We live in a highly rhythmic world, and humans have developed a number of strategies for marking the passage of time by counting rhythms. Perhaps the most obvious is the 24 hour cycle of light and dark that corresponds to the time it takes for the earth to make a full rotation along its axis (“one day”). From a single point on earth, we experience it as the predictable rising and setting of the sun. The time required for the earth to make a full revolution of around the sun is called “one year” – tracked at first in human history by observing the repetition of constellations’ position in the sky but more obviously experienced as the cycling through the seasons. The time for the moon to revolve around the earth is roughly 29 days, and is the approximate basis for measuring a “month” – this rhythmic process induces observable alterations of high and low tide, as well as the changing phases of the moon.
Photoperiod (plotted on the y-axis) varies by latitude (how far north or south you are on the globe). Each curve plots calculations for a particular latitude in the northern hemisphere, (see legend at top-right). The variation of photoperiod by latitude is the reason for seasonal "white nights" near the north and south poles. But photoperiod also shows rhythmic variation depending on the time of year (see x-axis).
Changes in photoperiod (see above) are one aspect of annual environmental rhythms that humans mark as the passing of "seasons." In addition to the duration of daylight (photoperiod), other ecological features such as light intensity, temperature, and humidity all vary with season. Seasons are ultimately due to the earth's rotation around its tilted axis as it orbits the sun.
Credit: NASA and NOAA
Long before humans developed words to label and keep track of such rhythms, each of these rhythms was a basic concern for most life on earth. With the passage of a year comes changes in the environment that we experience as seasons, which (over most of the globe) seriously affects the availability of resources and influences many organisms’ nutrition. The lunar rhythm of months, and the associated rhythm of tides, is important to marine life, especially in coastal regions. And for many organisms, the 24-hour rhythm of a day is of supreme importance, as it determines the availability of light – a decisive source of energy and nutrition for all photosynthetic organisms, and thereby for the entire biome. But the earth’s daily schedule is much more complex than just a switch between day and night. Temperatures change throughout the day, as does the quality of light, as the sun passes overhead. These rhythmic environmental changes lead to a cascade of rhythms among terrestrial life. The old saying that “the early bird catches the worm” offers only a hint at the temporal patterning of life on earth. The early bird catches the worm that stayed at the surface too late, and a bird of prey might just as easily catch the “early bird,” if it lingers too late. Just as much of modern human life is busy-busy-busy, rushing from one appointment to the next, so likewise many organisms make a living on earth by doing the right thing at just the right time.
Human methods of keeping track of time have attained tremendous precision using hard-won technical knowledge. Modern time-keeping methods, like the atomic clock, rely on physical principles that were only understood in the middle of the 20th century. But long before we were able to build an atomic clock, humans (and many other organisms) benefitted from having a biological mechanism, a circadian clock, which also enabled them to keep rather precise time from day to day. A clock is circadian insofar as it enables an organism to reliably track a length of time which is about the length of a day (a rotation of the earth) – the term comes from the Latin circa (about) + diem (day). A biological clock mechanism could evolve only by tracking this sort of natural, geophysical rhythm: there are no biological clocks that track a “week,” since this human unit of time has no basis in our geophysically rhythmic world. To say that this biological mechanism is a clock is to say that it keeps time in a reliable fashon, and enables the organism to anticipate when repeating environmental events will occur.
For us today, the evolutionary logic of why an organism might benefit from having a circadian clock is straightforward and compelling. With a functioning clock, the organism can “plan ahead” for environmental changes that reliably recur, instead of just “waiting to see” what will happen and only taking action then. If a photosynthetic organism like a tree, for example, could predict when the sun would rise, then it could get ready in advance, initiating metabolic processes to prepare for photosynthesis. If instead the tree starts up these processes only once the sun has risen, it will miss out on the opportunity to harness the energy of the sun during this preparation interval and thereby reduce nutritional efficiency. Likewise, the “early bird” benefits from being able to predict when worms will be at the surface (though perhaps not as much as the worm would benefit from predicting when the bird will appear).
So there is at least one good evolutionary rationale for why organisms should have an internal sense of time. However, demonstrating that an organism has an internal clock is no trivial matter. Mimosa plants (and many others) have a daily rhythm in the state of their leaves: during the day, the leaves are spread open but they fold up at night. This rhythmic pattern of opening and closing repeats every 24 hours. But why should we believe that this may be controlled by an internal circadian clock, and what evidence would we need to establish that it is? How might we show that the plant is predicting when day will occur, and opening up its leaves in anticipation, as opposed to simply reacting to some environmental cue, and opening up its leaves in response? Perhaps the daily environmental rhythm of light and dark, or increasing and decreasing temperatures, simply causes the plant’s leaves to open and close, without any intrinsic mechanism operating in the plant to anticipate day-night cycles. In 1729, French astronomer Jean Jacques d’Ortous de Mairan began to systematically examine Mimosa pudica plants. Placing a specimen of the plant in a constantly dark corner of his cupboard, de Mairan noticed that the rhythmic pattern of leaf movements continued to occur in the absence of light cues. This simple experiment – verifications of which have been repeated thousands of times since then in different species and different cell types – is recognized as the intellectual foundation establishing one of the fundamental principles of chronobiology: the internal circadian clock.
If a circadian rhythm of an organism’s activity is controlled by a circadian clock, and thus arises from an anticipation of environmental changes (rather than simply responding to them as they happen) then the rhythmicity of the organism’s behavior ought to show persistence in constant conditions. In other words, when rhythmic inputs from the environment are removed – in this case daily fluctuation in light and dark – any continuation in the rhythm of the organism cannot result from that missing environmental input. de Mairan’s study provides an initial illustration: the Mimosa plants did not cease exhibiting a circadian rhythm in leaf-opening and leaf-closing when they were no longer causally affected by a light-dark cycle. This persistence suggests that the plant’s rhythmicity is not a simple response to environmental rhythmicity in light, but rather occurs in anticipation of it, and thus that it is genuinely controlled by a circadian clock.
While de Mairan’s study establishes that leaf-movements are not programmed by variations in light, his findings are not decisive for showing that Mimosa plants have a circadian clock, because it was not technically possible to establish constant conditions for all environmental variables. For example, the ambient temperature varies on a 24 h basis with generally higher temperatures during the day and lower at night: perhaps de Mairan’s plants were merely responding to changes in temperature. About a century later, in 1832, French-Swiss botanist Alphonse de Candolle verified de Mairan's findings that the light environment was not driving the leaf movements, but he provided another piece of evidence that supports the claim that Mimosa pudica plants rely on an internal circadian clock. de Candolle’s key observation was reported in his Physiologie Végétale (1832), in a chapter on the movement of plants, and in a section entitled “On the sleep of leaves.” Here de Candolle uses the common name for Mimosa pudica as “sensitive plant:”
“When I exposed the sensitives to continuous light, they had, as in the ordinary state of things, alternative sleep and waking [of their leaves]; but each of the periods was a little shorter than usual, the acceleration was on various stalks an hour and a half or two hours daily” (de Candolle 1832, Vol II, pp.860-61).
Based on this report, it is often said that de Candolle determined that in constant light, the Mimosa plants would fold their leaves once every “22-23 hours.” In the discussion that follows, we will refer to the period, or cycle length of this rhythm, as 22.5 h. This periodicity suggests that the plants could not be responding to ANY environmental rhythm such as temperature (or humidity or other unknown variables) because there is no environmental cue that fluctuates on a 22.5 h basis. If leaf movements were programmed by envronmental cues, then the leaf-folding should have matched the period of the myriad environmental cues of the natural day - i.e., every 24 h. Instead, the leaf-movements showed their own, non-24-hour period. This discovery led de Candolle to hypothesize that the Mimosa plant’s leaf movements were coordinated by an endogenous, or internal mechanism. In present-day chronobiology, it is recognized that an organism’s circadian clock does not, on its own, keep track of precisely 24-hour periods. Rather, when we “free” the clock from any environmental input, we are able to measure its free-running period (FRP), which is usually slightly less or slightly more than 24 hours (again: circa-dian, about a day). We will say more about FRPs later.
According to this hypothesis, when the Mimosa plants were placed into constant light, their internal clock continued to “tick,” but no longer reliably tracked the 24 h day. The plant’s clocks were running a little faster than the natural day (or, as circadian biologists say, the clock had a period of less than 24 h). de Candolle’s findings would later receive additional support that de Mairan did not have readily available. Not too long after de Candolle’s experiments, Charles Darwin would publish the Origin of Species (1859). As evolutionary theory gained ground in the biological sciences, de Candolle’s hypothesis would appear more and more attractive. An organism that had evolved a biological “clock” as an adaptation would be better able to respond to selection pressures, and would gain in fitness; if the clock were heritable, then one would expect such an evolutionary innovation to quickly become widespread. Darwin himself later performed his own experiments with plants, and promoted this hypothesis (Darwin & Darwin 1880, see esp. pp.407, 560). But unlike Darwin’s flagship cases of adaptive traits (e.g., bird beak-sizes and beak-shapes), it was difficult to work out what a circadian clock could be, as a trait of an organism. As a result, a few scientists remained unconvinced, and thought that some as-yet-unknown, external cue might be responsible for circadian rhythmicity, (although they had no very good argument for why the periods were not exactly 24 h).
Despite not knowing where clocks were or how they worked, by the 1950s, most circadian researchers were confident in the internal clock model as an explanation for the periodicity exhibited by so many organisms’ behaviors (but see Brown’s remarks in Brown, Hastings & Palmer 1970). In this basic review, we will not discuss the wealth of later empirical details establishing the molecular and genetic mechanisms that make up circadian clocks in different organisms (but see Dunlap, Loros & DeCoursey 2004, esp. ch.7). Our goal is rather to provide further introduction to the kinds of “phenomenological” evidence that has led biologists to assert the existence of circadian clocks in the first place. In doing so, we are largely following in the footsteps of Colin Pittendrigh and Jürgen Aschoff, who are widely regarded as the founders of circadian biology.
The foregoing provides an illustration of two key features of circadian clocks. As is illustrated by the initial findings of de Mairan and de Candolle, organisms exhibit some forms of rhythmic behaviors and activities that are best accounted for by the hypothesis that the organism has an endogenous circadian clock that keeps track of time persistently (without external cues), as evident in its free-running period (FRP). The hypothesis states that this clock controls, schedules, or programs the organism’s activities. The clock can thus be considered an endogenous, self-sustained oscillator (Pittendrigh & Bruce 1957). To these, two further features of circadian clocks must be added.
Often in science, convincing and decisive evidence for an hypothesis is most effectively obtained under somewhat artificial conditions. For example, de Marain and de Candolle showed that an organism has an endogenous circadian clock with its own, non-24-hour free-running period (and is not just responding to the environment), by putting the organism in constant conditions. But this is a highly artificial condition for most terrestrial organisms, and putting a Mimosa plant in constant darkness or constant light is a distinctively human manipulation. This kind of manipulation enables us to see that an organism has a clock, but it raises some real questions about how the clock normally operates. Normally, an organism’s internal clock is adjusted to match its external environment: when Mimosa plants are in the local day-night cycle, their rhythms of leaf-opening and leaf-closing do in fact track the 24-hour daily cycle, even though their free-running period (as revealed under constant conditions) is 22.5 hours. How do we make sense of this?
The interplay between clock and environment is an important part of why circadian clocks are adaptive. On the one hand, if the organism has no capacity to anticipate changes, using its own clock, then it cannot exploit environmental regularities – it will only be able to wait and see what happens and respond after the fact. On the other hand, if the organism keeps time only using its non-24-hour free-running period, then it will not be able to properly predict events in the actual environment, where events do in fact occur on a strict 24-hour rhythm. It is crucial that the organism be able to synchronize or entrain its clock to the local 24-hour cycles. As explained further below, entrainment can occur if light or some other environmental stimulus adjusts or resets the rhythm to make up for the 1.5 h difference between the free-running period and the exactly 24 h day. As such, a third defining feature of any natural circadian clock is that it must have a capacity for entrainment.
There is one final defining feature of circadian clocks: their operations are temperature compensated. We often forget that basic biology is dictated by basic chemistry, and that in chemistry, temperature is all-important. Many chemical reactions run faster at higher temperatures, and this basic rule affects a great many biological processes as well. But Erwin Bünning (1964) showed that the circadian clock manages to maintain its free-running period despite significant changes in ambient temperature. In other words, clocks do not generally run more slowly in winter than in summer, whereas other biological processes do (e.g., think of growth rates in bacteria or heart rates in frogs). Again, the evolutionary logic of temperature compensation is straightforward and compelling: a clock that is not insulated from changes in ambient temperature would not benefit many organisms, because temperature can vary widely depending upon the time of day, organisms' own current activity, or even depending upon whether the organism is in direct sunlight or in shadow.
This brief introduction has described the four defining principles of circadian clocks. An organism’s circadian clock is defined as an endogenous and temperature-compensated mechanism, which (if left to its own) counts a day as occurring over the length of the free-running period (FRP) (as is revealed by putting the organism in constant conditions), but which is normally susceptible to entrainment to the local 24-hour cycle. A circadian clock is functionally defined in terms of these four features: a circadian clock is defined as whatever it is inside an organism that enables it to keep time in this way. Again, our focus here is not on the details of the diverse biological mechanisms that in fact play this role in any given organism.
In what follows, we will first (Part II) introduce the reader to the conventions of data-gathering and data-representation that chronobiologists use to conclude that the existence of a circadian clock is a suitable explanation for an organism’s rhythmic activities. To introduce these methods and conventions, we will stick to a fairly basic sketch of circadian clocks. We will then (Part III) supplement this by illustrating some complexities of circadian clocks.