the ray-finned fishes.

Making phylognetic trees based on genetic data using MEGA software: With 28S rRNA sequence dataset constructed, students are brought to a computer laboratory facility during a lab period to learn how to align sequences and construct phylogenetic trees using two basic algorithms (neighbor joining (a genetic distance method) and parsimony) using the MEGA v.4.0 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.

Performance Assessment: Students are asked to collect sequences from a different gene (Cytb) of bony fishes from Genbank on their own to create a new dataset, align sequences, and generate another phylogenetic hypothesis based on this gene sequence. Some classroom time is spent demonstrating how students can access these sequences through Genbank.

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., Qiagen), 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: 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 and are not overwhelmed by having many samples to do.

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 (MacLab based in San Francisco, UC Berkeley, or any large university, will analyze shipped sampled with a PO from your department or credit card). With MacLab we had to wait only 2 days for the results-although it's nice because they send the data electronically and you create chromatograms right on your computer. If you are in a small class, students can do this too (software needed for this is free and available through any of these core facility websites). I showed the students the chromatograms a few sequences 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).


Student understanding of the concepts, performance of techniques, and attitudes were assessed by the following methods. These assessment measurement tools have not been validated by education assessment specialists, but they should serve as examples to give instructors a place to start when thinking about a portfolio of assessement objectives and tools that can be adapted for any version of this module that is used in the classroom.

A. Knowledge assessment
- Take-home assignment given at the end of the module, with short answer questions about basic concepts in systematics, phylogenetics, morphological vs. molecular characters (pros and cons), molecular evolution, neutral theory and molecular clocks, homology and analogy, homoplasy, and basic algorithms used in phylogenetics (distance and parsimony). Module Take-home assignment (Microsoft Word 54kB Jun23 09)
- Question asked on the final exam at the end of the semester that asks students to apply knowledge of what they've learned in this module to a novel biological question.

B. Skills/performance assessment
- Pre-post module survey of experience/comfort with bioinformatic and phylogenetic tools and skills
Skills Pre Module Survey (Microsoft Word 41kB Jun23 09)
Skills Post Module Survey (Microsoft Word 73kB Jun23 09)
- Homework assignments: print-outs from MacClade and MEGA analyses learned in class to be turned in and checked
- Take-home assignment: Students were asked to critically assess the merit of the hypotheses generated by the 28S rRNA 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.; Students also had to conduct an other analysis (using the model of the 28S rRNA sequence we did together during class) by downloading cytochrome b gene sequences from Genbank for similar species (plus additional ones if students are so inclined since this gene is sequenced for so many species by fish phylogeneticists). Comparisons between phylogenetic hypotheses are made. Students also critique a published phylogenetics paper that incorporates morphology and molecular analyses (that they have not seen before) to see if students can apply the knowledge of the methods/assumptions of phylogenetic analysis that they've learned. (see link in Knowledge Assessment)

C. Attitudes assessment
- Pre-post module survey of their views/opinions about bioinformatics, genomics, and phylogenetics.
Attitude Module Assessment Survey (Microsoft Word 54kB Jun23 09)
- Question on take-home assignment asking students about their views on interdisciplinary nature of the module

- Question given at the end of the semester asking students about the value of inclusion of phylogenetics in a Comparative Anatomy course

References and Resources

Software and related resources used in this module:
Molecular Evolutionary Genetics Analysis (MEGA) (available for Windows, DOS, MAC, Linux)-very easy to use for undergraduates although some downloads contained bugs. Freeware.

Phylogenetic Trees Made Easy: A How-To Manual, 3rd Ed. Barry G. Hall. Sinauer Assoc. Press
*A great manual on how to use MEGA.

MacClade v.4.08 for MAC, by David Maddison and Wayne Maddison
MacClade is the computer program for phylogenetic analysis that is best for tree construction from character matrices and character evolution--especially morphological/developmental characters, but also could be used with DNA and amino acid sequences (but more powerful and versatile algorithms/software is typically used for phylogeny reconstruction). Must purchase this.

Texts and Readings
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