Natural variation, including the continual selective pressures that lead to speciation, is one of the more dynamic aspects of biology. However, traditional instruction on the topic is often passive in nature, leaving little opportunity for scientific inquiry. In this laboratory exercise, we use a statistics-based, guided-inquiry approach to engage students in natural variation. Students are introduced to speciation and classification by using a dichotomous key to identify various common local trees on the basis of leaf characteristics. Once the students have learned characteristics useful for identification, they are given two leaf samples, a sugar maple and an “unknown.” They are asked to choose characteristics and collect quantitative data in order to determine whether the unknown is a sugar maple. Before data collection, students form hypotheses related to the identity of their unknown, followed by statistical comparison of means to support or refute their original hypotheses. In this way, students gain an appreciation for the activities undertaken by taxonomists that are related to natural variation and classification.

The general intent of the introductory biology laboratory is to reinforce basic biological concepts while teaching skills that are necessary for conducting science. Traditional laboratory exercises have been expository or “cookbook” in nature rather than inquiry-based. The diversity of life and its classification, including the continual selective pressures that lead to speciation, has been no exception. Although the topic is one of the more dynamic aspects of biology, laboratory exercises have tended to be “show-and-tell,” presenting students with lists of characteristics that distinguish earthworms from planarians, green algae from conifers, and bacteria from archaea. During lab activities designed to coincide with lecture presentations, students are typically shown representative examples of taxa, often preserved specimens, with the occasional dissection or live specimen. Seldom is the scientific method used, and so there is very little opportunity for scientific inquiry.

While there is evidence that inquiry-based labs result in increased understanding (e.g., Lord & Orkwiszewski, 2006; Rissing & Cogan, 2009), it can be difficult to implement them (e.g., Sundberg & Armstrong, 1993; Brainard, 2007). The activity presented here is an intermediate approach, called a “guided inquiry” by D'Avanzo (1996) and Grant and Vatnick (1998), in which students are given a research question and resources, but they design and conduct an open-ended investigation. This method not only gives the students some ownership and experience with the scientific method, it also provides guidance and is somewhat easier to manage logistically. We use a statistics-based, inquiry approach to engage our students in natural variation and classification, in this case by learning to classify species of common local trees on the basis of leaf characteristics. Once the students have identified characteristics useful for classification, they are given two leaf samples, a sugar maple and an “unknown” sample. They are then instructed to use the scientific method to address the following question: Is the unknown a sugar maple? Before they collect data, the students form hypotheses related to the identity of their unknown. They then choose characteristics for comparison, collect quantitative data, and conduct statistical comparison of means to support or refute their original hypotheses.

This lab is designed to be completed during one 3-hour session of our introductory biology course for majors. It addresses many of the central themes of biology outlined in BIO2010 as critical for understanding the unity and diversity of life, specifically those concerning heritable genetic variation and speciation (National Research Council [NRC], 2003). Furthermore, this lab follows the BIO2010 recommendation that students be engaged by developing project-based laboratory activities with discovery components. In addition, many National Science Education 9—12 content standards are addressed (Table 1), and this lab could certainly be modified for use at the high school level (NRC, 1996).

Table 1.

National Science Education Standards addressed by this lab.

Standard (9–12)Fundamental Abilities & Concepts
Content Standard A: Science as Inquiry
  • Abilities necessary to do scientific inquiry

  • Understanding about scientific inquiry

 
  • Design and conduct scientific investigations.

  • Use technology and mathematics to improve investigations and communications.

  • Formulate and revise scientific explanations and models using logic and evidence.

 
Content Standard C: Life Science
  • Biological evolution

 
Biological classifications are based on how organisms are related. 
Content Standard E: Science and Technology
  • Understanding about science and technology

 
Scientific inquiry is driven by the desire to understand the natural world. 
Content Standard G: History and Nature of Science
  • Nature of scientific knowledge

 
  • Science distinguishes itself through the use of empirical standards,logical arguments, and skepticism.

  • Scientific explanations must be consistent with experimental and observational evidence.

 
Standard (9–12)Fundamental Abilities & Concepts
Content Standard A: Science as Inquiry
  • Abilities necessary to do scientific inquiry

  • Understanding about scientific inquiry

 
  • Design and conduct scientific investigations.

  • Use technology and mathematics to improve investigations and communications.

  • Formulate and revise scientific explanations and models using logic and evidence.

 
Content Standard C: Life Science
  • Biological evolution

 
Biological classifications are based on how organisms are related. 
Content Standard E: Science and Technology
  • Understanding about science and technology

 
Scientific inquiry is driven by the desire to understand the natural world. 
Content Standard G: History and Nature of Science
  • Nature of scientific knowledge

 
  • Science distinguishes itself through the use of empirical standards,logical arguments, and skepticism.

  • Scientific explanations must be consistent with experimental and observational evidence.

 

Background

Organismal variation can be genetic or environmental, and many traits are influenced by both genes and environment. Genetic variation results when individuals inherit different alleles for certain traits. Heritable variation is crucial because it provides the raw material on which natural selection acts. Populations that do not vary genetically cannot evolve. Heritable variation is also useful for classification. Variation helps us identify different species of organisms. For identifications to be reliable, we need to use characters that have a genetic basis, rather than ones that are easily influenced by environmental conditions. Correct identification of organisms has practical consequences in spider and snake bites, in ingestion of plant parts by children, and in deciding whether to hike through vegetation that may or may not be poison ivy.

Millions of species inhabit the earth. Although each species has some unique identifying traits, different species may still closely resemble each other. Thus, at-a-glance identification requires investing much time (often years) to learn the similarities and differences among the species of interest. However, identifications in a reasonable length of time are possible without becoming an expert on a group.

Taxonomists study known organisms, distinguish within-group variation from variation that exists among groups, and decide which groups merit recognition as species. Characters that vary among (but not within) groups are useful for separating and identifying these groups. Using these features, taxonomists write identification keys that make identification possible by nonexperts. Keys let users identify an organism from among the possible alternatives through a step-by-step process of elimination. Virtually any group with observable variation can be the subject of a key.

How do scientists separate natural variation into groups? Two related fields deal with this issue. Taxonomy is the science of classification, whereas systematics involves using phylogenetic information (i.e., information about evolutionary relationships) to identify and classify organisms. Taxonomists attempt to classify all the organisms on earth. Biological taxonomy is important for three major reasons. It allows us to catalogue and comprehend the great diversity of life on Earth. Organizing all of life into a series of a few categories makes it easier to study. Second, taxonomy assists communication. Having named categories allows different people to refer to them in an understandable fashion. Third, when a systematic approach is used, classifications have a predictive value. If the two-spotted bumblebee stings you and it is painful, you can predict that the sting of its relative, the white-faced bumblebee, will also be painful, even though you've never been stung by one.

Modern taxonomists attempt to use a natural system of classification that reflects the evolutionary relationships of the organisms being classified. This results in natural groups in which all the organisms are more closely related to each other than to members of any other group. “Relatedness” implies a common origin for members of a group at some time in the past; the more recently that two species shared a common ancestor, the more closely they should be classified. Since taxonomists cannot read evolutionary history directly, they must infer relatedness. Taxonomists use all available evidence (e.g., morphology, chemistry, behavior, fossils, etc.) to determine how taxa are related. DNA sequence data are often the best indicator of relatedness, but such data have only recently become easy to obtain. As more and more DNA data become available, old classifications are revised to reflect new knowledge, making modern taxonomy a dynamic field.

Anatomical similarities often indicate evolutionary relatedness. For classification, it is important to use traits that are evolutionarily significant — that is, traits that are similar because of shared ancestry. One cannot determine ahead of time which traits these will be. Taxonomists observe and compare all features of their study groups to identify useful characteristics. Multiple traits are required for classification, because traits distinguishing group A from group B may not differentiate A and C. For example, tail length distinguishes bobcats and mountain lions but is useless for separating jaguars and leopards. Statistics and graphs can help us identify taxonomically useful traits, particularly at the level of species. Statistics such as the t-test and analysis of variance are used to test whether the differences measured between two groups are statistically significant.

Materials

Computers equipped with statistical analysis software (we use SPSS 16.0 for Windows)

  • Protractors

  • Rulers

  • Laminated leaves for use with the identification key. We maintain at least 10—12 laminated specimens of each. Specimens are laminated following drying and pressing (see below). Specimens may vary, depending on the area; these are the examples we use (labeled A—I):

    • A = buckeye (Aesculus glabra)

    • B = dogwood (Cornus florida)

    • C = hackberry (Celtis occidentalis)

    • D = redbud (Cercis canadensis)

    • E = white ash (Fraxinus americana)

    • F = black locust (Robinia pseudoacacia)

    • G = Osage orange (Maclura pomifera)

    • H = hickory (Carya sp.)

    • I = sugar maple (Acer saccharum)

  • Pressed specimens labeled with different colors for use with the inquiry exercise (specimens may vary depending upon area). These are the examples we use:

    • Teal label = sweet gum (Liquidambar styraciflua)

    • Green label = sugar maple

    • Purple label = silver maple (Acer saccharinum)

    • Pink label = box elder (Acer negundo)

    • Red label = red maple (Acer rubrum)

    • Yellow label = Norway maple (Acer platanoides)

When necessary, depending on wear and tear, we collect approximately 5—10 branches 12—24 inches long of each of the following: red maple, silver maple, Norway maple, box elder, and sweet gum, and approximately 12—14 branches 12—24 inches long of sugar maple. Collected specimens are immediately processed, or stored at 4°C and sprayed daily with water for 2 to 3 days to keep them fresh until processing. Leaves are pressed and dried for 48 hours in a professional plant press; however, this could also be accomplished by using an inexpensive homemade plant press, such as one made of newspapers, cardboard, blotter paper, and wood held together with two straps.

Procedure

During the first part of the lab, students working in groups of three or four are given laminated leaf specimens of different species of local trees. Using an identification key (Figure 1), the groups try to identify the genus of each specimen. Diagrams of common leaf characteristics are provided (Figure 2), in addition to a glossary of terms commonly used to describe these characteristics (Figure 3). Once they have identified each species, the instructor checks the identifications for correctness. If a correct identification is not achieved, the students try again until all specimens have been correctly identified. This trial-and-error portion of the lab allows the students to gain experience with the types of leaf characteristics that are useful in separating species, providing background for the design of the inquiry exercise during the remainder of the lab.

Figure 1.

Key to genera of common wild trees of northern Kentucky and southern Ohio, based on leaf characteristics.

Figure 1.

Key to genera of common wild trees of northern Kentucky and southern Ohio, based on leaf characteristics.

Figure 2.

Diagrams of common leaf morphology characteristics used to classify trees.

Figure 2.

Diagrams of common leaf morphology characteristics used to classify trees.

Figure 3.

Glossary of common leaf morphology characteristics used to classify trees.

Figure 3.

Glossary of common leaf morphology characteristics used to classify trees.

Each group is then assigned an “unknown” to work with. Students begin by carefully comparing several stems of the unknown with several stems of their known species, in this case the sugar maple. The scientific question being addressed is this: Do your specimens look similar enough to belong to the same species, or do you think they belong to different species? The answer to this question is used to formulate a testable hypothesis about the similarity (or difference) of the two sets of specimens.

Once the hypothesis has been developed, each group will study the specimens to assess their similarity, using a dissecting microscope if necessary. The students are instructed to choose quantitative (measurable), not qualitative, characters. Some examples of measurable characters are blade length, petiole length, ratio of petiole length to blade length, number of teeth on the margin, angle between the midvein and first major vein to branch off, ratio of blade length to width, angle between the first and second lobes, and total number of lobes. Using leaves from at least three stems of each type of specimen, the students take measurements from up to eight mature leaves and record the data. Once the data have been collected, statistical analysis is conducted for each character. The students are instructed to enter their data into four columns of a spreadsheet, the first column being the independent variable (e.g., sample — sugar maple vs. unknown) and the remaining columns the dependent variables (e.g., characters — petiole length). Students are then instructed to conduct independent-sample t-tests to compare the means between the sugar maple and the unknown for all measured characters. Students then use the P values generated by their data analysis to support or refute their original hypotheses, using P < 0.05 as the critical value to indicate a significant difference between the means.

Discussion & Conclusions

This lab addresses the following objectives:

  • Understand the relationship among heritable genetic variation, natural selection, and speciation

  • Use a taxonomic key for identification based on natural variation

  • Develop testable hypotheses to address a scientific question

  • Analyze data from two or more groups, comparing means, to test hypotheses related to classification

  • Interpret data analysis

The data collected during this experiment allow students to gain an appreciation of the inquiry activities undertaken by taxonomists in classifying organisms. Specifically, students gain hands-on experience in the critical nature of the selection of characters that allow for the identification of between-group variation to distinguish species, rather than within-group variation of the same species.

For example, the initial observations by students comparing sugar maple with the unknown, when the latter is the sweet gum, tend to lead to the development of hypotheses that the two species are not the same, because these species appear to be quite different (e.g., opposite vs. alternate arrangement of leaves). The blades of the leaves of these species are distinguishable as well (Figure 4). Characters selected by students tend to support this hypothesis, in that comparisons of means typically exhibit significant differences between samples (P < 0.05). Figure 5 represents an example of data analysis conducted by students, comparing the sugar maple to the “unknown” sweet gum in terms of the angle of the first vein to branch off the midvein, the ratio of the blade to petiole length, and the length of the petiole. In this example, with rather dissimilar species, the significant differences generated by the statistical analysis illustrates between-group variation.

Figure 4.

Photographs of typical sugar maple and sweet gum leaves.

Figure 4.

Photographs of typical sugar maple and sweet gum leaves.

Figure 5.

Comparison of selected characters comparing sugar maple and unknown (sweet gum). Error bars represent mean 6 standard error of the mean (SEM). Asterisk indicates a value significantly different from that of sugar maple (Student's t-test; P < 0.05).

Figure 5.

Comparison of selected characters comparing sugar maple and unknown (sweet gum). Error bars represent mean 6 standard error of the mean (SEM). Asterisk indicates a value significantly different from that of sugar maple (Student's t-test; P < 0.05).

Interestingly, when students compare the sugar maple with the unknown and the latter is another sugar maple, the hypotheses they develop are usually divided between the two species being the same or being different. This provides an excellent teaching and learning opportunity. Figure 6 represents an example of data analysis of the same characters as described above, when the unknown was another sugar maple. In this case, only one of the three selected characters showed a significant difference between the samples. The students are forced to interpret the data. With further discussion and perhaps additional comparisons, they come to the conclusion that the character that is significantly different in this case is an example of within-group variation, which is not useful in the classification of separate species.

Figure 6.

Comparison of selected characters comparing sugar maple and unknown (also sugar maple). Error bars represent mean ± standard error of the mean (SEM). Asterisk indicates a value significantly different from that of sugar maple (Student's t-test; P < 0.05).

Figure 6.

Comparison of selected characters comparing sugar maple and unknown (also sugar maple). Error bars represent mean ± standard error of the mean (SEM). Asterisk indicates a value significantly different from that of sugar maple (Student's t-test; P < 0.05).

In addition to in-class discussion, students are assessed in this lab on the basis of their ability to answer questions related to the exercise, including:

  • What types of characteristics and methods are most useful for identifying organisms?

  • What consequences does natural variation have for classification?

  • How can the results of statistical analyses be used to test hypotheses?

This guided-inquiry exercise meets multiple objectives regarding content and skills, while also preparing students for future open-ended investigations. We've utilized species of trees that are endemic to the deciduous forests of the Ohio River Valley near our campus, but the lab could certainly be modified to use other types of plant species endemic to other areas. In fact, this lab could easily be reworked for study of natural variation not only in plants, but also in protists, fungi, animals, or any group of organisms for which quantitative characters can be identified.

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