The choice of the scientific method to be used depends on the question to be investigated, the type of study being performed, and the maturity of the particular subdiscipline. I review the scientific methods frequently used in biology since Darwin, the aspects of the nature of science relevant for teaching and learning about evolution, and some recent studies that tested the theory of evolution and some of its features. I also present some guidelines for teachers, within an inquiry-based instructional framework, to facilitate students’ understanding that hypothesis-driven and observation-driven studies are equally important and responsible for the advancement of scientific knowledge in the field of biology, both in the past and in the present.
An understanding of the nature of science (NOS) is important in the development of scientifically literate citizens and facilitates learning of specific scientific subject matter (Lederman, 2007). Therefore, the National Science Teachers Association (NSTA) recommends that NOS should be a focus of science classes (NSTA Board of Directors, 2000) and that all K–16 teachers should facilitate students’ learning through scientific inquiry, which is central to the learning of science and reflects how science is done (NSTA Board of Directors, 2004). Aspects of NOS can be learned both through explicit, reflective instruction and implicitly through doing science (Lederman, 2007). Here, I offer a contribution to facilitating teachers’ implementation of NOS and evolutionary concepts in structured-, guided-, or open-inquiry science classes.
Charles Darwin's work shows us the complexity of the natural world and of the science – biology – that studies natural phenomena that are known to be parts of complex systems. Doing biology is a complex activity, described by different scientists in different ways, and therefore helping students understand how biology is done is a difficult task. Various models of inquiry-based instruction are available for teachers to use, including an empirically based “activity model” (Harwood, 2004) that consists of 10 activities that scientists perform, done in any order and with as much repetition as necessary: (1) make observations (as a starting point and/or repeated several times), (2) ask questions, (3) define the problem (limit the area of study), (4) form the question (develop the question for research), (5) investigate the known (literature and experts), (6) articulate an expectation (hypothesis and prediction), (7) carry out the study, (8) examine the results, (9) reflect on the findings, and (10) communicate with others (Figure 1).
The model in Figure 1 describes how (some) biologists do their work on a daily basis – including the idea of science as a process open to many paths, a question-driven process, not always producing new theories – and provides the teacher a framework for planning, implementing, and evaluating inquiry instruction, focusing on one or more of these aspects and activities. Here, I address the crucial question of how to “carry out the study” and emphasize aspects of NOS by focusing on Darwin's work and on recent findings that support his theory of evolution. Given that translation of NOS concepts into practice has been described as mediated by teachers’ acquired NOS understanding (Wahbeh & Abd-El-Khalick, 2014), this article constitutes an “instant update” that aims to facilitate development of lessons that help students perform both inductive and deductive scientific reasoning in their daily lives.
Darwin contributed to biology by establishing the fact of evolution, based on a huge amount of observations, and then proposing a theory to explain one of the most common mechanisms of evolution, which he called “natural selection.” Facts and theories are not steps in any ladder of increasing certainty; according to Gould (1981), facts are the world's data, and theories are structures of ideas that explain and interpret facts. Darwin's work enlightened this difference between the two.
Alfred Russel Wallace (1860) considered that Darwin created a new science and a new philosophy. In one of Darwin's first published studies, “The Structure and Distribution of Coral Reefs,” one can see his role in changing the view of the natural world: “In an old-standing reef, the corals, which are so different in kind on different parts of it, are probably all adapted to the stations they occupy, and hold their places, like other organic beings, by a struggle one with another, and with nature; hence we may infer that their growth would generally be slow, except under peculiarly favourable circumstances” (Darwin, 1842). In summary, Darwin contributed to the change in our view of the natural world, in aspects such as diversity, adaptation to the environment, intraspecific competition, and gradual phenomena.
Scientific Method(s) & How to Carry Out a Scientific Study
Karl Popper (1902–94), one of the most influential philosophers of the 20th century, proposed the use of the hypothetico-deductive method as the only scientific method, rejecting the inductivism proposed by Francis Bacon (1561–1626) and the methods of induction proposed by John Stuart Mill (1806–73). According to the hypothetico-deductive method, any hypothesis is considered a scientific hypothesis only if it is falsifiable. Furthermore, this method is characterized by two main phases, such as formulating a hypothesis and then testing it. Therefore, this practice is often characterized as “the” scientific method and is historically associated with scientists such as Blaise Pascal (1623–62), physicist such as Isaac Newton (1624–1727) and biologists, Claude Bernard (1813–78), Louis Pasteur (1822–95), and Gregor Mendel (1822–84). The last three were biologists and contemporaries of Darwin, and some authors consider that Darwin also followed the hypothetico-deductive method (Ayala, 2009), though this is a controversial issue.
Darwin, however, claimed in his autobiography that he had followed the inductivist method “on true Baconian principles and without any theory collected facts on a wholesale scale” (Barlow, 1958), accumulating related observations so that a universal statement (theory or hypothesis) could eventually emerge from them. In On the Origin of Species, Darwin (1859) highlighted the importance of the accumulation of observations (Figure 2, left side), and he also recognized that his work on the structure and distribution of coral reefs (Barlow, 1958) was the most deductive of his studies, in that he wrote the theory on the basis of a hypothesis made before observing any corals (Figure 2, right side).
In summary, Darwin, like all scientists, asked revelant questions that guided his work; however, different epistemic goals and the nature of the phenomenon being studied will lead to different questions, and different questions are addressed using different kinds of scientific investigation. Figure 2 is a simplified scheme that focuses on the different ways research may be performed as part of a scientific process that is question driven, creative, and open to many paths of discovery – not necessarily a step-by-step or linear procedure. Darwin was an inductivist who also used the hypothetico-deductive method to test his theory, performing several studies in a way that can be summarized as on the left side of Figure 2 (and following the dashed arrow line), on subjects such as corals (Darwin, 1842), live and fossil barnacles (Darwin, 1851), orchids and their fertilization by insects (Darwin, 1862), and insectivorous and climbing plants (Darwin, 1875a, b). Darwin thus influenced other scientists not to consider the hypothetico-deductive method as the only correct way to do science.
Observation-Driven Investigation vs. Hypothesis-Driven Investigation
Scientists use multiple research methods (experiments, observational research, comparative research, and modeling) to collect data. According to several authors, life sciences can be divided into two cathegories: historical (or descriptive) and experimental, each using a different scientific methodology – observation driven and hypothesis driven, respectively (Meyer et al., 2013). Historical hypotheses, common in paleontology and geology, are formulated by observing natural phenomena (Figure 3) and constitute explanatory hypotheses (left side of Figure 2) that cannot be replicated and tested in any laboratory, mainly because of the time scale of the phenomena (Cleland, 2001). For example, a meteor impact was first hypothesized as the cause of the dinosaurs’ extinction after deposits of iridium (a rare component in Earth's surface) were discovered in the Cretaceous–Tertiary boundary (Alvarez et al., 1980). Several other pieces of evidence later corroborated this hypothesis, such as the discovery of the crater of impact and dinosaur fossil records related to deposits of iridium and shocked quartz (Alvarez et al., 1995).
Currently in biology, when studying a subject for which there is little information available or when studying highly complex systems, it is common to formulate a hypothesis and then collect huge amounts of data that are subsequently analyzed in an attempt to uncover any correlations that have not yet been described (Figure 3, bottom left). Figure 3 is a simplified classification using dichotomy, manipulation versus nonmanipulation, and observation versus hypothesis testing; however, this is more complicated, because one can find continuities between these categories.
Current computational resources and statistical methods allow the development of descriptive studies, such as the characterization of the genome, proteome, and metabolome of model species or of an individual, as well as the characterization of a pathological or environmental change condition. Such studies are often followed by hypothesis-driven research on a specific gene or molecular mechanism (Figure 3, upper right). These experiments can be performed in different settings – in the laboratory, in the field, or through virtual (digital) modeling. Generally we think of descriptive work as being based on observations, with no manipulation of nature (Figure 3, bottom left), but if one intends to study a dependent variable, then one changes and/or controls independent variables and observes the consequences (Figure 3, upper left).
It is desirable to use multiple lines of evidence, obtained by different tests or different fields of study, to evaluate any scientific idea. Therefore, it is important that students understand the distinction between experimental work and descriptive work and that both types of studies are useful and equally important for the advance of scientific knowledge.
Foundations of Modern Biology: Theories, Laws, Principles & Models
The main foundational theories of modern biology are evolutionary theory and cell theory. The major contributions to developing them were made in the 19th and 17th centuries, respectively.
A scientific law is a descriptive statement of relationship among observable phenomena (Abb-El-Khalick, 2012). Laws of biology are derived from empirical observations that are not absolute and come with exceptions due to unknown aspects of the system (e.g., codon bias in the genetic code); thus, they formalize consistent observations but do not explain them. Relatively few laws of biology have been described, including the Mendelian inheritance laws and Kleiber's law of metabolic scaling (Dhar & Giuliani, 2010).
Darwin was the main contributor in defining two laws presently accepted by biologists: the first states that all known properties of living organisms follow the laws of physics and chemistry, and the second states that all biological processes and all the characteristics that distinguish different species have evolved by natural selection (as summarized by Wilson, 2006). Darwin (1837) used the first law to hypothesize about the origin of life from the combination of chemical compounds. He wrote: “The intimate relation of Life with laws of chemical combination, & the universality of latter render spontaneous generation not improbable.”
In 1973, Van Valen proposed an evolutionary law by showing that there is a constant probability of extinction for any lineage through the history of life and that any improvement (in fitness) in one lineage is counteracted by improvements in others; thus, there is a continuous, coevolutionary “arms race” (Morris & Lundberg, 2011).
Recently, McShea & Brandon (2010) proposed the “zero-force evolutionary law,” formulated as follows: “[I]n any evolutionary system in which there is variation and heredity, there is a tendency for diversity and complexity to increase, one that is always present but may be opposed or augmented by natural selection, other forces, or constraints on diversity or complexity.” This law correlates diversity, complexity, and evolution by natural selection or other factors, and includes a probabilistic increase of diversity and complexity that is present even in the absence of natural selection. According to the authors, this law is a useful generalization that arises from the properties of variation in nature (e.g., complexity increases during ecological sucession).
Increasing amounts of empirical data from next-generation sequencing and novel analytical approaches have contributed to the understanding of the genetics of speciation and are the foundations of the new field of speciation genomics (Seehausen et al., 2014). New laws will likely be established by this field in the future.
A fundamental principle according to Morris & Lundberg (2011) is that life is a game (i.e., an evolutionary challenge), such that strategies for growth, reproduction, and survival are played against each other and the evolutionary solutions are contingent on the frequency and density of alternative strategies.
A scientific model is an idea or a set of ideas that explains what causes a specific natural phenomenon. For example, the punctuated equilibrium model of how evolution occurs (Figure 4B) predicts that the majority of evolutionary change occurs in short periods and in association with speciation events, which are rare because of evolutionary stability or stasis (Eldredge & Gould, 1972). A model can be represented by equations (Figure 4C); by graphs, such as Jacques Monod's model of the growth of bacterial cultures (Figure 4A); by 3D structures (e.g., the DNA double helix model; Figure 4D); or by drawings (e.g., the tree of life model; Figure 4E, F). Figure 4 depicts explanatory models that are used to predict the behavior of a specific biological system.
Experimentally Testing Darwin's Theory of Evolution
Popper is often associated with the notion that Darwin's theory is “not a testable scientific theory but a metaphysical research programme” (Popper, 1976). However, in 1978 Popper recognized that the theory of evolution was difficult to test but not impossible to test, saying, “I have changed my mind about the testability and logical status of the theory of natural selection….” In terms of possible tests, he mentioned industrial melanism as an observable natural phenomenon and some easy experimental tests involving the adaptation of bacteria to a new environment with penicilin (Popper, 1978).
It is now possible to test several features of Darwin's theory. Recently, Wasik et al. (2014) performed the first artificial selection for structural color on butterfly wings, and their study is an excellent example for classroom discussion of the continuum between manipulative description (descriptive study) and manipulative hypothesis testing (experimental study). These authors used butterflies of the genus Bicyclus in the genus Bicyclus that exhibit predominantly brown color along marginal eyespots, whereas some other species in the genus present transverse bands of bright violet–blue on the dorsal surface of the forewings. Bicyclus anynana, the study species, does not exhibit the violet color in natural conditions, so Wasik et al. (2014) tested whether it could be made to evolve the same violet–blue color of other species via artificial selection and then tested how the color is generated and how it has evolved. They artificially selected the most extreme individuals of each sex by measuring their reflectance spectra from the region of the dorsal forewing, associated with violet–blue color in other Bicyclus species, and then interbred individuals displaying ultraviolet (UV) reflectance peaks nearest 400 nm during six consecutive generations (i.e., <1 year). This procedure led to a gradual increase in the reflectance peak wavelength in the selected population. The study demonstrated that laboratory populations of B. anynana had significant additive genetic variation that controlled reflectance peak wavelength allowing the rapid evolution of a novel scale color. This coloration plays important roles in fitness and diversity in natural populations of two other species of the same genus with reflectance peaks of 400–450 nm (Wasik et al., 2014).
In the second part of the study, individuals that exhibited significantly increased reflectance in the wavelength range of 400–500 nm were selected to study how the violet color has evolved. The results showed that changes to the ground scales, caused by increased thickness of the lower lamina of these scales, were primarily responsible for the evolution of violet color in this artificial-selection experiment. And in a descriptive part of the study, Wasik et al. (2014) observed, by scanning electron microscopy, the presence of violet color in natural populations of certain Bicyclus species and showed that the natural evolution of this color was caused by the same mechanism via similar scale modifications; therefore, the violet color seems to constitute an evolutionary trend from brown-pigmented ancestors with UV structural color.
Wasik et al. (2014) concluded that in natural populations of these buterflies, one can find genetic variation, including the potential for violet color of the wings, and that natural or sexual selection may be primarily responsible for this color's presence or absence. Structurally colored wing patterns have been described as species-recognition signals and as sexually dimorphic signals involved in female mate choice, and it has been shown that under low light conditions some of the butterflies’ invertebrate predators (e.g., spiders) perceive signals in the UV range (Stevens, 2005).
An ongoing experiment started in 1988 by Lenski (2014) began with 12 populations (from the same ancestror) of Escherichia coli living in flasks containing 10 mL of DM25, a minimal medium containing 25 mg/L glucose as the limiting resource. Each day, 0.1 mL of each culture was transferred to a new flask with 0.9 mL of media. These bacteria have been evolving for >60,000 generations in identical, controlled, and constant environmental conditions. The founding strain is strictly asexual; thus, populations have evolved by natural selection acting on variation generated by spontaneous mutations that occurred during this long-term experiment (Blount et al., 2008). As a species, E. coli is characterized as not being able to grow on citrate under oxic conditions; however, in this experiment, the authors confirmed their hypothesis that these cells, when propagated in a medium containing abundant citrate, would evolve a new characteristic: some individuals of one population have evolved to efficiently use the abundant citrate in their environment, while continuing to use glucose, and have shown a huge increase in population density after 31,500 generations (Blount et al., 2008, 2012).
Testing Darwin's Concept of the Universal Common Ancestor
As first suggested by Darwin (1837–38), all organisms are descendents of a single species from the distant past. In other words, they share a common ancestor, which he identified at the bottom of the evolutionary tree with the number 1. The concept of universal common ancestry (UCA) is one of the central pillars of modern evolutionary theory; however, both the status and the nature of UCA have been questioned by several authors (Yonezawa & Hasegawa, 2010).
Theobald (2010) conducted the first formal test of UCA, not assuming that similarity in DNA sequence indicates a genealogical relationship. Using model selection theory to identify the hypothesis closest to reality, he found that UCA is the most accurate and parsimonious hypothesisis – at least 100× more probable than the competing hypotheses of independent or parallel origins of different taxa in the three domains of life (Eukarya, Bacteria, and Archaea). There is the possibility that separate populations could have merged, at some time point, by exchanging genes laterally, eventually leading to a single species that was ancestral to us all. For discussion of several features of science with students, I suggest using the figures in Theobald (2010) and some parts of his subsequent paper (Theobald, 2011), including the following text: “It is always possible that a biological model may be proposed in the future that explains the data better than the UCA models. I emphasize…that I have not provided absolute ‘proof’ of UCA. Proof is for mathematics and whiskey; it is not found in science. Nevertheless, these results provide strong evidence for UCA, given the hypotheses and sequence data currently available. As it stands, UCA explains the data best by far.”
Wang et al. (2011) defined the “last universal common ancestor” (LUCA) as the life-form from which all extant life-forms were derived – though not as the first life-form that emerged on Earth – and found that its occurrence seems to have been simultaneous with the appearance of oxygen in the atmosphere ~2.9 billion years ago (as estimated by a molecular clock), which enabled the emergence of aerobic metabolism; subsequently, enhancement of protein biosynthetic functions and of the cell membrane enabled the split into three domains of life, giving rise to the huge biodiversity seen today.
Novel Evidence of Precambrian Life, Evolutionary Stasis & “Living Fossils”
The Precambrian was a phase in the history of life that Darwin was not able to study. However, recent research has demonstrated the biogenic origin of Precambrian stromatolites (Greek: “layered rock”), which was controversial until the beginning of the 21st century (Allwood et al., 2006). The cyanobacteria that formed the stromatolites are thus the earliest life form found in the fossil record, estimated to have lived 3.4 billion years ago (Schopf, 1993). These photosynthetic organisms lived in colonies in shallow water. They produced stromatolites by the precipitation of calcium carbonate and sediments accumulated layer by layer, usually curved so that there was maximum exposure to sunlight. These patterns are found both in fossils (Figure 5A) and in extant rare living forms (Figure 5B) found in some regions, such as Western Australia and the Bahamas. Therefore, we might consider stromatolites as evidence of evolutionary stasis or “living fossils” (Schopf, 1994).
Living fossil is an ambiguous term with no consensual definition; however, there are some common features, such as the retention of ancestral morphology, apparent stasis over geologic time, and resemblance to ancient fossil forms. Therefore, living fossils are representatives of otherwise extinct groups and are often common in the fossil record; examples include the coelacanth and the lungfish, both described by Darwin, and the ginkgo tree, among others (Werth & Shear, 2014). Currently, some biologists consider that the evolutionary success of such species is due to their being ecological generalists, whereas others describe them as specialists that possess a key innovative characteristic that gives them a competitive advantage (Schopf, 1994).
Other microorganisms can be found in the Precambrian ecosystem, such as two deep-water, sediment-inhabiting, sulfur-cycling bacterial communities that have been found in the fossil record in Western Australia, one from the 1.8 Ga Duck Creek Formation and the other from the 2.3 Ga Turee Creek Group (Schopf et al., 2015). These authors found similarities between both fossil communities and modern sulfur bacteria (found in the coast of Chile) in morphology, habitat, and physiology (producing the same sulfur isotopes and pyrite as a metabolic product). Therefore, the living sulfur-cycling biota constitutes evidence of evolutionary stasis, described by Schopf et al. (2015) as the “greatest absence of evolution ever reported” in an environment unchanged over billions of years, although no molecular study was performed; because DNA sequences and proteins are not available for fossil species, the detection of any change in genes (e.g., affecting the efficiency of sulfur uptake) is not possible. I suggest discussing with students the two opposite views (see Appendix): “evolution's null hypothesis” (Schopf et al., 2015) and zero-force evolutionary law (McShea & Brandon, 2010; Coyne, 2015).
Implications for Science Teaching
The priorities for science education have been defined as developing students’ abilities to interpret and apply scientific information in the present and in the future as voters and decision makers (Moreno, 2007). However, misconceptions about science and the process of science are frequently found among undergraduate students – such as “It's not science unless it's an experiment”; “There is only one way to do science”; “Science is a linear, straightforward process”; and “Doing science does not require creativity – it is tedious and boring, not fun” – and these have been described as influencing students’ attitudes toward science and how students appreciate and value science as a way of knowing (Egger, 2009). Therefore, five aspects of NOS were defined as fundamental for any science curriculum (Moreno, 2007):
Science does not proceed in a linear fashion (see Figure 1)
Science is based on questions
Scientific knowledge is tentative (it may change, in the light of a new finding or new interpretations)
Some teaching strategies promote deep understanding of how science works (e.g., inquiry instruction)
Students should also understand that socioscientific context affects the questions that scientists choose – or are allowed – to ask. That context includes communication with peers in their own research group; communication between research groups, both informal (mail, e-mail, phone, social networks) and formal (conferences, scientific journals); and interchanges with the larger society. Darwin defined evolution as descent with modification from a common ancestor, an idea that has always been controversial, even though it neither implies the nonexistence (or existence) of God nor speak about God's role in the origin of life or the universe. Recently, the Vatican communicated that students should be able to learn about science independently of their religious beliefs (Singer, 2009), which is in accordance with the NSTA's official stand of excluding nonscientific explanations from science classes (NSTA Board of Directors, 2000), as well as with the Next Generation Science Standards (NGSS Lead States, 2013) and Dobzhansky (1973).
In biology, many laws and theories are associated with the probability of the occurrence of a phenomenon, so one should refer to strong evidence and not to proof. Evidence of these laws is the result of research conducted using scientific methods. The choice of the scientific method to be used depends on the question to be investigated, the type of study being performed, and the maturity of the particular subdiscipline. Furthermore, students should understand that hypothesis-driven and observation-driven studies are equally important and responsible for the advancement of scientific knowledge, both in the past and in the present.
Web Resources on NOS & Evolution for K–16 Classes
Understanding Science. Information about how science works.
Understanding Evolution. Information about evolution.
Long‐term Evolution Experiment. Richard Lenski's project site on experimental evolution using E. coli; includes ongoing results and publications.
Survival Game Who Wants to Live a Million Years? Interactive game for students to play online, applying the concepts of variation, mutation, and natural selection and the importance of diversity in a population to avoid extinction.
Avida‐ED Digital Evolution System for Education. Application that helps students learn about evolution and the scientific method by allowing them to design and perform experiments to test hypotheses about evolutionary mechanisms using evolving digital organisms.
Special thanks to the ones who inspired me. The views expressed in this paper are my own. I thank the anonymous reviewers for useful comments and suggestions and Faculdade de Ciências, Universidade do Porto, Portugal, for financial support.
Suggestions for K–16 Classes on Evidence of Evolutionary Stasis
The study entitled “Sulfur-cycling fossil bacteria from the 1.8-Ga Duck Creek Formation provide promising evidence of evolution's null hypothesis” by Schopf et al. (2015) got a great deal of attention in the press. In the week of its publication, one could find headlines such as “By not evolving, deep sea microbes may prove Darwin right” (Los Angeles Times) and “The mysterious 2 billion-year-old creature that would make Darwin smile” (Washington Post), as well as in blog posts by biologists, such as that of Jerry A. Coyne: “A new paper claims that evolution has stopped in a bacterial species. Is it true?” Therefore, I suggest exploring these resources with students in a classroom debate between two groups, using evidence from the literature to articulate the following two opposing claims:
“Evolution's null hypothesis” (Schopf et al., 2015): “Evolution is a result of organisms adapting to a changing physical or biological environment. The corollary to that is if the environment doesn't change, then you would predict the organisms wouldn't change, either” (http://www.nytimes.com/2015/02/10/science/unchanged-for-more-than-two-billion-years.html?_r=0).
“Zero-force evolutionary law” (McShea & Brandon, 2010): “Species probably change most rapidly when the environment is changing, but there's no reason why environmental change is a sine qua non for evolution…. Even in an unchanging environment, organisms can still evolve in significant ways. If new mutations arise that adapt the species better to that unchanging environment, then we will have evolution” (Coyne, 2015).
I recommend using Young (2006) in class to promote discussion by students on the characteristics of the Precambrian period, such as exceptionally slow (hypobradytelic) evolution of asexual, metabolically diverse, and ecologically versatile prokaryotes, of long-lived species, and of lineages that exhibit long-term stasis (Schopf, 1994).