Category Archives: Clock Classics

My new science post on the SciAm Observations blog – History of circadian genetics research

I wanted to write about this for years. Finally a good opportunity emerged: two new circadian papers provided the “news hook” for a blog post I wanted to write providing historical, philosophical, sociological, theoretical and methodological context for the findings in circadian genetics.

I also used the new tool – Dipity – to make timelines of key events in this history. The post is long, but serves as an Explainer, a “basics” post and a source of important references, so I hope people bookmark it for future reference.

I hope you have the time and patience to read it (perhaps save on Instapaper and read on your daily train commute):

Circadian clock without DNA–History and the power of metaphor

Then let me know what you think – comment there, share the link on social networking sites, respond on your own blogs, etc….

Postscript to Pittendrigh’s Pet Project – Phototaxis, Photoperiodism and Precise Projectile Parabolas of Pilobolus on Pasture Poop

ResearchBlogging.orgPostscript to Pittendrigh's Pet Project - Phototaxis, Photoperiodism and Precise Projectile Parabolas of Pilobolus on Pasture PoopThis is an edited, expanded, updated, revised and (hopefully) improved version of an old post. You can see the original here (or click on the “From The Archives” icon as usual).

Have you ever been out in the country visiting a farm? If so, you must have seen piles of manure, either stashed somewhere or just lying around the paddocks. And if that manure was a little older and starting to dry out and decompose, you likely saw some fine, white fuzz on its surface. Have you seen that? That fuzz is Pilobolus (not the dance troupe, but the fungus), one of a number of species in the genus. If you had a strong magnifying glass with you, and you trained it at the fuzz, you would have seen something like this:

Pilobolus has a portion of its life-cycle in which it has to pass through the digestive tract of a large herbivorous mammal. Since large mammals roam far and wide, this is a great way for the fungus to disperse. There is one problem, though: once excreted out with the feces, how do fungal spores get back into a large mammal again?

Unlike rabbits and some rodents, large mammals do not tend to eat their own manure. Actually, if you observe a field with a properly kept cow herd – a relatively small number of animals in a relatively large area, and rotated regularly between fields – you will notice that all the cows poop in one spot and no cow ever comes close to that spot to graze. So, what is a poor Pilobolus to do?

It gets ready, it aims, and it shoots!


Pilobolus assumes the position, builds a weapon, fills it with ammunition, aims and shoots. The position is on top of the pile of manure. The ammunition are spores, packaged tightly at the very tip of the filament. The weapon is the sporangiophore, a large swelled organ right below the tip.

The sporangiophore fills up with sap – osmotically active compounds – which builds up pressure until it is about 7 kilograms per square centimeter (100 pounds per square inch). There is also a line of weakness where the cap – the spore package – joins the sporangiophore vesicle. In the end, the pressure causes the sporangiophore to explode which sends the package of spores far, far away – if the wind is in the right direction, as far as 12 feet.

The goo from the sporangiophore goes with the spore package. It is very sticky, so wherever the spores land they tend to stay put. Ideally, that is on a blade of grass which is far enough from the manure pile to have a chance of getting eaten by a cow.

Here is a pretty picture of Pilobolus and a photomicrograph of the spore mass (crushed by the slide and slipcover):

[images from BioImages]

This is very cool (though wait for more coolness below), but also has an economic and environmental impact. Pilobolus spores themselves do not cause harm to their mammalian hosts, but some parasitic worms have evolved a neat trick – hitchiking on the Pilobolus spores right into the digestive tracts of large mammals.

While domestic cattle is regularly dewormed, the real problem is with wild ruminants, especially in places in which they do not have large areas to roam in, as in the elk in the Yellowstone Park. Here is a photograph of a Pilobolus harboring the Dyctiocaulus larvae:


So, Pilobolus shoots its spores really far away, by exerting enormous pressure on the ‘cap’. But, anyone who’s been in an artillery unit in the military will tell you that the distance is determined by angle. Soldiers manning the cannons know that an approximately 45 degree angle of the cannon will result in the greatest distance for the projectile. But a cannon projectile is a large, heavy object (also smooth and aerodynamic), so air resistance plays almost no part in this calculation – the force of gravity is the only force that the projectile needs to overcome.

A fungal spore is a microscopic object. At the small scale (pdf), physics works a little differently – gravity effects are minimal and the air resistance (drag) is the main determinant of maximal distance. Thus, 45 degrees is not neccessarily the optimal angle for achieving the greatest distance.

Frances Trail and Iffa Gaffoor, working with Steven Vogel at Duke University, made some calculations (which I have not seen and I do not think they got published, but I heard them from Dr.Vogel some years ago), looking at the shape and size of spore-caps of several species of Pilobolus (they published data on some other shooting fungi, though – you can read the paper here if you have access, sorry – not OA). The optimal angle for maximal distance ranges, in different species, between 9 and 30 degrees, the most common fuzz found on cow dung requiring about 15 degrees. The maximal distance, without wind, is about 6-7 feet. Quite right. Six feet is about as close as cows will come to a cowpie in well managed cattle establishments.

But does Pilobolus really shoot at 15 degrees? Well, what it does is it shoots towards the Sun. The way Pilobolus aims is through positive phototaxis. Like a sunflower, it follows the Sun in the sky and shoots at the Sun in the morning.

If you place Pilobolus in a box with light coming in only through a pinhole, all the fungi will shoot their spores at the pinhole:

How does Pilobolus see the light? Beneath the sporangium is a lens-like subsporangial vesicle, with a light-sensitive `retina’. It controls the growth and shape of the sporangiophore quite precisely. Thus, the packet of spores is always aimed at a light source:

So, the Pilobolus spores are found 6-12 feet away from the manure and they reproduce quite nicely even in the best managed cattle herds. So, they are probably shot at their optimal 15-degree angle. But they shoot at the Sun. Ergo, they shoot at the Sun when the Sun is about 15 degrees above the horizon.

One can think of two possible ways this can be achieved. One would be a mechanical sensor that triggers the explosion when the angle between the stalk and the cap is 15 degrees. This would work only if each individual was always standing upright on a flat surface, which is not the case on the rough relief of a manure pile.

The other strategy is to time the release so it coincides with the time when the Sun is about 15 degrees above the horizon. But, the trajectory of the Sun differs at different times of year.
In the middle of the summer in a high latitude, when the daylength is, let’s say, 18 hours, the Sun shoots straight up from the East and reaches the zenith right above exactly at noon. Thus, the Sun is around 15 degrees above the horizon about 90 minutes after dawn.

In winter, when the day may be only 6 hours long, the Sun traverses the sky low above the horizon from East to South to West, and may reach 15 degrees much slower (some Earth scientist in the audience can make a quick calculation here), e.g., 2 or even 3 hours after dawn.

How does the Pilobolus adjust to seasonal differences in Sun’s trajectory? By using its circadian clock, which entrains to different photoperiods with a systematically different phase:

Actually, the Pilobolus was the first fungus in which a clock was discovered. The effects of daylength on timing of spore-release was discovered back in 1948. The endogenous rhythmicity – meaning that the spores get shot every day even if there is no light present (in continous darkness) – was discovered in 1951. The major breakthrough was provided by (pdf) Esther-Ruth Uebelmesser in her dissertation:

At the same time that Schmidle published his findings, Esther-Ruth Uebelmesser (1954) dedicated her thesis work to the same subject. Her thesis is remarkable in many ways. Many of her experiments anticipated circadian protocols, frequently used in later years (different T-cycles and photoperiods, reciprocity, night interruption experiments, entrainment by temperature cycles, etc.). Although she did not fully exploit the richness of her experimental approaches in her interpretations, she must be considered a pioneer of the field and has certainly inspired Colin Pittendrigh to use Pilobolus as a circadian model system (Bruce et al., 1960). Probably, Pittendrigh abandoned this model system because of the unbearable smell penetrating the laboratory when the bovine dung media was prepared (Michael Menaker and Gene Block, personal communication, December 2000).


While in Neurospora accumulation of conidia (conidial bands) appears to be driven in these protocols with a constant phase angle in reference to lights-off (Fig. 2A), the phase angle of the spore-shooting rhythm in Pilobolus was systematically different with changing cycle lengths (Fig. 2B), possibly reflecting circadian entrainment. Closer investigation, however, revealed that the Pilobolus sporulation rhythm is also driven by the LD cycle, but unlike in Neurospora, by lights-on. Sporulation in Pilobolus is triggered by light, and the spores mature for approximately 28 h before they are shot (see arrows in Fig. 2B and C). The maturation time represents a kind of memory capacity for prior events. This is seen in experiments in which the fungi were released to DD (e.g., from LD 4:4 shown in Fig. 2C). The rhythm, synchronized to a given light cycle, persists for another 28 h until the endogenous circadian control takes over. Thus, depending on conditions, the production of asexual spores in Pilobolus is controlled both by the clock (phase angle) and by light (a driven spore release once per LD cycle).

[images from Roenneberg and Merrow 2001]

What this all means is that a circadian clock in this fungus is entrained by the dawn (not dusk) and it integrates photoperiodic information in a manner that is consistent with the need to shoot spores towards the Sun at the time of the morning when the Sun first reaches 15 degrees (actually, the tracking movement of the spore lags the Sun by about 20 minutes – fungi are slow to move – but even that is probably compensated for by the circadian clock).

Moreover, Pittendrigh’s students discovered that the Pilobolus clock is extremely sensitive to light (both intensity and duration of light). Its clock requires only a millisecond of light to be completely reset.


In a more recent paper, the explosive ejection of the spores was filmed with an ultra-high-speed video camera and in their subsequent calculations derived from the images, the “launch speeds ranged from 2 to 25 m s−1 and corresponding accelerations of 20,000 to 180,000 g propelled spores over distances of up to 2.5 meters.” You can see the video (turn on the volume – it is set to music) here:

What next?

This is where the story ends, for the time being. But there are still gaps.

For instance, I am not sure if it was ever tested in the laboratory that Pilobolus actually shoots at 15 degrees. This is, according to Dr.Vogel, relatively easy to do, by placing the fungi on a manure-based medium at the center of one of those transparent semi-spheres used by exhibitors at various product fairs (e.g., technology fairs). The ejected spores stick to the inside of the transparent plastic and can be seen from the outside. Measuring the length of the arc from the desk to the spore (and knowing the radius) is all one needs to calculate the angle.

Second, we still do not know for sure if the Pilobolus cues in to the season-specific photoperiod (more likely) or the season-specific Sun trajectory (less likely). One can, in the laboratory, dissociate these two factors by exposing groups of fungi to summer-specific photoperiod and winter-specific trajectory (using a strong flashlight attached to a string and mounted on an arc-shaped wire, attached to a little motor) or vice-versa, as well as season-specific photoperiod with diffuse (instead of focused) light source.

Finally, an evolutionary question. Horses are not as picky as cows concerning the distance from the manure at which they will graze. Pilobolus lives in our horses and shows up in the manure all the time. Is there relaxed selection for the populations (species?) that live in horses? Is their timing off? Is their angle-determination lousy? This would be an easy head-to-head test in the lab (and field) as well. And if there is such a difference between species, looking at molecules – dynamics of gene expression patterns and protein-protein interactions – can perhaps teach us something more about the ways simple parts can accomplish complex tasks in these organisms.

But, if you’d rather learn all of the above in a Dr.Seuss-like poem, go ahead, it’s right here.


Bruce, V., Weight, F., & Pittendrigh, C. (1960). Resetting the Sporulation Rhythm in Pilobolus with Short Light Flashes of High Intensity Science, 131 (3402), 728-730 DOI: 10.1126/science.131.3402.728

TRAIL, F., GAFFOOR, I., & VOGEL, S. (2005). Ejection mechanics and trajectory of the ascospores of Gibberella zeae (anamorph Fuarium graminearum) Fungal Genetics and Biology, 42 (6), 528-533 DOI: 10.1016/j.fgb.2005.03.008

Fischer, M., Stolze-Rybczynski, J., Cui, Y., & Money, N. (2010). How far and how fast can mushroom spores fly? Physical limits on ballistospore size and discharge distance in the Basidiomycota Fungal Biology, 114 (8), 669-675 DOI: 10.1016/j.funbio.2010.06.002

Roenneberg, T., & Merrow, M. (2001). Seasonality and Photoperiodism in Fungi Journal of Biological Rhythms, 16 (4), 403-414 DOI: 10.1177/074873001129001999

Uebelmesser E-R (1954) Über den endogenen Tagesrhythmus der Sporangienbildung von Pilobolus. Arch Mikrobiol 20:1-33.

Yafetto, L., Carroll, L., Cui, Y., Davis, D., Fischer, M., Henterly, A., Kessler, J., Kilroy, H., Shidler, J., Stolze-Rybczynski, J., Sugawara, Z., & Money, N. (2008). The Fastest Flights in Nature: High-Speed Spore Discharge Mechanisms among Fungi PLoS ONE, 3 (9) DOI: 10.1371/journal.pone.0003237

Clock Classics: It all started with the plants

I was wondering what to do about the Classic Papers Chellenge. The deadline is May 31st, and I am so busy (not to mention visiting my dentist twice week which incapacitates me for the day, pretty much), so I decided to go back to the very beginning because I already wrote about it before and could just cannibalize my old posts: this one about the history of chronobiology with an emphasis on Darwin’s work, and this one about Linnaeus’ floral clock and the science that came before and immediately after it.
In the old days, when people communed with nature more closely, the fact that plants and animals did different things at different times of day or year did not raise any eyebrows. That’s just how the world works – you sleep at night and work during the day, and so do (or in reverse) many other organisms. Nothing exciting there, is it? Nobody that we know of ever wondered how and why this happens – it just does. Thus, for many centuries, all we got are short snippets of observations without any thoughts about causes:

“Aristotle [noted] that the ovaries of sea-urchins acquire greater size than usual at the time of the full moon.”(Cloudsley-Thompson 1980,p.5.)
“Androsthenes reported that the tamarind tree…, opened its leaves during the day and closed them at night.”(Moore-Ede et al. 1982,p.5.)
“Cicero mentioned that the flesh of oysters waxed and waned with the Moon, an observation confirmed later by Pliny.”(Campbell 1988, Coveney and Highfield 1990)
“…Hippocrates had advised his associates that regularity was a sign of health, and that irregular body functions or habits promoted an unsalutory condition. He counseled them to pay close attention to fluctuations in their symptoms, to look at both good and bad days in their patients and healthy people.”(Luce 1971,p.8.)
“Herophilus of Alexandria is said to have measured biological periodicity by timing the human pulse with the aid of a water clock.”(Cloudsley-Thompson 1980, p.5.)
“Early Greek therapies involved cycles of treatment, known as metasyncrasis….Caelius Aurelianus on Chronic and Acute Diseases…describes these treatments.. .”(Luce 1971, p.8.)
“Nobody seems to have noticed any biological rhythmicities throughout the Middle Ages. The lone exception was Albertus Magnus who wrote about the sleep movements of plants in the thirteenth century” (Bennet 1974).

de%20Mairan%20face.jpgThe first person to ask the question – and perform the very first experiment in the field of Chronobiology – was Jean-Jacques d’Ortous de Mairan, a French astronomer. What did he do?
In 1729, intrigued by the daily opening and closing of the leaves of a heliotrope plant (the phenomenon of ‘sleep in plants’ was well known due to Linneaus), de Mairan decided to test whether this biological “behavior” was simply a response to the sun. He took a plant (most likely Mimosa pudica but we do not know for sure as Linnean taxonomy came about a decade later) and placed it in a dark closet. He then observed it and noted that, without having access to the information about sunlight, the plant still raised its leaves during the day and let them droop down during the night.
However, De Mairan was an astronomer busy with other questions:

“….about the aurora borealis, and the relation of a prism’s rainbow colors to the musical scale, and the diurnal rotation of the earth, and the satellites of Venus, and the total eclipse of the sun that had occurred in 1706. He would waste no time writing to the Academy about the sleep of a plant!”(Ward 1971,p.43.)

de%20Mairan%20paper.jpgHe did not wanted to waste his time writing and publishing a paper on a mere plant. So his experiment was reported by his friend Marchant. It was not unusual at that time for one person to report someone else’s findings. Marchand published it in the Proceedings of the Royal Academy of Paris as he was a member, and the official citation is: De Mairan, J.J.O. 1729. Observation Botanique, Histoire de l’Academie Royale des Sciences, Paris, p.35.
In the paper Marchant wrote:

“It is well known that the most sensitive of the heliotropes turns its leaves and branches in the direction of the greatest light intensity. This property is common to many other plants, but the heliothrope is peculiar in that it is sensitive to the sun (or time of day) in another way: the leaves and stems fold up when the sun goes down, in just the same way as when touches or agutates the plant.
But M. de Mairan observed that this phenomenon was not restricted to the sunset or to the open air; it is only a little less marked when one maintains the plant continually enclosed in a dark place – it opens very appreciably during the day, and at evening folds up again for the night. This experiment was carried out towards the end of one summer, and well duplicated. The sensitive plant sense the sun without being exposed to it in any way, and is reminiscent of that delicate perception by which invalids in their beds can tell the difference between day and night. (Ward 1971)”

Marchant and de Mairan were quite careful about not automatically assuming that the capacity for time measurement resides within the plant. They could not exclude other potential factors: temperature cycles, or light leaks, or changes in other meteorological parameters. Also, the paper, being just a page long (a “short communication”), does not provide detailed “materials and methods” so we do not know if “well repeated” experiments meant that this was done a few times for a day or two, or if the same plants were monitored over many days. We also do not know how, as well as how often and when, did de Mairan check on the plants. He certainly missed that the plants opened up their leaves a little earlier each day – a freerunning rhythm with a period slightly shorter than 24 hours – a dead giveaway that the rhythm is endogenous.
The idea that clocks are endogenous, residing inside organisms, was controversial for a very long time – top botanists of Europe were debating this throughout the 19th century, and the debate lasted well into the 1970s with Frank Brown and a few others desparately inventing more and more complicated mathematical models that could potentially explain how each individual, with its own period, could actually be responding to a celestial cue (blame Skinner and behaviorism for treating all behaviors as reactive, i.e., automatic responses to the cues from the environment).
The early 18th century science did not progress at a speed we are used to today. But the paper was not obscure and forgotten either – it just took some time for others to revisit it. And revisit it they did. In 1758 and 1759 two botanists repeated the experiment: both Zinn and Duhamel de Monceau (Duhamel de Monceau 1758) controlled for both light and temperature and the plants still exhibited the rhythms. They used Mimosa pudica, which suggests to us today that this was the plant originally tested by de Mairan.
Suspecting light-leaks in de Mairan’s experiment, Henri-Louis Duhamel du Monceau repeated the same experiment several times (Duhamel du Monceau 1758). At first, he placed the plants inside an old wine cave. It had no air vent through which the light could leak in, and it had a front vault which could serve as a light lock. He observed the regular opening and closing of the leaves for many days (using a candle for observation). He once took a plant out in the late afternoon – which phase-shifted the clock with a light pulse. The plant remained open all night (i.e.., not directly responding to darkness), but then re-entrained to the normal cycle the next day. Still not happy, he placed a plant in a leather trunk, wrapped it in a blanket and placed it in a closet inside the cave – with the same result: the plant leaves opened and closed every day.
So, he was convinced that no light leaks were responsible for the plant behavior. Yet he was still not sure if the temperature in the cave was absolutely constant, so he repeated the experiment in a hothouse where the temperature was constant and quite high, suspecting that perhaps a night chill prompted the leaves to close. He had to conclude: “I have seen this plant close up every evening in the hothouse even though the heat of the stoves had been much increased. One can conclude from these experiments that the movements of the sensitive plant are dependent neither on the light nor on the heat” (Duhamel de Monceau 1758). He did not know it at the time, of course, but he was the first to demonstrate that circadian rhythms are temperature compesated – the period is the same at a broad range of constant temperatures.
The research picked up speed in the 19th century. Augustus Pyramus de Candolle repeated the experiments while making sure not just that the darkness was absolute and the temperature constant, but also that the humidity was constant, thus eliminating another potential cue. He then showed that the period of diurnal movements of Mimosa is very close to 24 hours in constant darkness, but around 22 hours in constant light (using a bank of six lamps). He also managed to reverse day and night by using artificial light to which the plants responded by reversing their rhythms (De Candolle 1832) after the initial few days of “confusion”.
Another astronomer, Svante Arrhenius argued that a mysterious cosmic Factor X triggered the movements (Arrhenius 1898). He attributed the rhythms to the “physiological influence of atmospheric electricity”. Charles Darwin published an entire book on the Movement of Plants in 1880, arguing that the plant itself generates the daily rhythms (Darwin 1880).
The most famous botanist of the 19th century, Wilhelm Pfeffer, started out favouring the “external hypothesis”, arguing that light leaks were the source of external information for de Mairan’s and Duhamel’s plants (Pfeffer 1880, 1897, 1899). But his own well-designed experiments (as well as those of Darwin) forced him to change his mind later in his career and accept the “internal” source of such rhythmic movements. Unfortunately, Pfeffer published his latter views in an obscure (surprisingly, considering the short and catchy title) German journal Abhandlungen der Mathematisch-Physischen Klasse der Königlich Sächsischen Gesellschaft der Wissenschaften, so most people were (and still are) not aware that he changed his mind on this matter.
In the early 20th century, Erwin Bunning was the first to really thoroughly study circadian rhythms in plants and to link the daily rhythms to seasonality. He and many others at the time mostly studied photoperiodism and vernalization in plants, two phenomena then thought to be closely related (we know better today). For the rest of the century, animal research took over and only recently, with the advent of molecular techniques in Arabidopsis, has the plant chronobiology rejoined the rest of the field.

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