Local Population Structure and Behavior of the Wood Frog, Rana sylvatica

Derek Dean, Williams College, based on an original activity by David Smith, Williams College
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The purpose of this exercise is to assess the relatedness of wood frog tadpoles within and between two local ponds. With this information, we will determine the population structure (are the tadpoles in the two ponds related), genetic health (level of inbreeding), and even behavior (paternity of egg clusters, sibling aggregation) of the local wood frog population.

Learning Goals

1) Students will gain a familiarity with the molecular biology skills used in population genetics, such as PCR, fragment length analysis, and microsatellites. They will also learn to use and interpret fundamental equations of population genetics, such as Hardy-Weinberg and F-statistics.

2) Students will learn to appreciate a multidisciplinary approach to biology, in this case combining population genetics and behavior in one study.

3) In our version of the exercise, students will design and execute their own behavioral experiments, integrate their data in small groups, lead a discussion of the critical analysis of the data, and then revisit this critical analysis by completing a problem set on their own.

Context for Use

At Williams College, this lab is used for a second semester introductory biology class. Class size is approximately 150-170, with lab sections typically consisting of 20-24 students. It could easily be adapted for a smaller upper level college biology course by perhaps increasing the independent element of the analysis or adding more sophisticated statistics.
3-4 standard lab periods (2.5-4 hours) would be required for completion of the exercise. Week 1, egg collection and examining the vernal pool environment. Week 2, behavioral analysis, DNA extraction, and PCR. Week 3, analysis of PCR results. Week 4, discussion/assignment.
At minimum, a PCR machine, standard molecular biology supplies (gloves, Pipetmen, etc.), and gel electrophoresis apparati would be required. We would recommend using this lab exercise after students have had at least several lab periods of experience with pipetting and centrifuging. It would also be desirable to preface the exercise with a mini-lecture that walks them through the goals of the lab (see the introduction to Part I of the procedures).

Description and Teaching Materials

-The following teaching materials have been attached

Lab exercise Rana pop gen lab exercise (Microsoft Word 2.5MB Jun21 09)

-Powerpoint slideshow used to introduce the experimental design Slideshow for intro lecture (PowerPoint 9.8MB Jun21 09)

-Final student assignment (i-iii done as a group on Week 4, the remaining questions are done one their own) Rana lab assignment (Microsoft Word 74kB Jun21 09)

-Sample Excel Data for assignment Sample Rana data (Excel 73kB Jun21 09)

Teaching Notes and Tips

-We held a discussion of the data on Week 4 of the 4-week module. We suggest you use this time to help them develop the skills necessary to do the final assignment. On week 4, using laptops and their data, students worked together to answer questions i-iii. This helped them prepare to answer the remaining questions on their own.

-After egg clusters are collected on Week 1, it is essential to place each egg cluster in a separate container while the tadpoles hatch so as to be able to identify siblings.

-Behavioral experimental designs we tried: (1) two siblings or non-siblings in a petri dish, measure distance between them after 15 minutes; (2) petri dish with permeable, plastic barrier in the middle, put a tadpole on one side and siblings or non-siblings or no tadpoles on the other side, measure distance the first tadpole is from barrier (3) students were encouraged to refine or explore other methods and to discuss their findings on Week 4. With our methods, we discovered no significant different between conditions, though in every case there was a trend towards sibling aggregation. We used this opportunity to discuss whether we believed the trend or not. If so, we discussed how would we make others believe it (e.g. blind testing, increased sample size, use a larger arena, take more data than one static measurement at one time).

-PCR protocols that we used are attached here. Rana lab PCR protocols (Microsoft Word 40kB Jun21 09) The WellRed-labeled primers are expensive, and some primers work better than others for a given population. Therefore, we recommend you survey various unlabeled primers (even trying ones from the references that we did not use) before you invest in the WellRed label.


Skills assessment can be found in the "Final student assignment" attachment under the Teaching Materials section.

Attitude assessment that we used is attached here. Attitude Assessment (Microsoft Word 31kB Jun21 09) We gave them these questions to answer anonymously on Weeks 2 and after they had turned in their final assignment.

References and Resources

Please see end of Lab exercise in Teaching Material section and the PCR protocols document in the Teaching Tips section for a list of helpful references.