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.

Cool New Cryo SEM

The electron microscopy facility that I work in here at UConn just got a new piece of equipment over the summer and I have some images from it to show off. 

The laboratory now has a cryo-stage for the scanning electron microscope! But let's take a step back in case this type of microscopy is new to you. Basically a scanning electron microscope (SEM) shoots electrons at a sample that is placed in a chamber under high vacuum. The electrons bounce off the sample and enable you to detect an image of the surface that is at a much higher magnification than you can see with a light microscope. The light (dissecting) microscope that I have in the lab magnifies 50-250X, whereas the SEM can magnify up to 200,000X! That is pretty awesome in and of itself, but the cryo-stage adds a whole other level to this equipment. Typically the samples that you look at have to be completely dry before placing them into the vacuum. This is a big issue for biological samples, which can be full of water. There are a number of ways to get rid of the water, but these processes often change the shape of the structures. For some studies this is not a major issue, but for other studies scientists are really trying to see what the plants or animals look like when they are hydrated as they would be when alive. 

Cryo-stage to the rescue! With this equipment a sample can be flash frozen in liquid nitrogen with all of the water in place and then placed into the microscope on the chilled cryo-stage. Then the sample can be viewed with the tissues fully hydrated.

I was out of town when they used some of my moss samples for a test run, so I didn't get to see the equipment in action, but here are some of the images that were taken.

This is a leafy gametophyte stem with a cluster of antheridia at the apex. We are looking down at the top of the stem and there is a second leafy gametophyte lying on its side in the background. The leaves are fully expanded and in an arrangement that you would see when hydrated. If they were dry they would be all folded and curled up on themselves.

Here is the cluster of antheridia and hairs at higher magnification. 

And at an even higher magnification. The hairs located in the antheridia clusters in the Funariaceae are characterized by having a large apical cell, which we can see here is fully hydrated. The filaments covering the hairs are probably fungs or bacteria. These mosses were grown in the laboratory but not in sterile conditions.

Some of the leaves were removed from the gametophyte to make for easier viewing. This image shows the inside of the leaf cells. The outline of the cell walls are visible and it is super cool that we can see the water filling each of the cells.

Congratulations to Dr. Cantino and colleagues on their successful National Science Foundation research grant that funded this new piece of equipment.

A Sporophyte Gone Wild

What happens when a moss sporophyte's calyptra does not detach properly? Really odd development! I came across this sporophyte in one of my Funaria hygrometrica cultures recently.

Calyptra Recap: The calyptra is a small cap of gametophyte tissue that covers the moss sporophyte apex during its development. It is necessary for proper capsule and spore formation in moss sporophytes. Studying the calyptra-sporophyte interaction was the focus of my dissertation research. (You can read more about my main findings here. The calyptra has a cuticle. - The calyptra cuticle develops early relative to the sporophyte cuticle.)

So, this really odd development. It looks like the calyptra did not detach from the rest of the leafy gametophyte properly. Usually there is a line of dehiscence at the bottom that allows the calyptra to separate as a distinct cap. In this specimen the calyptra appears to have split open down the side with the sporophyte continuing to grow. As usual, sporophytes without their calyptra on top do not develop a capsule at all and instead produce an obconic-shaped sporophyte.

On this sporophyte, the apical region and seta meristem both appear to have turned brown and died. Also, there is a funny little projection of tissue sticking off the right side of the thickened stalk. (There is a zoomed-in photo below.) I don't think that I have ever seen anything that looks like this on a sporophyte before!

What is it? Is it branching? Maybe. Is it the beginning of a leaf? Probably not. It would be really cool to section it and see what the internal anatomy looks like. Does it have  central strand tissue going out into it? It looks pretty small so probably not. I wonder if I saved this sample in the lab after taking some photos of it so that I can process it for some anatomical study. Either way, I think that it is some pretty cool morphology to think about!

 

On the Left: An close-up of the sporophyte breaking through the side of the not detached-calyptra. On the right: An up-close view of the tissue projecting from the sporophyte.

A Desktop Calendar Experiment

I change the background image on my computer once a month and I really like having a calendar on my computer desktop, since I don't have a wall calendar in the office. For the past several years I have been using the desktop calendars from Chocolate and Zucchini, a food blog that I follow. Unfortunately, she is no longer making the images with calendars. Thus I was left with a dilemma. I need to find a new place to get my desktop calendars. I didn't find any that I really wanted to look at for a month, so I decided to make my own. I have a lot of bryophyte images that I wouldn't mind looking at for a month and here is the result. 

If you are interested in downloading this desktop calendar follow the instructions below. 

1 - Single click on the image to open it up in a new window. (If you use the image directly from the blog post you will loose a lot of resolution.)

2 - Right-click (or ctrl-click) on the image, and chose the option that says, "Set as Desktop Background" or "Use as Desktop Picture". The wording may vary.  

3 - If the image does not fit your desktop neatly, you may have to adjust the image (Mac: System Preferences > Desktop & Screen Saver > Desktop; Windows: Control Panel > Display > Desktop) and choose "Fill screen" as the display mode of your background image.

I hope that the image comes through with enough resolution and that I positioned the calendar well so that it doesn't get cut off. Any issues or suggestions please let me know. This is totally an experiment and we shall see how it goes.

And I almost forgot the bryological information. These are leafy gametophytes of the moss Funaria hygrometrica (cord moss) that I grew in the laboratory. It is a population from Connecticut that I used for my PhD research. 

The blog is changing

You may have noticed some changes to the look of the blog recently. It is in the process of being migrated to the science blog network Field of Science. I decided that it might be a fun step in the evolution of my blog to join up with other scientists for more interactive discussions about science and mosses.

I am off to the Botanical Society of America's annual conference on Saturday to present a part of my dissertation research on the calyptra cuticle in the moss Funaria hygrometrica. Also I will be presenting  results from a group project in my lab on the systematics and morphological evolution in the Funariaceae.

Wish me luck it is my first time presenting TWO talks at a scientific meeting and they are on the same day!

The Calyptrae of Funaria hygrometrica have a cuticle.

 The first chapter of my PhD dissertation research has been published!

Jessica M. Budke, Bernard Goffinet and Cynthia S. Jones. 2011. A hundred-year-old question: is the moss calyptra covered by a cuticle? A case study of Funaria hygrometrica. Annals of Botany 

Below are some links for access to the publication courtesy of Oxford Journals.

Full Text: http://aob.oxfordjournals.org/cgi/content/full/mcr079?ijkey=nTMQI5rz8MP6cGB&keytype=ref

PDF: http://aob.oxfordjournals.org/cgi/reprint/mcr079?ijkey=nTMQI5rz8MP6cGB&keytype=ref 

Mosses Grow on a New Substrate. Whale!

Mosses grow on all sorts of substrates. Soil, tree bark, leaves, rocks, sand, dung, old socks abandoned in the woods, and now a whale! My labmates were out visiting the Seymour Marine Discovery Center at Long Marine Laboratory in Santa Cruz, California over spring break and they brought back these photos of some Funaria hygrometrica (cord mosses) growing on a whale skeleton outside of the center. When they first told me that they found mosses growing on a whale I totally did not believe them, but I was imagining a breathing swimming whale. They even got permission from the Marine Center to collect some of the moss for us to use in our research collection. The description of this collection location on the label is going to be great! Thanks Laura and Juan Carlos for the photos.

You can read more about 'Ms. Blue' the whale here on the Marine Center's website. She has a pretty interesting story that goes from finding a blue whale washed up on the shore to her most recent relocation.

Happy April Fools Day!

Berry Go Round #27

The latest edition of the plant carnival Berry Go Round has been posted at A Neotropical Savanna. This month there is a featured post about mosses from Justin Thomas at The Vasculum. He features a number of different mosses including a few in the Funariaceae (Funaria hygrometrica and Physcomitrium pyriforme to be specific). This is the moss family that I study. He has some really sharp photos and includes nice descriptions to help you identify the species. The rest of the posts focus on flowering plants, but despite their non-mossy-ness they have some great botanical information to share. Enjoy!      

For more about blog carnivals and my posts about the earlier editions of Berry Go Round, click here.

Happy St. Patrick's Day

Shamrocks and leprichauns are green just like mosses. To celebrate the day I dug through my digital photos and came up with some green mossy gems to share. Below is a photo of the moss species that I am working on for my dissertation research, Funaria hygrometrica.

This is another species in the Funariaceae, Physcomitrium pyriforme with sporophytes that have matured and are now brown.

All of these photos were taken a couple of years ago. I initially tried growing my mosses on soil in pots in the greenhouses we have on campus. Unfortunately the mist rooms kept them too moist and the mosses were overrun by cyanobacteria and algae. That is when I switched to growing them in little plastic terrariums on a light cart in my laboratory.

I am not sure which species is below. The leafy gametophytes of members of the Funariaceae all look very similar and I did not mark the photo.

There are a few more photos below the fold. Enjoy!

These are some hornworts that my labmate Juan Carlos had planted up in the greenhouse. From the almost readable label it looks like they might be in the genus Anthoceros.

An additional up close shot of the capsules and calyptra of Funaria hygrometrica.

In this batch of bryophyte images I also took a number of shots of the orchids that grow in our teaching greenhouses. Though they are gaudy angiosperms I thought that I would include a couple of them here.

Moss Stickers

I was cleaning out my office drawers and came across a mossy item that I had not seen in months. (My office drawers tend to become a disaster over the course of the semester.) It was a sticker book made from my moss photos, many of which have been used on this blog. The company that I used is at www.moo.com and is based out of the UK. I had heard about them from somewhere and really liked the idea of making a sticker book. The books come with 16 perforated pages and you can upload a large number of images. I think that I could have had every sticker in the book of a different photo. Instead I picked a set of 12 that I liked, because I knew I would be giving them away. I passed out sheets of these stickers to my labmates and many of them ended up on the fronts of their laboratory notebooks. It wasn't exactly where I had imagined them sticking, but they seem to have enjoyed them. I even have a couple of sheets left! Now I just have to decide where to stick them...

The Stickers shown in the photo from upper left to right: Polytrichum gametophyte stems with fall leaves, Polytrichum stems up close, and Funaria hygrometica. Second Row: Leucobryum mixed with Dicranum, Leucobryum tuft, and Leucobryum upclose. Lower sheet, top row: Anacamptodon capsule, Tetraphis gemmae cups, and Sphagnum. And finally Sphagnum leaf cross section, Tetraphis peristome teeth at the top of the capsule, and Tetraphis gemmae.

Moss Life Cycle 3

The spores land on a suitable substrate (either soil, tree bark or rocks, depending on the species) and grow to form a filamentous mat, called protonema (7 o'clock). These protonema filaments have been compared to both algae and rhizoid filaments that attach bryophytes to their substrate.

Each protonema mat can produce many leafy buds that will develop into grown mossy plants (9 o'clock). Thus you can have many individual gametophyte stems in the same patch that are genetically identical to each other. Think clones from your favorite sci-fi movie. All that is needed is a single spore to produce an entire mossy patch.

And there we have it. We have made it all the way through the moss life cycle, hopefully without to much brain strain and confusion. If you have any questions about the life cycle feel free to drop me a line in the comment section.

The Moss Life Cycle 2

The last we heard form our moss life cycle we had arrived at fertilization. This process produces a diploid sporophyte that has two sets of chromosomes per cell. The sporophyte starts out as a small embryo (12 o'clock photo) that grows (2 o'clock photo) and grows (4 o'clock photo). The sporophyte consists of a stalk that elevates the capsule, also called a sporangium, "high" into the air. (Height is relative. The stalk is only a few centimeters tall, but it is much taller than the green leafy gametophyte.)


Inside the capsule, spores (6 o'clock) are produced. They are formed by the cell division process of meiosis. This process takes diploid sporophyte cells and produces haploid spores. Basically it takes the number of chromosomes in a parent cell and decreases them by half in the child cells. These spores leave the capsule flying on the wind and are the part of the moss life cycle that is the main dispersal unit.

They land on a suitable place to grow and... (stay tuned for the continuing adventures of the moss life cycle.)

The Moss Life Cycle 1

If you remember anything about plants from biology class you might recall learning about life cycles. Typically this is a challenging and dreaded concept for students to learn. Life cycles involve a lot of new terminology and there are different cycles for every group of plants.

Personally I really like life cycles and I think that they are critical to understanding plant biology. The life cycle of mosses is something that I think about on a daily basis, but I know that is a little out of the ordinary. Below, I introduce the moss life cycle using the moss species that I study, Funaria hygrometrica, so that those of you who aren't as intimately involved with plants would have a good summary of how it all works.

I am going to break this topic down into a few posts since it is a lot of information to digest at once.


Starting on the far left (9 o'clock) is an image of the leafy green gametophyte (aka. the moss plant). This portion of the life cycle is haploid, meaning that it has one set of chromosomes per cell. It is different from the large photosynthetic portion of most plants which is diploid with two sets per cell. It is photosynthetic, capturing sunlight water and carbon dioxide to make sugars.

The function of the gametophyte in the life cycle is to make gametangia. Gametangia (antheridia- male & archegonia - female) are the sexual reproductive structures. Thus the gametophyte is the sexual stage of the life cycle.

At 11 o'clock are two images of these sexual reproductive organs that are produced by the leafy gametophyte. To the far left are the antheridia and below toward the right is an archegonium.

The antheridia are the dark brown structures that each produce hundreds of sperm. The single thin structure is an archegonium which contains only one egg per. The sperm and egg cells are also called gametes.

So we have gametophytes (mossy plant) that make gametangia (antheridia & archegonia) which produce gametes (sperm & egg). All of these structures are haploid and are produced by mitosis. In this process of cell division there is no change in the number of chromosomes per cell .

If you have any tips or comments on learing about the life cycle of mosses, feel free to share in the comments section. Stay tuned for the next installment of the life cycle.

Mosses in the Azores

Mosses are everywhere! Connecticut, Japan, and the Azores (a group of islands off the coast of Portugal) just to name a few. Information came out over Bryonet recently about an online biodiversity portal that has been developed for the plants and animals of the Azores. The website can be viewed in Spanish, Portuguese and English. They break the database down into the specific groups of plants and animals, with an entire database devoted to the bryophytes. There is a species list that you can browse through or you can search for your favorite genus.

I ran a little search and discovered that my research moss Funaria hygrometrica is located on two of the nine islands. When I clicked on a particular island a 500 X 500 meter distribution map is displayed, which is quite helpful for narrowing down hunting areas when searching for moss.

Click here to check out the Azorean Biodiversity Portal.

Microarthropods Help to Disperse Sperm

Cronberg, N., R. Natcheva, and K. Hedlund. 2006. Microarthropods Mediate Sperm Transfer in Mosses Science 313: 1255.

I was tidying up my computer desktop today and came across a really cool article about moss sperm. Yes, mosses have sperm. They are flagellated and get around by swimming in water. This fact can limit the distance that sperm can travel since they need a film of water to swim through. This is not an issue for plants such as pine trees and dandelions, because their equivalent dispersal units are pollen. Pollen is more easily dispersed since it can be transported long distances via wind or animal pollinators.

There are some interesting ways that bryophytes can disperse their sperm. One of them is a type of liverwort that explosively sends its sperm into the air, thus sending it farther from the parent plant. I discussed this Airborne Sperm Dispersal and the associated video in a previous post. Click here for a link to that blog post.

Getting back to the paper at hand, researchers hypothesized that sperm could be dispersed via small arthropods such as springtails and mites. Animals act as pollinators for many flowering plants, maybe they interact with the sperm of mosses as well?

They tested this hypothesis in an experiment where they placed the male and female mosses at different distances from each other and with or without microarthropods. They found that the mosses that were separated without a film of water connecting them could not reproduce. The sperm could not travel to the eggs without the water and no sporophytes were produced. When the microarthropods were added to containers under these same conditions ... (drumm-roll) ... sporophytes were produced! They are not sure exactly how, but the sperm were able to catch a ride on the arthropods and to be transported from the male to female mosses. It is a pretty amazing feat if you ask me and I think that it would be great to see a SEM photo of the sperm attached to the microarthropods.

In case you have never before seen one, this is a photo of some moss antheridia of Funaria hygrometrica that I took. Sperm are made inside of the brown antheridia. My former officemate always describes them as 'corn dog-shaped' when teaching. The green structures intermingled with them are hairs with swollen apical cells.

Check out the original article too see their data and figures. It is a short, but good, read.

Wild Moss Video

I decided to search on YouTube this afternoon to see if there were any interesting videos of mosses and I came across this one. Its title is Spinning Plant Thing and the video along with comments regarding it can be seen here. It is a pretty entertaining video and a fun example of people observing the world around them, but having no idea what they are looking at. My favorite part is when they hypothesize that it is an alien!

What they are actually observing is a moss sporophyte, which consists of a stalk and capsule at the top. The moss species is most likely Funaria hygrometrica. (You can see the leafy gametophyte of this species in my post from October 5th.)

I can also explain the spinning. Funaria hygrometrica's common name is the cord moss, because the stalk that holds the capsule is very twisted when dry, like a cord of rope. When they add water it is absorbed into the cells and they straighten out and untwist the stalk. As the moss dries back up it twists again. This phenomenon is due to the arrangement of the cells in the stalk. The sporophyte is actually attached to the leafy green gametophyte part of the moss that it sticking out of. That part is hard to see in the video but they notice it is growing out of a patch of moss. I don't blame them for thinking that they were looking at two different plants the sporophyte and gametophyte of mosses look very different. One is leafy and green, while the other is leafless and usually yellow or brown. The part that people usually think of when they think of a moss is the leafy green portion.

Why are Mosses cool?

Have you ever been walking through the woods and noticed a patch of green on the side of a tree, rock or fallen log? Well you might have spotted a moss. Mosses are plants, typically small, and come in a variety of shapes and shades of green. One of the great things about them is that they are more and more interesting the closer that you get to them. What appears to be a swatch of green at a distance is actually a miniature forest up close. This is an image of a colony of Funaria hygrometrica, the cord moss, that I have growing in the laboratory. The mosses growing outside in Connecticut are currently not very photogenic due to the drought and heat wave we are having. But don't worry, mosses are quite resilient and most types can come back to life after drying to a crisp.