41stSaas‐Fee course from Planets to Life 3‐9 April 2011 Lecture 9A. The probability of acquiring life elsewhere and the origin of eukaryotes

• Are there enough known parameters to construct a probability equaon for an origin of an Earth life? • What is required to get a eukaryoc cell? • What drives mulcellularity? • Are these extremely rare events? 41stSaas‐Fee course from Planets to Life 3‐9 April 2011 Lecture 9 B – The origin of eukaryotes

• The endosymbiosis theory for the origin of eukaryotes • Symbiosis today – a couple of examples • The early stages in the formaon of eukaryotes is not a rare event Important differences between eukaryotes and prokaryotes • Unity of biochemistry and genec code • Eukaryotes have mulple chromosomes, prokaryotes have a singular circular chromosome • Eukaryotes genomes are full of non‐coding DNA and genes encoding RNA • Eukaryoc translaon and transcripon are separate, prokaryotes they happen concomitantly • Eukaryoc informaon genes are Archaea, operaonal genes are Bacteria • Most eukaryote genes have no known prokaryoc homologues • Archaea are adapted to energy stress and can make due with lile free energy; Bacteria and eukaryotes reproduce using reliable energy sources Important differences between eukaryotes and prokaryotes • Unity of biochemistry and genec code • Eukaryotes have mulple chromosomes, prokaryotes have a singular circular chromosome • Eukaryotes genomes are full of non‐coding DNA and genes encoding RNA • Eukaryoc translaon and transcripon are separate, prokaryotes they happen concomitantly • Eukaryoc informaon genes are Archaea, operaonal genes are Bacteria • Most eukaryote genes have no known prokaryoc homologues • Archaea are adapted to energy stress and can make due with lile free energy; Bacteria and eukaryotes reproduce using reliable energy sources If this tree is correct and there was extensive genec exchange (lateral gene transfer) then it is likely that there was only one tree of life since any new genec innovaon that increased survival was rapidly transmied to other “organisms” What is lateral gene transfer and symbiosis?

A reculated version of the tree of life, displaying at its boom the last common community (LCC) instead of the last common organism. Implicaon is that lateral gene transfer and symbiosis were (and sll is) a dominant mechanism for creang diversity and complexity (from Boa, 2004) The first stages in the development of eukaryotes • A fusion of archaea and bacteria Nature 431, 2004

Four schemes of natural order in the microbial worlds. a. The three‐domain proposal based on the ribosomal RNA tree, as rooted with data from anciently duplicated protein genes. b. The two empire proposal, separang eukaryotes from prokaryotes and eubacteria from archaebacteria, c. The three domain proposal with connuous lateral gene transfer among domains. d. the ring of life, incorporang lateral gene transfer but preserving the prokaryote divide Rivera and Lake – fusion of bacteria and archaea to form a eukaryote • What kind of bacteria and archaea fused? The three domain The three domain tree Euryarchaeota tree shows eukaryotes and root archaea are

Eubacteria Crenarchaeota Archaea separate groups that share a common ancestor Eukaryotes to the esclusin of The Eocyte tree the eubacteria Euryarchaeota root Eubacteria Crenarchaeota

The “eocyte” tree has eukaryotes originang within the archaea and Eukaryotes sharing a common Cox et al., 2008 used the sequence of 53 proteins from all ancestor with the three domains involved in transcripon, translaon and Crenarchaeota (eocyte) replicaon to show the eocyte tree is favored The second phase of eukaryoc development • The acquision of the mitochondria and the chloroplast – early symbiosis – The mitochondria are gram‐negave bacteria that probably evolved around 2.5 Ga – The chloroplasts are cyanobacteria that evolved around 2.7 Ga

The evoluon of oxygenic photosynthesis (Calvin Bensen Cycle) and the mitochondria (controversy about the original mirochondria but all mulcellular organisms use mitochondria for respiraon involving oxygen and the Tricarboxylic Acid Cycle (TCA) From Hedges et al., Evol. Biol., 2001

Summary diagram showing relationship between timing of evolutionary events (Table 2) and that of Earth and atmospheric histories. Time estimates are shown with ± 1 standard error (thick line) and 95% confidence interval (narrow line). The phylogenetic tree illustrates the radiation of extant eubacterial lineages (blue), and dashed lines with arrows indicate the origin of eukaryotes (BK-o) and origin of mitochondria (BK-m). The earliest divergence (last common ancestor) was not estimated but is placed (arbitrarily) just prior to the AK divergence. The increasing thickness of the eukaryote lineage represents eubacterial genes added to the eukaryote genome through two major episodes of horizontal gene transfer. The rise in oxygen represents a change from <1% to >15% present atmospheric level [34,52], although the time of the transition period and levels have been disputed [19,53]. Yeast cell (~5µm) Centric diatom (~10 µm)

The mitochondria and chloroplasts come from symbiosis with cyanobacteria and an alpha‐ proteobacteria Previously Proposed Models

i. genome fusion

ii. unidenfied host cell acquires nucleus from archaeon, mitochondria from bacterium

iii. archaeon engulfs bacterium

iv. protoeukaryote (genome with lineage disnct from archaeal or bacterial) engulfs bacterium

Model i aempts to explain genomic evidence

Models ii‐iv are theories on the origin of the nucleus and/or mitochondria Poole and Penny (2007) Bioessays Koonin, Senkevich, Dolja 2006. Biology Direct. Symbiosis is a very common phenomenon in nature Animal symbiosis

• Definion of symbiosis • Examples of symbiosis – A look at our future – Light for all reasons • Symbiosis at vents – Riia symbiosis – aquiring the symbiont – Other animals • Concluding comments Expanded terminology on organism interactions with other organisms  • Symbiosis: An associaon between two or more species • Endosymbiont: A symbiont that lives inside of its host, oen within host cells (intracellular symbiont) • Facultave mutualist: A beneficial symbiont that associates with the host, but can also live apart from it. Examples include Rhizobium species that associate with legumes, but also have a free‐living stage in their life cycle. • Obligate mutualist: A beneficial symbiont that lives exclusively with its host and depends on the host for survival. Examples include many nutrional symbionts of insects, which cannot survive outside the insect host cell. These associaons are reciprocally obligate when the host cannot live without the endosymbiont. • Parasite: A symbiont that has a negave effect on host fitness, in contrast to a mutualist that enhances host fitness • Reproducve parasite: a symbiont that manupulates host reproducon to its own benefit. Reproducve parasites usually bias offspring to infected females PNAS, Nov. 2008

The sea slug Elysia chloroca acquires plasds by ingeson of its algal food source Vaicheria litorea. Organelles are sequestered in the mollusc’s digesve epithelium, where they photosynthesize for months in the absence of algal nucleocytoplasm. This is perplexing because plasd metabolism depends on the nuclear genome for >90% of the needed proteins. The findings indicate that the sea slug provides the essenal plasd proteins. Genes supporng photosynthesis have been acquired by the animal via horizontal gene transfer and the encoded proteins are retargeted to the plasd. The source of the photogenec Elysia chloroca genes in the sea slug is from V. litorea. Predaon, horizontal gene transfer results in a “green animal”. Next step is “green humans”. Rowher and Thurber (2009) state that the presence of viruses in both the chloroplast and the nucleus provides a possible mechanism for HGT of photosynthec genes to the host. Viruses would have two roles: They dramacally alter the slug’s life history, and they are probably the vector for HGT between an animal and a plant Two very different sea slugs have evolved ways of using the ability of plants to convert the sun's energy into sugars and other nutrients. In simple terms they have become “photosynthec”.

The aeolid nudibranch Pteraeolidia ianthina which "farms" colonies of brown single‐celled algae (zooxanthellae) in its The sacoglossan Placida cf. dendrica body. showing the green network of ducts which contain the green chloroplasts from its algal food. In a symbioc mutualism, the clownfish feeds on small invertebrates which otherwise potenally could harm the sea anemone, and the fecal maer from the clownfish provides nutrients to the sea anemone. The clownfish is addionally protected from predators by the anemone's snging cells, to which the clownfish is immune. Marine animals that use luminescent as part of their behavior and survival

Most involve symbioc luminescent bacteria Taningia danae ‐ a female 7 m long filmed off the coast of Japan at 240‐940 m depths

Photograph of Watasenia scinllans taken by its own light, showing The Hawaaiian bobtail squid (Euprymna luminescing organs of arms and body scolopes) (ventral view) This blackdevil angler fish, Melanocetus johnsonii, has a luminescent lure that she uses to attract prey and to identify herself to potential mates. Flashlight fish refer to a family of fish, the Anomalopidae, also known as the lantern‐eye fish. Most are in the genus Photoblepharon and the one in the picture is P. steinitzi. The bacterial symbiont is a Photobacterium species.

Photographs of Photoblepharon congregated along the reefs on the Gulf of Elat by night. The night photograph was taken by the light being emied from an aggregaon of about 30 Photoplepharon. The coordinated light emission can scare away prey. Some of the aggregaons consist of more than 100 fish and the light emied can cover more than one meter. Luminescence and animal behavior • Camouflage: Pony fish and the squid Euprymna scolopes match the light from above by ventral luminescence. • Avoid predaon: Schooling fish such as the flashlight fish can coordinate their light emission so as to create a zone of light that is bigger than their predators. A species of shrimp shoot a luminescent substance at their prey. • Lure prey such as the lantern fish • Communicaon: Fireflies signal each other with light during courtship; The flashlight fish also use light to communicate. • Apparently, the flashlight fish use their luminescence to see. The man in the picture had twenty buckeuls of water pored over his head (le) from Phosphorescent Bay in Puerto Rico, a bay well known for abundant bioluminescent dinoflagellates. When light were exnguished, the man was literally “glowing in the dark” (right). From Naonal Geographic, 1960.

Riia pachypla “Giant tube worm” Size: tube length up to 1.5 m Distribuon: Galapagos spreading center, EPR, Guaymas Basin Biology: Forms clusters on rocks in zones of diffuse flow vents. Feeds only on internal symbioc, sulfide oxidizing bacteria Ridgeia piscesae Size: Maximum tube length 1900 mm; diameter 3‐13 mm Distribuon: Explorer Ridge, Juan de Fuca Ridge, Gorda Ridge Biology: Grows gregariously in clusters at diffuse flow vents; nutrients from sulfur‐oxidizing endosymbioc bacteria

Symbiosis in Riia

Transport of nutrients

Vesimentum and collar involved in tube secertion

Houses the trophosome

Juvenile Riftia pachyptila removed from its tube Nature 441:345348 (2006)

1. The larvae of Vesmenfera are symbiont‐free and possess a transient digesve system 2. Each generaon of tubeworm must be newly colonized with its specific symbiont 3. New model indicates bacterial symbiont colonizes the developing tube of the seled larvae and enters through the skin and this process occurs before the development of the trophosome 4. In later juvenile stages there is massive apoptosis of host epidermis muscles and undifferenated mesodermal ssue, which ws coincident with the cessaon of the colonizaon process. Tubeworm arficial selement cubes (TASCs). A. Riia pachyple, Oasisia alvinae and Tevnia jerichonana on TASCs aer one year of development at 9°N (EPR). B. Close‐up of one seled juvinile tubeworm 400 µm in length with trophosome (tr). All three genera of tubeworms have the same symbiont and acquire it the same way. mo = mouth a = anus fg = foregut mg = midgut hg = hindgut b = brain te = tenticles tr = troposome ve = vestimentum dv = dorsal blood vessels vv = ventral blood vessels op = opishosome or = obturacular region developing from vestimentum

Schematic drawings of animals reconstructed from serial sections from early larval stages to adult and the portal for entry of symbionts and development of the troposome (a); and schematic cross-sections in the region of symbiont uptake and troposome development (outlined with double arrow in drawing) (b). (c) FISH with symbiont‐specific probes labelled with Cys (red) and counter stained with with DAPI (blue); Aposymbioc larvae (le), juvenile with labeled symbionts confined to the trophosome (le). (d) TEM micrograph of aposymbioc larva showing midgut cells with degrading prosts and remaining tests (arrows). (e) cells as in (d) containing degrading bacteria (stars) surrounded by myelin bodies (arrows) Some conclusions about endosymbiont transmission in vent tubeworms (connued) 1. In later juvenile stages there is massive apoptosis of host epidermis muscles and undifferenated mesodermal ssue, which is coincident with the cessaon of the colonizaon process. Apoptosis is a process of deliberate life relinquishment by a cell in a multicellular organism. It is one of the main types of programmed cell death (PCD), and involves an orchestrated series of biochemical events leading to a characteristic cell morphology and death. The apoptotic process is executed in such a way as to safely dispose of cell corpses and fragments.

Programmed cell death is as needed for proper development as is mitosis. Examples:

* The resorption of the tadpole tail at the time of its metamorphosis into a frog occurs by apoptosis. * The formation of the fingers and toes of the fetus requires the removal, by apoptosis, of the tissue between them. * The sloughing off of the inner lining of the uterus (the endometrium) at the start of menstruation occurs by apoptosis. * The formation of the proper connections (synapses) between neurons in the brain requires that surplus cells be eliminated by apoptosis Some conclusions about endosymbiont transmission in vent tubeworms • Early larval stages of tube worms feed on prosts and bacteria • Symbiont along with other marine bacteria enter larva through the skin and the symbiont is found in both the epidermis and in regions that eventually become the trophosome • In larvae, an extracellular substance apparently released by special glands formed a mucous coat in which newly seled animals were embedded. Diverse environmental bacteria including the symbiont colonized the mucous coat. • Aer the trophosome is formed, tube worms no longer take up bacteria through the skin and lose their mouth and anus. • It is not yet understood the mechanisms by which tubeworms idenfy their symbiont and select it while eliminang all other bacteria How does the tube worm make a living from bacterial symbiosis? • Bacteria produce organic‐nitrogen compounds including amino acids and nucleode bases (the organic backbone of nucleic acids)

• Tube worm can encapsulate and digest bacteria

The giant clam (Calyptogena magnifica) found on the East Pacific Rise and at the Galapagos; note that C. magnifica has a iron hemoglobin, whereas most marine invertebrates have copper hemes Eye

Erythropsis

Warmonia

The unicellular dinoflagellate Erythropsis pavillardi showing an eye organelle

Eye organelle of the unicellular dinoflagellates Erythropsis and Warmonia. (A) Erythropsis. (B) Erythropsis. (B) Eye organelle of Erythropsis. (C) Warnomia. (D) Eye organelle of Warnomia. (E) Nucleus and eye organelle of Warnomia. (F) Birefringence, the rena‐like structure detected in polarized light in Warnomia. (G) Ultrastructure of the eye organelle of Warmonia. (H) Ultrastructure of the rena‐like structure with stacked membranes and large pigment granules Two hypotheses for the origin of the metazoan photoreceptor cells (Gehring, 2005)

The symbiont hypothesis: based on the observaon that the eye organelle in flagellates like Volvox is located in the chloroplast, suggests that light percepon goes back to cyanobacteria (proteorhodopsin gene found in Nostoc and Pyrocycs). This hypothesis is also supported by idenficaon of photoreceptor organelles of some dinoflagellates which are as elaborate as the human eye but assembled in a single cell. They consist of a cornea‐ like surface layer, a lens‐like structure, a rena‐like structure with stacked membranes, and a pigment cup ‐ all in a single cell. Summary • The formaon of eukaryotes is easy and probably occurred many me – cell/cell fusion, cell ingeson of other cells without digeson is very common, and symbiosis are ancient processes and common today • The ming of the emergence of eukaryotes was determined by the evoluon of cyanobacteria and the mitochondria symbiont • The origin of eukaryoc characteriscs not shared with bacteria and archaea have mostly unknown origins

The Drake Equaon

N = R* fp ne fl fi fc L

N = The number of communicave civilizaons R* = The rate of formaon of suitable stars (stars such as our Sun) fp = The fracon of those stars with planets. (Current evidence indicates that planetary systems may be common for stars like the Sun.) ne = The number of Earth‐like worlds per planetary system fl = The fracon of those Earth‐like planets where life actually develops fi = The fracon of life sites where intelligence develops fc = The fracon of communicave planets (those on which electromagnec communicaons technology develops) L = The "lifeme" of communicang civilizaons Thinking about probability equaons for the formaon and evoluon of life (High and Low “rare” probabilies) • Parameters for an origin of life – Earth life • Liquid water; Rocky, metal rich core; Tectonism, Physical and chemical “cycles” • “Ribofilms” • Hirisontal gene transfer mechanisms – Life different from Earth life • Parameters for an evoluon of life into more complex life and mulcellularity – Earth life • Metabolically diverse microbial life (archaea and bacteria) – including photosynthec microbes (the chloroplast) and microbes that make energy using O2 (the mitochondria) and heterotrophic microbial communies (digest parculate carbon for food) • Symbiosis – Life different from Earth life