We describe an alternative to the kinds of observation-based lab exercises that are often used to cover animal and plant evolution with respect to transitioning from aquatic to terrestrial habitats. We wrote this activity to address these objectives, but also to model the process of scientific inquiry and to require students to collect and analyze quantitative data. Additionally, we designed this activity so that students must consider the evolution of plant and animal traits in an integrated fashion.

Introduction

Context

We created this activity for students in an introductory biology course for biology majors. These students should be able to be complete the activity in a single 3-hour laboratory section. Prior to this lab, we have introduced students to plant and animal cells and tissues and they have considered the characteristics and functions of these cells and tissues.

Rationale

We designed this activity to meet AAAS recommendations for Core Competencies and Disciplinary Practice (Brewer & Smith, 2011). In particular, it addresses the following recommendations:

  1. Ability to Apply the Process of Science: Biology is evidence based and grounded in the formal practices of observation, experimentation, and hypothesis testing.

  2. Ability to Use Quantitative Reasoning: Biology relies on applications of quantitative analysis and mathematical reasoning.

We also wrote this activity to challenge students to consider the evolution of plant and animal traits in an integrated fashion, rather than as distinctly separate, sequential topics. Many traditional introductory biology courses that focus on organismal biology tend to cover each type of organism (plants, animals, bacteria, fungi, etc.) separately, which may inhibit students from considering the common problems of all organisms in similar habitats and the solutions to those problems that have evolved. In the course in which this lab activity is used, we make a concerted effort to draw parallels between basic physiological models used by all organisms (adapted from Modell, 2000). Furthermore, we use this comparative approach because interleaving can improve inductive learning (Kornell & Bjork, 2008; Birnbaum et al., 2013).

Student Learning Objectives

At the conclusion of this activity, students will be able to

  1. explain why animals and plants that evolved mechanisms for limiting water loss in terrestrial habitats were more successful;

  2. describe how the structure of leaves and skin varies, depending on the habitat for which an organism is adapted; and

  3. apply a scientific approach to measure the thickness of the outer tissue layers of leaves and statistically analyze the data with respect to habitat.

Teaching Materials & Methods

To address the learning objectives, we modeled this investigation after the scientific method as described by McPherson (2001). First, students make initial observations from prepared microscope slides. The slides are cross sections of animal skins (amphibian and mammalian) and of plant leaves (hydromorphic, mesomorphic, and xeromorphic). The two animal skins are on separate slides; the three leaf cross sections are a composite on a single slide. We provide each lab table with a set of these three slides.

On the basis of their initial observations, students make predictions about each of the five specimens (amphibian, mammal, hydromorphic plant, mesomorphic plant, and xeromorphic plant) and the habitat for which they are most well adapted (dry, wet, or temperate). Then, focusing on the plant specimens, students generate informed hypotheses based on their observations and predictions. To test these hypotheses, they next make measurements of the outer tissue layers of the three leaves to see whether there is a correlation with the habitat in which the plant type is found. To ensure accuracy and diversity among these measurements, we provide each lab table a set of 36 photomicrographs (six examples of each of the three leaf types at two magnifications, 100× and 400×) made from the plant specimens using a Canon Rebel XSi DSLR camera and a Motic BA300 compound microscope. We let the students choose which photomicrographs to measure, and they collect and graph the quantitative data. Finally, using P values from an analysis of variance (ANOVA), they draw conclusions about the relationship between habitat and the morphology of the outer tissue layers of the plants.

See Table 1 for a summary of the materials needed, and Figure 1 for examples of the photomicrographs. We also give students a worksheet, which helps guide them through the investigation (reproduced below).

Table 1.
Materials needed for the activity.
Description
Cross section of amphibian skin (Carolina Biological Supply no. 314522) 
Cross section of mammalian skin (Carolina Biological Supply no. 314486) 
Composite of three leaf cross sections (Carolina Biological Supply no. 293214) 
Compound light microscopes 
One set of photomicrographs per lab table* 
Rulers for measuring photomicrographs 
Internet-connected laptops or tablets for conducting ANOVA and constructing graphs 
Description
Cross section of amphibian skin (Carolina Biological Supply no. 314522) 
Cross section of mammalian skin (Carolina Biological Supply no. 314486) 
Composite of three leaf cross sections (Carolina Biological Supply no. 293214) 
Compound light microscopes 
One set of photomicrographs per lab table* 
Rulers for measuring photomicrographs 
Internet-connected laptops or tablets for conducting ANOVA and constructing graphs 
*

If the equipment to make your own photomicrographs is not available, please contact the authors for a full set of the color photomicrographs.

Figure 1.

Examples of the photomicrographs provided to the students for measuring (annotations do not appear on those used by students).

Figure 1.

Examples of the photomicrographs provided to the students for measuring (annotations do not appear on those used by students).

Student Preparation

Prior to the day of the lab, we give the students a reading assignment that reviews some of the basic terminology associated with plant and animal tissues. When the students arrive for lab, we ask that they be able to identify and define specific tissues and structures (Table 2).

Table 2.
Terms with which students should be proficient prior to the activity.
CuticleStomataGuard cellsPore
Mesophyll Epithelium Basal lamina Stratum corneum 
Epidermis Dermis Hypodermis  
CuticleStomataGuard cellsPore
Mesophyll Epithelium Basal lamina Stratum corneum 
Epidermis Dermis Hypodermis  

Student Worksheet

Introduction

The chemical reactions of life take place in an aqueous solution. This makes water indispensable to living organisms. For this reason, terrestrial animals and plants have evolved mechanisms for limiting evaporative water loss. Depending on the outside environment, evaporation can be a major source of water loss for terrestrial organisms. Several factors play a role in how quickly water can evaporate, including

  • the SA:V ratio of the organism/structure;

  • the thickness of the outer covering of the organism, which separates the internal environment from the outside world; and

  • the relative humidity of the air outside the organism.

Before continuing, you and your group should consider exactly how these factors affect evaporative water loss. Does a higher SA:V ratio mean faster or slower water loss? Does lower relative humidity mean faster or slower water loss?

Observations

You will now examine five cross sections, three of plant leaves and two of animal skins. The three leaf cross sections (labeled H, X, and M) are all located on a single slide, so you will need to scan the cover slip from top to bottom to locate all three. The two cross sections of animal skins (labeled A and B) are located on separate slides. On the two sections of animal skin, the area above the section represents the “outside” world, while the area below the cross section represents in “internal” environment of the organism. Figure 2 can be used as a guide.

Figure 2.

Diagrammatic representation of animal skin cross section.

Figure 2.

Diagrammatic representation of animal skin cross section.

On the three sections of plant leaves, the orientation is somewhat different than that of the animal skin, because the entire leaf is shown. Therefore, the areas of the slide above and below the section represent the “outside” world, while the middle of the tissue section represents the “internal” environment of the plant. Figure 3 can be used as a guide.

Figure 3.

Diagrammatic representation of plant leaf cross sections.

Figure 3.

Diagrammatic representation of plant leaf cross sections.

First, you will observe and make some sketches of each specimen. You should do this using both 100× and 400× magnification. Use a separate sheet of paper to make your sketches, being sure to include pertinent information and label important structures. Use images in your textbook and/or on reputable websites to assist you in labeling. As you are sketching and labeling, use Tables 3 and 4 to summarize your observations. At this point, it is OK to qualitatively describe what you see. You will make quantitative measurements later. Keep in mind, though, that the purpose of the tables is to compare/contrast the different types of leaves or skin. As such, words like “thinner” or “larger” or “more dense” aren't useful out of context. It would be preferable, for example, for you to state that the epidermis of Plant H is ______ compared to the epidermis of Plant M.

Table 3
Summary of observations about plant leaf slides.
Plant HPlant XPlant M
SA:V Ratio    
Thickness of Epidermis    
Thickness of Cuticle    
Density of Stomata    
Location of Stomata    
Plant HPlant XPlant M
SA:V Ratio    
Thickness of Epidermis    
Thickness of Cuticle    
Density of Stomata    
Location of Stomata    
Table 4.
Summary of observations about animal skin slides.
Animal AAnimal B
Overall Thickness of Skin   
Thickness of Dermis   
Thickness of Epidermis   
Animal AAnimal B
Overall Thickness of Skin   
Thickness of Dermis   
Thickness of Epidermis   

Based on these observations, you and your group should develop an explanatory hypothesis to relate the morphology of these organs to the relative amount of water available in the environment.

Identification

Of the three plant specimens you examined, one is adapted for dry habitats, one is adapted for wet habitats, and one is adapted for temperate habitats. Likewise, one of the animal specimens is adapted for dry habitats while the other is adapted for wet habitats. Based on your prior knowledge and your observations, come up with some predictions about each specimen and its habitat (Table 5). Once you have completed this table, check with your instructor to have your ideas confirmed.

Table 5.
Predictions about slide specimens.
SpecimenHabitatJustification
  
  
  
  
  
SpecimenHabitatJustification
  
  
  
  
  

Measurements

To complete this exercise, you and your group will be focusing on the plant specimens. In order to facilitate making accurate measurements of thickness/distance, you will be using photographs made from the same slides you examined earlier. Your group will measure the thickness of the “outer layer” of the leaf, which includes the epidermis and the cuticle. If you are unsure about where to start and stop measuring, ask your instructor or teaching assistant for some guidance.

You will need to make measurements on at least four different specimens of each plant type (H, X, and M). On a separate sheet of paper, create a data table and then populate the table with your measurements taken from the photomicrographs. Use the data in this table to construct a graph (with a graphing program such as Excel or Numbers) that best conveys the data. Make sure that your graph is properly formatted and labeled.

ANOVA

Now that you have made some measurements, the next step is to determine whether these numbers actually represent statistically significant differences in structure. We will use a statistical test called an “analysis of variance” (abbreviated ANOVA), which is used to compare three or more subject groups. This test results in the calculation of a “P value,” which indicates the probability that the differences among groups were a result of chance. In most biological applications, a P value of 0.05 or less is needed in order for any differences in the averages to be considered significantly different. If the P value is greater than 0.05, you cannot reject the null hypothesis that there is no difference among the groups.

Some additional background information, as well as an online ANOVA calculator, can be accessed at http://www.physics.csbsju.edu/stats/anova.html. Use the resources from this website to test your hypothesis, using the data you just collected.

Data Analysis & Conclusions

In table 6, complete the table with the results of your ANOVA and write a few sentences in which you draw some conclusions about the thickness of the outer covering of leaves with respect to their habitat.

Table 6.
ANOVA results.
Plant XPlant MPlant H
Mean    
Median    
Standard Deviation    
P = 
Conclusions: 
Plant XPlant MPlant H
Mean    
Median    
Standard Deviation    
P = 
Conclusions: 

Results & Feedback on the Activity

In general, our students were able to correctly identify each specimen to its respective habitat. They also were fairly successful at correctly stating the hypothesis and null hypothesis to be tested in the “Measurements” section. However, several typical questions and issues did arise.

  1. Which photomicrograph should be measured? We provide each lab table with photomicrographs made at two different magnifications – 100× and 400× (Figure 1) – to correspond with the fields of view that students observed with microscopes. The decision of which magnification to use depends on the measuring instruments available. With standard 12-inch rulers, only the 400× image can be measured easily. We used this question as an opportunity to discuss the concepts of accuracy and precision in the scientific method.

  2. Which layers should be measured? The hydromorphic plant (H) does not have a cuticle layer. We allowed the students to choose which layer(s) they would measure, so long as they could justify their choice. Some students measured the epidermal layer only, while others measured both the epidermal and cuticle layers. Interpretations in the conclusion should be consistent with the choice of layer(s) measured.

  3. Where should the depth of the layers be measured? We used this question as an opportunity to discuss variability and the importance of reproducible methods. The thickness of the epidermal layer varies along a leaf. We allowed students to decide how they would ensure that their measurements were reproducible. The typical solutions were to measure either the narrowest or the widest point on each photomicrograph.

  4. Microscope problems. Some students neglected to orient themselves to the entire specimen before zooming in, which resulted in their missing critical structures, such as stomata. Note that students should examine both the top and bottom of each leaf specimen for stomata, as the hydromorphic plant's stomata are on the top of the leaf.

  5. Graphing and quantitative problems. We expected students to draw graphs that summarized the data, such as bar graphs with means and error bars. Some students drew line graphs instead of bar graphs, connecting points between the H, M, and X categories. Some students did not summarize the data; rather, they graphed each data point as a separate bar. We suggest using more specific instructions on constructing the graph if students are inexperienced with creating summary graphs.

  6. Conversion problems. Some students did not convert from millimeters or centimeters to micrometers (i.e., they did not use the scale bar on the photomicrograph). We suggest working through an example to ensure that students are able to calculate the conversion correctly.

Further Considerations

  1. Depending on the available equipment and the prior knowledge and skill of the students, you can modify this activity so that students are making measurements directly from the slides, using ocular micrometers, rather than measuring photomicrographs that you make in advance.

  2. You could also expand this investigation to include predicting the response of organisms to climate change and comparing/contrasting short-term and long-term adaptations.

  3. Because the exercise does not allow students to interact with whole organisms, you could include an additional assignment in which students observe various plants in their natural habitat and make predictions about the plants' adaptations for that habitat.

  4. We used this exercise early in the semester, and therefore wanted the students to focus on data collection and analysis. If you use the exercise later in the term, you may want to also have the students practice communicating their findings in either a formal lab report or in a presentation to their classmates.

  5. If you would like students to have some additional practice in formatting and graphing data, you could have them sketch some hypothetical graphs of their predictions rather than using Table 5.

  6. Because we wanted to make this exercise useful to a wide audience, we used a free online ANOVA calculator, which does not allow for any post hoc ANOVA tests. These tests can be included if you have access to statistical packages such as minitab or JMP.

  7. One of the foci of this exercise is the use of statistics to test hypotheses. If your course does not emphasize the use of statistics, you could modify this exercise to focus more on the initial formation of explanatory hypotheses and predictions, in which the students measure the plant specimens and determine whether the measurements support their explanatory hypotheses using only the graph.

Grading Rubric

See Table 7 for a rubric to use for grading the exercise.

Table 7.
A rubric for grading the exercise.
PointsSketchesObservation TablesIdentification TableData TablesANOVA Results & Conclusions
  • Include all specimens at 100× and 400×

  • Sketches neat and realistic

  • Labels correct and organized

 
  • Observations correct, thorough, and pertinent to the investigation

  • Comparative descriptions always used

 
  • All specimens correctly identified

  • Explanations logically sound and use correct vocabulary

 
  • Tables neat and easy to read

  • Measurements accurate

  • Proper units employed

 
  • Calculations fully correct

  • Conclusions supported by data

  • Graph format correct

  • Proper vocabulary used

 
  • Include all specimens at 100× and 400×

  • Some stylization

  • Some labels missing

 
  • Some observations missing

  • Comparative descriptions often used

 
  • All specimens correctly identified

  • Explanations logically sound

  • Proper vocabulary mostly used

 
  • Tables mostly neat and easy to read

  • Measurements mostly accurate

  • Proper units employed

 
  • Calculations fully correct

  • Conclusions mostly supported by data

  • Graph format correct

  • Proper vocabulary mostly used

 
  • Some sketches absent

  • Appear to be redrawn from text or web

  • Some labels missing

 
  • Several observations missing

  • Comparative descriptions sometimes used

 
  • Most specimens correctly identified

  • Explanations not logically sound

  • Some vocabulary misuse

 
  • Tables disorganized

  • Some measurements accurate

  • Proper units sometimes employed

 
  • Calculations mostly correct

  • Some conclusions supported by data

  • Graph format correct but is made from raw data

  • Some vocabulary misused

 
  • Many sketches absent

  • Appear to be redrawn from text or web

  • Many labels missing

 
  • Many observations missing

  • Comparative descriptions seldom used

 
  • Some specimens identified correctly

  • Explanations not logically sound

  • Vocabulary misuse

 
  • Tables disorganized

  • Measurements not accurate

  • Proper units not employed

 
  • Calculations incorrect

  • Conclusions not supported by data

  • Graph format incorrect

  • Vocabulary misused

 
  • Sketches absent

 
  • Observations absent

 
  • Identifications and explanations absent

 
  • Data tables absent

 
  • ANOVA results and conclusions absent

  • Graph absent

 
PointsSketchesObservation TablesIdentification TableData TablesANOVA Results & Conclusions
  • Include all specimens at 100× and 400×

  • Sketches neat and realistic

  • Labels correct and organized

 
  • Observations correct, thorough, and pertinent to the investigation

  • Comparative descriptions always used

 
  • All specimens correctly identified

  • Explanations logically sound and use correct vocabulary

 
  • Tables neat and easy to read

  • Measurements accurate

  • Proper units employed

 
  • Calculations fully correct

  • Conclusions supported by data

  • Graph format correct

  • Proper vocabulary used

 
  • Include all specimens at 100× and 400×

  • Some stylization

  • Some labels missing

 
  • Some observations missing

  • Comparative descriptions often used

 
  • All specimens correctly identified

  • Explanations logically sound

  • Proper vocabulary mostly used

 
  • Tables mostly neat and easy to read

  • Measurements mostly accurate

  • Proper units employed

 
  • Calculations fully correct

  • Conclusions mostly supported by data

  • Graph format correct

  • Proper vocabulary mostly used

 
  • Some sketches absent

  • Appear to be redrawn from text or web

  • Some labels missing

 
  • Several observations missing

  • Comparative descriptions sometimes used

 
  • Most specimens correctly identified

  • Explanations not logically sound

  • Some vocabulary misuse

 
  • Tables disorganized

  • Some measurements accurate

  • Proper units sometimes employed

 
  • Calculations mostly correct

  • Some conclusions supported by data

  • Graph format correct but is made from raw data

  • Some vocabulary misused

 
  • Many sketches absent

  • Appear to be redrawn from text or web

  • Many labels missing

 
  • Many observations missing

  • Comparative descriptions seldom used

 
  • Some specimens identified correctly

  • Explanations not logically sound

  • Vocabulary misuse

 
  • Tables disorganized

  • Measurements not accurate

  • Proper units not employed

 
  • Calculations incorrect

  • Conclusions not supported by data

  • Graph format incorrect

  • Vocabulary misused

 
  • Sketches absent

 
  • Observations absent

 
  • Identifications and explanations absent

 
  • Data tables absent

 
  • ANOVA results and conclusions absent

  • Graph absent

 

The authors acknowledge the editors and anonymous reviewers for their thoughtful assistance with this article. We also thank students in Biol 112 at Presbyterian College for completing this activity and for providing the content and feedback on which this article was based.

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