When Evolution Goes Sideways – Sea slug hybrids, part 3

This is the third and final installment in our story of photosynthesis by an animal. E. chlorotica, a simple sea slug, has proven to be not so simple. We have seen that it is an exception to the "animals as heterotrophs" rule, as well as an exception to the "horizontal gene transfer is an activity of unicellular organisms" rule. Can we learn more from this mollusk? There must be more, otherwise the post would end right here.

Horizontal gene transfer has occurred between E. chlorotica and its algae food, V. litorea, with gain of function for photosynthesis by E. chlorotica. This is an exception to the current idea of how evolution works. More than 50 years before Darwin’s theory of natural selection, there was another natural philosopher (the term “scientist” didn’t gain popularity until the 1850’s) who had his own ideas about inheritance.

According to Lamarck, giraffes acquired their longer neck, then passed it on.

Jean Baptiste Lamarck was a retired French soldier who became a famous zoologist and botanist; he coined the terms "biologist" and "invertebrate" among other accomplishments. In the late 1700’s and early 1800’s, Lamarck suggested inheritance followed strict natural rules. His idea was called "soft inheritance" and was the first comprehensive theory of evolution. Lamarckism, or inheritance of acquired characteristics proposes that new or changed features that an organism acquired during its life would be passed on to its offspring. His classic example was the giraffe. The food was high in trees, so the giraffe strained its neck to reach the leaves, and its neck got longer. Therefore, its offspring were born with longer necks. There are two main features to Lamarckism: 1- mature organisms can change their characteristics permanently; and 2- these acquired changes are passed on to progeny.

Then came Charles Darwin. His voyage on the Beagle took place from 1831-1836 and he formulated his theory soon after that, but he found it hard to reconcile his religious beliefs with his scientific findings and theories. Therefore, he did not publish his work until 1859, when he learned that another scientist, Alfred Russell Wallace, had come to the same conclusion and was about to publish. After years of public debate and testing the theory, Darwin’s and Wallace’s theory of natural selection as the instrument of evolution won out over Lamarckism, and is now widely accepted.

The principle behind natural selection is that changes in organism characteristics are always occurring, but when changes in the environment result in a reproductive advantage for the organism that has a inherited a certain new characteristic, then that characteristic will be passed on to the next generation at a higher rate (the organism with that change is more likely to survive long enough to mate and produce offspring – if you don’t survive to mate, your genes aren’t passed on – duh!).

A prototypical example of natural selection and evolution is nylon eating bacteria. Nylon wasn’t invented until the 1940’s, so the ability to feed on it must be new as well. In the 1970’s, a strain of Flavobacterium was found producing enzymes to digest nylon. Even more amazing were the results of multiple experiments carried out with a non-nylon digesting bacterium, Pseudomonas aeruginosa. When this bug was placed in an environment where nylon was its only possible food source, it took only a few generations before every bacterium on the plate contained enzymes to metabolize nylon, and these were not the same enzymes as found in the Flavobacterium. It happened in trial after trial, showing that life can adapt to whatever the environment offers. There are many other concrete examples of natural selection in the scientific literature – it is one of the most solidly supported theories in all of science.

In an ironic twist of fate, scientists are now helping bacteria evolve the ability to process garbage into adipic acid, one of the two major chemicals in nylon. Man invented nylon, nylon-eating bacteria taught us about evolution, we used this knowledge to evolve bacteria to make nylon!






We know that natural selection takes place, causing species to diverge, converge, and move along ever changing paths. We have seen it take place and can follow the clues it has left behind. However, horizontal gene transfer has made following those paths more difficult. Using molecular methods, scientists have ways of tracking the rate of change in DNA over time. With this data they can put together family trees and charts (cladograms) to show the points at which different species diverged from one another. But if a certain organism can pick up one or more new traits in one horizontal transfer, it makes patterns for small changes harder to use in developing the evolutionary cladograms. Is a new trait the result of mutation and natural selection or horizontal gene transfer?

A simple cladogram for apes and hominids is on the left. It shows evolutionary relationships between species. The right cladogram shows things can be confusing when horizontal gene transfer (crossing arrows) takes place.




Other features have entered the debate on the nature of Darwinian evolution as well. Does natural selection proceed slowly, with very small changes over time adding up to a measurable change in some characteristic or species (called gradualism), or does a species remain stagnant for a long time, until a mutation brings some rapid, even instantaneous, change (called punctuated equilibrium)?

Gradualism has been the prevailing theory for decades now, but examples of horizontal gene transfer would argue for punctuated equilibrium, especially if it is occurring in higher eukaryotes. Instead of many small changes adding up to a measurable difference over time, horizontally transferred genes bring the potential for big changes from the time of transfer down through all subsequent generations.

Is the retention and function of chloroplasts in E. chlorotica an example of punctuated equilibrium? Definitely not - the instantaneous gain of photosynthetic ability is a sudden change, but remember, the chloroplasts themselves are not passed on to the next generation – this is not an example of horizontal gene transfer in itself. But the fact that photosynthesis-specific genes are found in the nucleus of the slug is evidence that horizontal gene transfer has taken place and that they are passed on vertically. This would argue for punctuated equilibrium over gradualism. So, could this be an exception to the commonly held idea of evolution?

What is more, the combination of heritable plant genes AND acquisition of photosynthetic ability in an already living individual smells a lot like evolution by acquired characteristic – Lamarckism! There is a change in the individual (kleptoplasty of chloroplasts) and this acquired ability is passed on to future generations (via germ line inheritance of the photosynthetic genes). That seems like a more complex, two-step version of Lamarck’s giraffes.

If true, a blow will be dealt to neo-Darwinian evolutionary theory - but not a fatal blow. Darwin himself acknowledged that a mechanism of acquired characteristic inheritance or loss of disused characteristics might exist. He called it pangenesis. His idea was deeply flawed in its proposed mechanism; Darwin thought that cells would throw off small particles (pangenes) that would carry the essence of the change throughout the body and pass the characteristic to the next generation. It sounds silly to us now, but remember that changes in environment can initiate communication between cells that are far apart, and can affect DNA function. This is called epigenetics, and we may have an opportunity to talk about it in the future. However, in the case at hand, it might turn out that Darwin was right to hedge his bet against Lamarck.

It would be nice if the story were this simple – O.K., it’s already not simple – but there has been a catch. Even though the genes for some photosynthesis elements are present in the slug’s nucleus, scientists had not been able to show that they were doing anything. Logic says they must be, since the chloroplasts are functional for so long. One could track this is based on the fact that DNA genes are converted to RNA messages before being translated into protein. If the nuclear genes are making proteins, you should be able to see their RNA transcripts - but scientists went a long time without seeing the transcripts. They surmised that the transcripts were short-lived or present in low amounts that couldn't be detected.

All this speculation has been put to rest by recent evidence from Case Western Reserve University. Investigators in late 2011 studied the transcriptosome of E. chlorotica. Whereas the genome is the sum of all genes present in an organism, a transcriptosome represents all the mRNAs present in an cell or organism, but only at a specific point in time or under a specific set of conditions.

This is real difference between genomes and transcriptosomes; genomes are basically the same in all somatic cells under all conditions. But different cell types need different gene products, and reacting to different conditions will call for changes in which genes are transcribed to mRNAs and then translated into proteins. Therefore, the transcriptosome will be different for different cells and at different times

The scientists in the Case Western study isolated mRNA from whole E. chlorotica organisms afaer they had been starved for 2 months and then exposed to sunlight for two hours. Using starved animals ensured that the dose of sunlight would stimulate expression of as many photosynthesis genes as possible.

In all 111 chloroplast transcripts from 52 different genes were identified, many encoded by the stolen cholorplasts, but many others that represent nuclear genes - once found only in the nucleus of the E. chlorotica's algal food, but now found in the nucleus of the sea slug.

This is the first direct evidence of functional lateral gene transfer in a kleptoplastic organism - and think, they only had to study a mere 98,238,204 separate sequences and 8.9 billion nucleotides of code in order to make this discover. This suggests that the copy number and the transcription rate of laterally transferred genes are low, but who cares. We now have proof of horizontal gene transfer  and the production of a true plant/animal hybrid!



Pierce, S., Fang, X., Schwartz, J., Jiang, X., Zhao, W., Curtis, N., Kocot, K., Yang, B., & Wang, J. (2011). Transcriptomic Evidence for the Expression of Horizontally Transferred Algal Nuclear Genes in the Photosynthetic Sea Slug, Elysia chlorotica Molecular Biology and Evolution, 29 (6), 1545-1556 DOI: 10.1093/molbev/msr316


For more information on Jean Baptiste Lamarck, Charles Darwin, gradualism and punctuated equilibrium or pangenesis, as well as web-based activities and experiments, go to:

Jean Baptiste Lamarck –

www.victorianweb.org/science/lamarck1.html
www.ucmp.berkeley.edu/history/lamarck.html
www.lamarck.cnrs.fr/?lang=en
http://necsi.edu/projects/evolution/lamarck/intro./lamarck_intro.html
www.britannica.com/blogs/2009/02/the-rebirth-of-lamarckism-the-rise-of-epigenetics/
http://videosift.com/video/Darwinism-vs-Lamarckism-cute-short-about-evolution
www.indiana.edu/~ensiweb/lessons/lam.darw.html
www.sciencenetlinks.com/lessons.php?DocID=387
www.liftminds.com/lesson/60/Mechanisms_of_evolution_lamarckism_principles
www.clas.ufl.edu/users/ufhatch/NSF-PLANS/4-1_ORIGIN.htm
http://thinkwell.mindbites.com/lesson/3915-biology-contrasting-lamarck-and-darwin
www.imarksweb.net/book/lamarck+vs++darwin+worksheet/
www.iteachbio.com/Life%20Science/Evolution/Earlyevoltheory.doc

Charles Darwin –
www.aboutdarwin.com/
www.blupete.com/Literature/Biographies/Science/Darwin.htm
www.darwinday.org/
www.pbs.org/wgbh/evolution/educators/lessons/lesson2/act1.html
http://teacher.scholastic.com/activities/explorations/adaptation/
www.tes.co.uk/teaching-resource/Charles-Darwin-Cartoon-Strip-activity-6077894/
http://peer.tamu.edu/curriculum_modules/ecosystems/module_1/activity.htm
www.biologyinmotion.com/evol/index.html
http://serendip.brynmawr.edu/sci_edu/waldron/
http://science.pppst.com/evolution.html
http://ats.doit.wisc.edu/biology/ev/ns/ns.htm
www.accessexcellence.org/AE/AEC/AEF/1995/wartski_natural.php
www.mhhe.com/biosci/genbio/virtual_labs/BL_12/BL_12.html

Gradualism and punctuated equilibrium –
http://necsi.edu/projects/evolution/evolution/grad+punct/evolution_grad+punct.html
www.blackwellpublishing.com/ridley/a-z/Phyletic_gradualism.asp
www.talkorigins.org/faqs/punc-eq.html
www.indiana.edu/~ensiweb/lessons/peek.html
www.youtube.com/watch?v=Islx7y62I6M
www.zartistry.net/pgvspe/index.html
www.lessonplanet.com/lesson-plans/punctuated-equilibrium
www.truthinscience.org.uk/tis2/custom/Fossils%20Lesson%20Plan.pdf
http://evolution.berkeley.edu/evosite/evo101/VIIA1bPunctuated.shtml

Pangenesis -
www.answersingenesis.org/home/area/cfol/ch2-pangenesis.asp
www.neoteny.org/2009/12/04/gemmules/
www.chemistrydaily.com/chemistry/Pangenesis
www.softchalk.com/lessonchallenge09/lesson/genetics/an_introduction_to_Mendelian_genetics_print.html

When Amazing isn’t Enough- Sea Slug Hybrids, part 2

As you undoubtedly remember, last time we talked about a fascinating exception in biology, an animal that can perform photosynthesis. The sea slug, Eylsia chlorotica, eats algae and places the intact, functional chloroplasts in its tissues by a process called kleptoplasty. From that point on, the animal can turn light and CO₂ into carbohydrates – it no longer needs to eat. You might also recall that I hinted that the mere ability to perform photosynthesis isn’t the most amazing thing about this animal. So let us jump in right there.

The average life span of our sea slug of interest is ten months. Not enough time to read War and Peace, but forever compared to the mere 24 hours allotted to the mayfly. So E. chlorotica has roughly a year to make hay while the sun shines. However, the life span of the proteins that are needed for photosynthesis is much shorter.

RuBisCO, a complex protein of photosynthesis

The single most abundant protein on earth is called RuBisCO (Ribulose-1,5-bisphosphate carboxylase oxygenase). This protein adds carbon from CO₂ to the growing carbohydrate during photosynthesis, and has a turnover rate of about 5 days. This is an abnormally long life time for a protein. Chlorophyll can have a turnover rate of a mere 10 hours in some plants. Proteins get old fast, they start to work poorly or just stop working altogether. This is especially true for proteins that work in photosynthesis, since light can damage the very proteins that harness its energy.

Scientists, under laboratory conditions, have kept E. chlorotica alive for 14 months using just water and sunlight. The take home message is that there is active photosynthesis in sea slugs for months and months, when the proteins that make photosynthesis work may need replacing in just a few hours. It makes one wonder how E. chlorotica maintains active chloroplasts for so long.

Science has considered three main possibilities, but there might be more. First, there is something unique about the V. litorea (the algae E. chlorotica eats) photosynthetic proteins that makes them extremely long-lived. This is a tenable possibility, as a few plants have chlorophyll that might never be replaced. But even with immortal chlorophyll, these plants have hundreds of other photosynthetic proteins that must be constantly replaced. So this idea must take a back seat.

Second, there might be something unique about E. chlorotica that keeps the proteins from degrading. This would be amazing, since the sea slug’s own proteins degrade just as in other animals and are replaced regularly. Again, not the strongest hypothesis. Third, E. chlorotica has managed to find a way to make photosynthetic proteins. Intriguing possibility, isn’t it?

An animal that makes RuBisCO or chlorophyll takes the idea of a plant/animal hybrid to a whole new level. It isn’t just the ability to selectively save chloroplasts from digestion and then make use of them. It would be as if the sea slug bought an old motor (the chloroplasts) and but produces replacement parts by itself. But to make the replacement parts, the instructions must be there, and this means DNA.

For our sea slug to have the proper DNA, the plant genes must be consumed, avoid digestion, and be transported to the animal cell nucleus. What is more, the genes must be incorporated into the animal's chromosomes. This is a tall order.

Chloroplasts do have some of their own DNA, since they used to be their own organism (remember endosymbiosis?), but biologists know that many of the hundreds of photosynthesis genes have been transferred to the plant nucleus and are no longer housed in the chloroplast. Therefore, just maintaining functional chloroplasts is not sufficient to produce the proteins needed to keep them active.

Perhaps the slug retains the algae nucleus after feeding. This would provide all the genes needed to produce the proteins needed for photosynthesis, as long as the animal cell can reach and read the plant DNA. Since the chloroplast is not digested, perhaps neither is the nucleus. This would be a good idea, except that scientists have starved E. chlorotica for months, and then searched the slug for plant nuclei. They haven’t found any, so it is probable that the nuclei aren’t retained.

This leaves us with the possibility that the plant genes needed for photosynthesis have been donated by the algae and added to the animal’s cell chromosomes. Don’t laugh, this happens all the time in bacteria. It is called lateral (or horizontal) gene transfer, and it can account for things like antibiotic resistance and sex change in gut bacteria (yes, bacteria can change sex). Even viruses can help accomplish horizontal gene transfer. Viruses can insert their own DNA into the infected cell’s DNA and when they cut themselves back out, they may bring more than they put in. The next infected cell is then the recipient of DNA it may not have had previously.

In vertical transmission, all DNA in the offspring

comes from the parent. In horizontal gene transfer,

the movement is between two different organisms

of the same generation; the recipient cell now has

DNA it did not have before.

Lateral gene transfer can also occur in eukaryotes, but it is usually at the primitive end of the scale. The transfer of some chloroplast and mitochondrial genes to the nucleus millions of years ago is an example of horizontal gene transfer. Horizontal gene transfer with passage of the new genes to the next generation is easy in bacteria or lower eukaryotes because they don’t reproduce through sex. In fungi, even though some progeny are produced by mating, the DNA transferred to the progeny is still the same DNA that was laterally transferred.

Sex on the other hand, means sex cells. The DNA in sex cells (gametes) is the only DNA that gets passed on to the progeny (you get half your DNA from Mom’s egg and half from Dad’s sperm). For DNA to be passed on through horizontal gene transfer, the new DNA must be transferred into either an egg or sperm, and that has to be the particular egg or sperm that participates in fertilization. This is especially difficult to imagine for E. chlorotica, as the algae is eaten, and the chloroplasts are put into the gut cells. Nothing about this leads to algae nuclear DNA getting anywhere near the sex cells. It doesn’t seem very likely - but this is exactly what happens.

Pea aphids have incorporated fungal genes to

help them blend in to their surroundings.

Scientists have found several photosynthesis-specific genes in both mature E. chlorotica that have been starved for algae for months and in immature veligers that have never fed on algae. This can only mean that the genes have been passed vertically, from parent to child, and this means that the plant DNA has entered the gamete cells. The only similar instance I can think of is the transfer of a fungal carotenoid (pigment) gene to pea aphids (ant cows, a neat story on their own) that changed the aphid’s color to match their environment, giving them a camouflage advantage. This is itself a biological exception, the only instance of an animal that produces carotenoid pigment.

Lets summarize. We have an animal that can do photosynthesis – amazing. This same animal has taken up DNA from algae, and has incorporated the new genes into its germ line cells so that they are passed on to its offspring – more amazing. Next time, we’ll talk about how one of the greatest ideas of science might be run aground by a sea slug. Could it be that a discarded version of evolution might be correct?

For more information on horizontal gene transfer, as well as web-based activities and experiments, go to:


http://www.psrast.org/hrtrintr.htm
www.genomenewsnetwork.org/articles/05_01/Lateral_gene_lit.shtml
http://amrls.cvm.msu.edu/microbiology/molecular-basis-for-antimicrobial-resistance/acquired-resistance/acquisition-of-antimicrobial-resistance-via-horizontal-gene-transfer
http://mbio.asm.org/content/2/1/e00005-11.full

Biological Hybrids – The diet to end all diets?

It’s easy to tell a plant from an animal. Plants make their food from sunlight and carbon dioxide (CO₂), while animals have to consume plants and other animals in order to get the nutrients and energy they need. In fancy science talk, plants and certain bacteria are autotrophs (“own nourishment”), while animals and most bacteria are called heterotrophs (“different nourishment”).

Algae, plants, and certain bacteria (cyanobacteria) can make their own carbohydrates (sugars) by fixing CO₂. “Fixing” a molecule means to convert it from an unusable form to a usable form. To fix CO₂) from the air, it is incorporated into a solid form, using the energy of sunlight to add carbons to an existing 5-carbon sugar called ribulose bisphosphate. This bigger molecule is then broken into two 3-phosphoglyceric acid molecules before building glucose from these building blocks. We all know this process by another name: photosynthesis.

Animals, including humans - to McDonald’s everlasting delight - can’t perform photosynthesis. They have to get their carbohydrates and other building blocks the old fashioned way - they steal them from something else. By eating and digesting plants and other animals, heterotrophs make use of the plant’s hard work, or piggyback on another animal’s use of plant-produced carbohydrates. Downstream from photosynthesis or eating, the process is the same for both plants and animals; the chemical energy in the bonds that hold the carbons of sugars together are converted into a chemical currency that cells can cash in: mostly ATP.

So, plants are plants and animals are animals, and never the twain shall meet (or meat, in this case)…… EXCEPT for a certain group of sea slugs. Believe it or not, there is a type of sea slug that can perform photosynthesis. Drought-induced food shortages? No problem for these guys. Increasing CO₂ and greenhouse gases? The more the merrier for our little friends. Imagine the possibilities if humans could do this; never again would the sink be piled high with dirty dishes!

Nudibranches are the stars of the sea slug world.

So who is this tree-wanna-be? “Sea slug” is a catchall name for many types of fresh- and saltwater mollusks; underwater snails without the shell (or at least without a visible shell). He’s a homeless gastropod - all wriggly and jelly-filled. Nudibranches are the most numerous and colorful of these groups, but in total there are nearly 4000 species that might be called sea slugs.

E. chlorotica, our sea slug of interest.

Elysia chlorotica is the flavor we are interested in, from the order Sacoglossa, the family Elysiidae. E. chlorotica lives in the shallow saltwater marshes and tidal pools of the U.S. East Coast. Elysdiae are a hearty bunch, being found as far north as Nova Scotia as well as in the warm waters of Florida. They are medium sized for a sea slug, about 2-4 cm in length, but can grow to up to 6 cm.

The key to E. chlorotica’s success as an apprentice plant lies in its ability to make the most of its few meals. It is a picky eater, dining on only two species of algae, Vaucheria litorea and Vaucheria compacta. Using its radula, a sort of serrated tongue, the sea slug punctures the algae and sucks out the contents. But, instead of digesting the chloroplasts, they are taken inside the cells of the slug’s gut cells and they stay there. What’s more, they still work! One organism co-opting another organism for its own gain- the audacity!

However, this is far from first time something like this has occurred. About 1.5 billion years ago, when all life consisted of single celled organisms, one organism ate another organism; just like every other day since time began. But like E. chlorotica with chloroplasts, for some reason the meal wasn’t digested. The two organisms came to an arrangement, and the internal organism stayed and divided when his captor divided. This was called endosymbiosis, and is thought to account for mitochondria, nuclei, and even the chloroplasts of plants themselves.

E. chlorotica’s trick is a little different though, it doesn’t retain the entire algal cell, just the chloroplasts (or plastids as they are sometimes called). This requires a different name be devised for the new process, and I love the one they came up with – kleptoplasty. Literally, the sea slug is a kleptomaniac for the algal plastids; it has a deep-seated need to steal the belongings of another. I picture parental slugs going down to the police station to bail out their teenage offspring after a wild night at the kelp beds.

To me, kleptoplasty is much more amazing than endosymbiosis, because it is selective. Most of the algae is digested, but what is it about V. litorea chloroplasts that allows them to be maintained? No one knows how this occurs, but when they do figure it out, they will still have some issues with which to deal. For instance, after the chloroplasts are separated from the rest of the algae parts, they move inside the gut cells of the sea slug and stay there for the life of the animal! Just how did that come to be?

One matter that can be resolved is how a chloroplast in the gut can help a slug perform photosynthesis. E. chlorotica is a skinny fellow; turn him sideways and have him stick out his radula and he looks like a zipper. So, even though the chloroplasts are in the gut wall, they are still close enough to the surface to receive sunlight. This placement is helpful in another way as well.

Is it animal or vegetable?

Look at the image of E. chlorotica at the right. He is oval shaped, and because of the chloroplasts inside, he is green. Scientists believe this is important for the protection of the animal; a very convincing costume to make him look like a leaf that has fallen into the water. Even his digestive tract helps out, as it fans out to parts of the slug’s body, it looks like the veins in a leaf.

When the slug is born, it is in an immature form called a veliger. It feeds for about a week, and places the chloroplasts in its tissues. From that moment on, the sugary products of photosynthesis in the chloroplasts are exported into the gut of the slug, and distributed through its body to be used for fuel, just as if he had continued to eat and digest food. The animal doesn’t have to feed for the rest of its life, as long as there is sunlight and dissolved CO₂ in the water. Since it lives in water no more than 0.5 m deep, getting sunlight isn’t a problem and seawater is 15.1% dissolved CO₂, as compared to only 0.033% of air (but your soda has 80X more CO₂ than seawater, at least until you open the can). The result of this kleptoplasty is that even in times of food scarcity, E. chlorotica is a happy camper, just laying around, looking like a leaf, avoiding trouble, while at the same time soaking up the sun and turning it into food. That’s the life.

Unfortunately, it isn’t that simple. This sea slug has had to pull some even niftier tricks to hang onto its photosynthetic ability. We’ll talk about another amazing aspect of this animal’s plant envy next time. Stealing a chloroplast is one thing, but apparently E. chlorotica is willing to accept charity as well.

For more information on photosynthesis or endosymbiosis, as well as web based activities and experiments, go to:


Photosynthesis-
http://www.emc.maricopa.edu/faculty/farabee/biobk/biobookps.html
www.phschool.com/science/biology_place/biocoach/photosynth/intro.html
www.youtube.com/watch?v=C1_uez5WX1o

www.pbs.org/wgbh/nova/nature/photosynthesis.html

www.science-class.net/Biology/Photosynthesis.htm
www.the-aps.org/education/k12curric/activities/pdfs/carswell.pdf
http://local.brookings.k12.sd.us/biology/teacherlinks/photosynactivities.htm
http://nhscience.lonestar.edu/biol/bio1int.htm


Endosymbiosis –
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/E/Endosymbiosis.html
www.fossilmuseum.net/Evolution/Endosymbiosis.htm
www.sumanasinc.com/webcontent/animations/content/organelles.html
http://evolution.berkeley.edu/evolibrary/article/endosymbiosis_01
www.youtube.com/watch?v=RaAM8qQcs6E
www.astrobio.net/pressrelease/3223/endosymbiosis-timeline
www.accessexcellence.com/AE/AEC/AEF/1995/everson_endosymbiosis.php
http://wps.prenhall.com/esm_biology_belk_1/13/3353/858395.cw/index.html