Exploring Calyptra Function: A dissertation saga in summary

In mosses, the calyptra is a small cap of maternal tissue (1N - gamtophyte) that covers the top of the offspring (2N - sporophyte) during development. A long-held old hypothesis (from 1884!) is that the calyptra prevents the underlying tissues of the sporophyte from drying out. Think of it this way, the top of the moss offspring is made of young tissues that are sensitive to drying out. The idea is that the maternal plant provides a little cap on the top of its offspring to keep it safe from the harsh, cruel world. Similar to a mother sending her kids out to play in the snow with fuzzy hats to keep them warm. However, the idea with the calyptra is that it is a reverse shower cap, envision a old-fashioned shower cap keeping the water off your grandmother's perm, but rather than keeping the water out, it traps the water inside. With this little cap the apex is kept moist and can finish developing.

So this is a great idea and I have told you a nice tale, but this is a science blog and you came here for some evidence based findings, did you not. My PhD research focused on the hypothesis/idea that the function of the maternal moss calyptra is to prevent the apex/top of the offspring sporophyte from drying out as it grows and matures. 

Below are the highlights of my findings and how they connect to the examination of this hypothesis. Check out the figures and the summary statements in bold if you only have a moment. 

First the study organism - This is the moss Funaria hygrometrica, commonly called the cord moss. It is a plant that can be grown in the laboratory and is great for using in experiments.

Figure 1 from Budke et al. 2011 - Funaria hygrometrica
A. Moss sporophyte offspring.
B. Single sporophyte with calyptra on the top.
C. Small sporophyte covered by maternal calyptra.  

My first step was to examine the calyptra to look for features that would help in protection against dehydration. Plants are covered by an external layer of waxes and polymers (the plant cuticle) that prevents water loss from their bodies. I measured the thickness of the cuticle layers on two regions of the calyptra (rostrum, inflated base), the sporophyte, and leafy gametophyte (results in Figure 2).

Figure 2 from Budke et al. 2011
Cuticle thickness quantified

The cuticle covering the calyptra (both the rostrum and inflated base) are thicker than the cuticle on the leafy gametophyte and sporophyte. I also discovered that the calyptra rostrum has cuticular pegs, specialized cuticle thickenings that reinforce the cuticle in regions where the cells come together and may be leakier. These pegs were not found on any of the other structures that I examined. Both thicker cuticle layers and the presence of pegs are evidence supporting the hypothesis that the calyptra has a specialized cuticle that functions in preventing dehydration of the sporophyte apex. 

Budke JM, B Goffinet, and CS Jones. 2011. A hundred-year-old question: is the moss calyptra covered by a cuticle? A case study of Funaria hygrometrica. Annals of Botany 107: 1259-1277.

Part 1 summary - The calyptra has waxy layers that are significantly thicker than the leafy parts of the maternal plant, supporting the hypothesis that it is specialized structurally for preventing water loss. 

My second step was to examine the waxy cuticle (developmentally) to determine when the calyptra cuticle reaches maturity. I predicted that early during development the young sporophyte would have a thin cuticle and thus need protection from drying out. If the calyptra is providing protection, then I predicted that its cuticle would reach maturity early. Check out the figure to see how the moss changes size and shape during development. They start out so small, only a couple of millimeters tall, fractions of an inch.

Figure 1 from Budke et al. 2012
Moss sporophytes at nine developmental stages. 
All scale bars = 1 millimeter

  So I sliced and diced both calyptra and sporophytes at 9 different ages from young to old to figure out when the waxy cuticle develops on both the maternal calyptra and the offspring sporophyte. I found that all 4 layers of the calyptra cuticle were fully developed and thick at the earliest developmental stage, whereas the sporophyte is only covered by 1 or 2 thin layers at early developmental stages. Only later is the sporphyte covered by 4 thicker layers.

Figure 7 Budke et al. 2012
Diagram showing the four cuticle layers
present on the calyptra (c) at all 9 stages
and the wave of layers that are added
from the bottom to the top as the
offspring sporophyte (s) expands.

At early stages the maternal cap is fully protective with all 4 layers, whereas the sporophyte is covered by only 1 or 2 layers when young. This supports the idea that the calyptra is providing protection and the sporophyte requires protection. 

Part 2 summary - The calyptra is covered by four, thick cuticle layers at all developmental stages. The sporophyte is covered by only 1 or 2 layers early and more layers do not develop until later. This evidence supports the hypothesis that the maternal calyptra has the structural ability to protect the offspring sporophyte when it is young.  

Budke JM, B Goffinet, and CS Jones. 2012. The cuticle on the gametophyte calyptra matures before the sporophyte cuticle in the moss Funaria hygrometrica (Funariaceae). American Journal of Botany 99: 14-22.

Fig 5 Budke et al. 2013
A. Cuticle showing all layers present.
B. Cuticle after experimental removal of outer layer.

My third step was to carry out an experiment that tested the dehydration hypothesis to see if the waxy layers of the maternal calyptra are really necessary for sporophyte offspring success. I experimentally removed the waxy layers of the calyptra (a challenging task since all of the moss bits are so small) and then exposed the plants to a stressful dehydration event. This experiment showed that without the waxy cuticle on the calyptra sporophytes had lower levels of survival, they developed slower, and produced fewer spores per capsule. Some of them were even malformed and unable to open to release the spores. Remember that the spores are the part of the life cycle that disperses on the wind and arrives new places for the mosses to grow.

Part 3 summary - Under dry conditions, without the waxy layers, the maternal  calyptra is unable to protect the offspring sporophyte. Without the protective calyptra they are negatively affected. Fewer survive and they make fewer spores per capsule. This is another piece of evidence supporting the hypothesis that the maternal gametophyte calyptra is critical for protecting the offspring sporophyte from dehydration. 

Budke JM, B Goffinet, and CS Jones. 2013. Dehydration protection provided by a maternal cuticle improves offspring fitness in the moss Funaria hygrometrica. Annals of Botany 111: 781-789.

After my research we now have the scientific evidence to support the idea that the maternal moss calyptra is functioning to prevent the top of the young sporophyte offspring from drying out. No longer just a tale or hypothesis alone, there is now evidence to back up these ideas!

Stay tuned for additional parts of the calyptra story. I am working on a review paper summarizing and discussing the historical literature and experiments that focus on the moss calyptra and its function. Also, I am studying the calyptra cuticle comparatively in species that have small and large calyptra and small and large sporophytes.