Why aren't mitochondria and plastids considered symbiotes of eukaryotic cells?

Why aren't mitochondria and plastids considered symbiotes of eukaryotic cells?

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Mitochondria and plastids have their own DNA, their own membranes, and their reproduction is not tied to the reproductive cycle of the host cell. However, they are considered to be organelles rather than a separate species in symbiosis with eukaryotes. Granted, mitochondria and plastids are incapable of living outside their parent cells, and likewise eukaryotes are incapable of surviving without the help of mitochondria and plastids. But this is also true of many other symbiotic pairs in Eukarya. So where does the distinction lie? What makes mitochondria and plastids organelles rather than separate organisms?

I would say it has to do with the amount of mitochondrial or sequence that has been transferred to the host genome. As a consequence of all this information stored in the host genome, mitochondria cannot reproduce without the host. In this way, they are not their own organisms, but rather organelles.

Over evolutionary time, the line between organelle and intracellular endosymbiont gets blurry, but in their current state, mitochondria are organelles.

The same goes for for plastids, in general.

Hypothesis and the Origin of Eukaryotic Cell | Biology

The following points highlight the top six types of hypothesis with respect to origin of eukaryotic cell. The hypothesis are: 1. Independent Hypothesis 2. Endogenous Theory (Filiation Theory) 3. Chimera Hypothesis 4. Endosymbiotic Theory 5. Serial Endosymbiotic Theory (Set) 6. Syntrophy Hypothesis.

Type # 1. Independent Hypothesis:

The unique nature of the eukaryotic nucleus with structurally com­plex chromosomes is thought to be derived inde­pendently from pre-prokaryotes, neither from archaebactria nor from eubacteria however there is no concrete evidence in support.

Type # 2. Endogenous Theory (Filiation Theory):

Eukaryotic cells originated from ‘Proto-eukaryote’, a large anaerobic bacterium, that formed nucleus, mitochondria, chloroplasts by invagi­nation of plasma-membrane and enclosed gene­tic material inside double membrane.

Type # 3. Chimera Hypothesis:

According to this concept, eukaryotic cells originated as chimera of two or more prokaryotic cells.

Though there is no intermediate organisms between prokary­otes and eukaryotes, eukaryotes are more closely related to archaebacteria in certain respects, particularly to thermophilic archaebacteria of hot spring which do not possess cell wall, looking like amoeba, with cytoskeleton-like structure, having sulphur compound based energy metabolism, Fe 3+ or Mn 4+ acting as respiratory oxidants, with aerobic respiration.

One sugges­tion may be that eukaryotes originated as a chimera between an archaebacterium and a eubacterium (Fig. 2.21).

Of the different chimera hypotheses, fusion model and engulfment model are mechanistically problematic (Fig. 2.22). By contrast, symbiotic model relies on intimate rela­tionships over extended periods of time that allowed symbionts to co-evolve and become dependent on each other.

Type # 4. Endosymbiotic Theory:

The more well docu­mented and generally accepted theory for the origin of eukaryotic organelles is endosymbiotic theory. Recent evidences justify that organelles have originated from the endosymbiotic association of ingested aerobic and photosynthetic prokaryotes, the precursors of mitochondria and chloroplast respectively.

Molecular data have played an important role in supporting xenogenous origin (from outside of cell) rather than autogenous origin (from within the cell) of organelles. Recent phylogenetic analyses reveal that many eukary­otic organellar and nuclear genes whose prokaryotic ancestry can be pinned down are of bacterial origin.

Phylogenetic analyses reveal that many eukaryotic orgnallear and nuclear genes whose prokaryotic ancestry can be pinned down are of bacterial origin. In the case of endosymbiosis one type of cell (symbiont) entered into another type of cell (host) through phagocytosis.

The ingested cell under some circumstances could survive and repro­duce within cytoplasm of the host cell. The rela­tionship is stabilized by their mutual benefits of metabolic symbiosis and becomes obligatory.

Horizontal gene transfer from symbiont to host genome causes the loss of corresponding protein synthesizing ability of the symbiont and is likely to be selectively favoured. The development from symbiont to organelle is completed by the loss of its independent survival ability.

This idea is based on the fact that organelles like mitochondria and chloroplasts:

(i) Are replicators, i.e., can divide indepen­dently.

(ii) Carry genetic information, i.e., DNA.

(iii) With protein synthesizing machinery, i.e., ability of transcription and translation.

(iv) Have own ribosomes of prokaryotic type, i.e., 70S type.

The evidences supporting bacterial origin of mitochondria and chloroplasts are convincing.

a. Mitochondria and Chloroplasts contain their own DNA

(i) DNA simple, closed circular supercoiled dsDNA with single origin point.

(ii) DNA controls the synthesis of their rRNA 2 and tRNA, ribosomal proteins and certain proteins of respiratory chain (mitochon­dria) and similar genes for PSI, PSIl, cytochrome of complex, ATP synthase and ribulose bisphosphate carboxylase of choroplastids.

b. They contain their own ribosomes:

(ii) Shine-Dalgarno sequence on 16S rRNA.

c. Antibiotic specificity:

Ribosomes are sensi­tive to chloramphenicol (SOS), streptomycin and tetracycline (30S) like bacteria but eukaryotic ribosomes are insensitive to these antibiotics.

d. Molecular phylogeny:

16S rRNA and tRNA sequencing have shown that chloroplasts and mitochondria are evolutionarily related to bacteria.

Type # 5. Serial Endosymbiotic Theory (Set):

Serial Endosymbiotic Theory, supported by Taylor 1974, Gray 1983, Doolittle and Daniels 1988, Margulis 1995, proposes the following steps of evolutionary origin of eukaryotic cell (Fig. 2.23).

SE I (Origin of Flagella):

A thermo acidophil, fermenting, Gram(-ve) bacterium merged with Spirochaete through phagocytosis to develop so- called undulipodium flagellated cells.

SE II (Origin of Nucleus):

The resulting pre- eukaryote went through secondary endosymbiosis by engulfing archaebacterium with membra­nous folds. The archaebacterium becomes nucle­us, losing cell membrane, while the membra­nous folds develop nuclear envelope and endo­plasmic reticulum. The genome of bacterium is transferred to the nucleus through membrane pores. Classical example of such eukaryote is Giardia lamblia.

SE III (Origin of Mitochondria):

Mitochon­dria is surrounded by a double membrane repre­senting outer and inner membrane of bacteria. The inner membrane is invaginated forming tubular or discoid cristae. The biochemistry of energy metabolism in mitochondria is very much similar to that of purple non-sulphur bacteria.

The theory implies that the aerobic bacterium established itself as a symbiont within an anae­robic fermenting proto-eukaryote and lost the ability of photosynthesis and become mitochon­drion (Fig. 2.24). Strombidium purpureum is an example, where mitochondrial rRNA sequence shows analogy to eubacterial rRNA.

The serial endosymbiotic theory postulated that the capture of an proteobacterial endosymbiont by a nucleus containing eukaryotic host resembling extant amitochondriate protists, results in the origin of mitochondria.

Giardiai like anaerobic primitive eukaryotes by engulfment of an aerobic Gram(-ve) eubac­terium like Paracoccus denitrificans resulted pro­tista (unicellular eukaryote) with mitochondria classical example is Pelomyxa palustris.

SE IV (Origin of Chloroplast):

Chloroplasts in mitochondria containing eukaryotic cell evolved by association of photosynthetic endosymbionts like photosynthetic bacteria or cyanobacteria (Mereschowsky). Plastid genes are strikingly similar to cyanobacteria in sequence organization and mode ‘of expression. Phylo­genetic analysis of rRNA and tufA sequences indicates cyanobacterial origin of all plastids.

A well-studied example of endosymbiotic cyanobacteria (cyanelles) is Cyanophora paradoxum. In cryptomonad flagellates and dinoflagellates chloroplasts represent a second generation endosymbiont. This type of secondary/tertiary endosymbiosis (Fig. 2.25) results in several sets of membranes around the chloroplast in which the outermost membrane represents the cell mem­brane of the latest endosymbiont.

Origin of Peroxisomes:

Peroxisomes may have been formed through endocytosis of prokaryotes with detoxifying capabilities.

Origin of CERL system:

Lysosomes are developed from invaginated vesicles with enzymes. Further extension of invaginations into the cytoplasm formed tubular network to form Golgi bodies and endoplasmic reticulum.

Type # 6. Syntrophy Hypothesis:

A novel symbiotic hypothesis states that eukaryotic cells arose through metabolic sym­biosis or syntrophy between eubacteria and methanogenic archaea.

The hydrogen hypothesis holds that eukaryotic cells originated through a symbiotic metabolic association in anaerobic environments between a fermentive α-proteobacterium that generated hydrogen and CO2 as waste products, and a strict anaerobic autotrophic archaeon that depended on hydrogen and might have been a methanogen.

The syntrophy hypothesis as proposed by Moreira and Lopez-Garcia (1998) is also based on symbiosis mediated by interspecies hydrogen transfer but the organisms involved were δ pro-teobacteria (ancestral sulphate-reducing myxo-bacteria) and a methanogenic archaea (Fig. 2.26 and 2.27).

Margulis (2000) proposed the origin of eukaryotic nucleus via symbiogenesis by syntrophic merger between a thermoacidophil archae­bacterium and heterotrophic swimmer eubac­terium under selective pressure of oxygen avoi­dance and speed swimming the former gene­rated hydrogen sulfide to protect the later, the chimera emerged was an amitocondriate protists with nucleus as a component of the karyomastigont.

Eukaryotic nucleus with introns and spliceosomes, originated through mitochondrial endosymbiont, created a strong selective pressure to exclude ribosomes from the vicinity of chromo­somes and forcing nucleus-cytosol compartmentalization — thus breaking the prokaryotic paradigm of co-transcriptional translation — allowing the proper maturation of mRNA.

Endosymbiotic Theory


The endosymbiotic theory is usually used to explain the origin of eukaryotic cells, but it can also be applied to bacterial cells. For example, Gram-negative bacteria could have evolved via an endosymbiosis between a clostridium and an actinobacterium, implying that their inner membrane is derived from the plasma membrane of the endosymbiotic bacterium, whereas the outer membrane originated from the plasma membrane of the bacterial host.

Darwin did not consider the significance of symbiotic associations in his theory of evolution. Moreover, endosymbiosis-mediated fusion of evolutionarily distinct lineages (netlike or reticulate evolution) contrasts with his idea of bifurcating divergence from common ancestors (treelike evolution). Thus, endosymbiotic associations are sometimes treated as examples of non-Darwinian evolution via the inheritance of acquired characteristics (e.g., acquisition of new genes and membranes) or macrogenesis involving ‘hopeful monsters’ (e.g., protozoans containing red or green algal endosymbionts). Nevertheless, each endosymbiotic association comes under natural selection, resulting in the survival (and reproduction) of only those best adapted to their environments.

Regardless of these general evolutionary considerations, available data clearly indicate that endosymbioses have had an enormous impact on the evolution of biosphere of our planet. It seems that a special role in this process was played by the mitochondrial endosymbiosis, which not only enabled the origin of the first eukaryotic cell but also facilitated a dramatic increase in the complexity of the eukaryotic world through the evolution of multicellularity.

Why aren't mitochondria and plastids considered symbiotes of eukaryotic cells? - Biology

Evidence for endosymbiosis

Biologist Lynn Margulis first made the case for endosymbiosis in the 1960s, but for many years other biologists were skeptical. Although Jeon watched his amoebae become infected with the x-bacteria and then evolve to depend upon them, no one was around over a billion years ago to observe the events of endosymbiosis. Why should we think that a mitochondrion used to be a free-living organism in its own right? It turns out that many lines of evidence support this idea. Most important are the many striking similarities between prokaryotes (like bacteria) and mitochondria:

    Membranes — Mitochondria have their own cell membranes, just like a prokaryotic cell does.

When you look at it this way, mitochondria really resemble tiny bacteria making their livings inside eukaryotic cells! Based on decades of accumulated evidence, the scientific community supports Margulis's ideas: endosymbiosis is the best explanation for the evolution of the eukaryotic cell.

What's more, the evidence for endosymbiosis applies not only to mitochondria, but to other cellular organelles as well. Chloroplasts are like tiny green factories within plant cells that help convert energy from sunlight into sugars, and they have many similarities to mitochondria. The evidence suggests that these chloroplast organelles were also once free-living bacteria.

The endosymbiotic event that generated mitochondria must have happened early in the history of eukaryotes, because all eukaryotes have them. Then, later, a similar event brought chloroplasts into some eukaryotic cells, creating the lineage that led to plants.

Eukaryotes, Origin of


Much of the evidence for the endosymbiotic theory comes from the structure and handling of these organelles’ genetic codes. Both mitochondria and plastids have DNA sequences in circles as that of bacteria. Their DNA also lacks histones (proteins that the DNA is wrapped around) which are present in eukaryotes and some archea. In addition, mitochondria and plastid transcription begin with the amino acid fMet (formylmethionine) as in bacteria, not Met (methionine) as in eukaryotes.

Ribosome sizes are questionable evidence for the endosymbiotic theory. Bacteria usually have ribosomes of 70s (Svedberg units) and eukaryotes usually have around 80s in their cytoplasm. While the mitochondrial and plastid ribosomes are usually of around 70s, they do in fact vary among species from around 60s to 80s, thus overlapping both bacterial and cytoplasmic eukaryote ribosome sizes.

Other evidence for the endosymbiotic theory comes from the two membranes usually surrounding these organelles. The inner membrane belongs to that of the original bacteria and outer membrane presumably a result from the original engulfment. The outer membrane has approximately a 1:1 protein–lipid ratio by dry weight, similar to many eukaryotic cytoplasmic membranes, while the inner membrane (which is made of two layers) has approximately a 3:1, similar to many bacteria. These organelles and bacteria also both utilize electron transport enzymes lacking elsewhere in eukaryotes.

Some of the best evidence for the endosymbiotic theory however comes from bioinformatics. Phylogenetic analyses of various bacteria, mitochondria from various hosts from various kingdoms, and nuclear DNA from those hosts usually place mitochondria as most related to a group of bacteria known as proteobacteria, often placed closest to Rickettsia and other α-proteobacteria. The α-proteobacteria as a group are almost entirely symbiotic or parasitic which may have predisposed the mitochondrial ancestor to an existence within its host. Chloroplasts are most often placed next to cyanobacteria and both contain thylakoids and chlorophyll a cyanobacteria are also involved in a number of symbioses including lichens and corals.

Section Summary

Like a prokaryotic cell, a eukaryotic cell has a plasma membrane, cytoplasm, and ribosomes, but a eukaryotic cell is typically larger than a prokaryotic cell, has a true nucleus (meaning its DNA is surrounded by a membrane), and has other membrane-bound organelles that allow for compartmentalization of functions. The plasma membrane is a phospholipid bilayer embedded with proteins. The nucleolus within the nucleus is the site for ribosome assembly. Ribosomes are found in the cytoplasm or are attached to the cytoplasmic side of the plasma membrane or endoplasmic reticulum. They perform protein synthesis. Mitochondria perform cellular respiration and produce ATP. Peroxisomes break down fatty acids, amino acids, and some toxins. Vesicles and vacuoles are storage and transport compartments. In plant cells, vacuoles also help break down macromolecules.

Animal cells also have a centrosome and lysosomes. The centrosome has two bodies, the centrioles, with an unknown role in cell division. Lysosomes are the digestive organelles of animal cells.

Plant cells have a cell wall, chloroplasts, and a central vacuole. The plant cell wall, whose primary component is cellulose, protects the cell, provides structural support, and gives shape to the cell. Photosynthesis takes place in chloroplasts. The central vacuole expands, enlarging the cell without the need to produce more cytoplasm.

The endomembrane system includes the nuclear envelope, the endoplasmic reticulum, Golgi apparatus, lysosomes, vesicles, as well as the plasma membrane. These cellular components work together to modify, package, tag, and transport membrane lipids and proteins.

The cytoskeleton has three different types of protein elements. Microfilaments provide rigidity and shape to the cell, and facilitate cellular movements. Intermediate filaments bear tension and anchor the nucleus and other organelles in place. Microtubules help the cell resist compression, serve as tracks for motor proteins that move vesicles through the cell, and pull replicated chromosomes to opposite ends of a dividing cell. They are also the structural elements of centrioles, flagella, and cilia.

Animal cells communicate through their extracellular matrices and are connected to each other by tight junctions, desmosomes, and gap junctions. Plant cells are connected and communicate with each other by plasmodesmata.

Additional Self Check Questions

1. What structures does a plant cell have that an animal cell does not have? What structures does an animal cell have that a plant cell does not have?

Results and Discussions

Symbioses of nitrogen fixing bacteria with sponges, corals and insects (invertebrates)

Marine sponges (Porifera) are evolutionary primordial invertebrates, which can harbour a variety of extra- and intracellular bacteria or bacterial communities [24–26]. However, the symbiotic character of these associations is well defined only in a few cases [27]. Symbioses with sponges have been described for many different groups of cyanobacteria [28], where the symbionts seem to provide their hosts with organic carbon, nitrogen or secondary metabolites [27, 29]. This might also be the case for the filamentous cyanobacterium Oscillatoria spongeliae, which is found to be host-specific in Dysidea spp. [30]. Cyanobacterial symbionts of Chondrilla australiensi s are thought to be vertically transmitted [31, 32], but an obligate status for these interactions has yet to be tested rigorously.

Corals in general are partners of endosymbiotic dinoflagellates (zooxanthellae), which provide photosynthetically derived carbon to their animal hosts [33], but nitrogen fixation by cyanobacteria is also a well-known feature of coral reefs and coral communities [34–36]. The metazoan coral Montastraea cavernosa is an example of a host harbouring symbiotic cyanobacteria [37]. In the Montastraea endosymbiosis, two symbiotic organisms, the zooxanthellae and cyanobacteria, share the same host compartment. Here, the nitrogen fixation by the cyanobacteria might be facilitated by the host providing energy rich compounds. If so, this would indicate a high degree of specificity association between all three partners [31].

Also higher invertebrates benefit from the metabolic capacities of nitrogen-fixing bacteria. The hindgut of wood-feeding termites is colonised by flagellate protozoa [38, 39], which facilitate digestion of lignocellulose [40]. The carbon-rich but nitrogen-poor nature of the termite diet requires nitrogen from other sources [41]. This is thought to be provided by intracellular bacteria associated with termite gut flagellates, such as Trichonympha agilis in Reticulitermes santonensi [42]. These are examples of permanent endosymbionts placed phylogenetically in a new phylum endomicrobia [42]. Interestingly, although the endomicrobia are symbionts of the flagellate protists rather than the termites, they might best be considered as animal endosymbiotic associations. More recently, free-living spirochetes of the termite hindgut have also been revealed to fix molecular nitrogen and provide their host with nitrogen metabolites [43]. A further interaction has also been identified in Tetraponera ants, which harbour a subset of different bacteria in a special organ ("bacterial pouch"), among them relatives of Rhizobium, Pseudomonas and Burkholderia [44]. However, although these symbionts are related to nitrogen fixing and/or root-nodule associated bacteria, it is only speculated that the insect host benefits from fixation of molecular nitrogen. More likely, nitrogenous waste secreted by the host is metabolised and recycled by the bacteria. This is also indicated by the high amount of Malphigian tubules in the pouch, which transport nitrogenous waste. Nevertheless, nitrogen fixing activity of the symbiotic bacteria of Tetraponera cannot be excluded as a possibility. The diverse symbiotic interactions between nitrogen fixing bacteria and insects described so far share some common characteristics.

These symbionts often inhabit specialised organs or regions of the host. This localisation in turn provides an optimal environment for their activity, without symbionts needing to reside inside host cells. This is in contrast to other well-known bacterial interactions with insects, like the Buchnera symbiosis [45]. Here, the symbionts reside within specialised host cells and show a remarkable degree of adaptation leading to an obligate and permanent level of interaction. One prerequisite for such co-evolution of both partners is stable vertical transmission of symbionts which usually takes place maternally, via infection of eggs or larvae [45, 46]. In contrast to endosymbionts, stable integration and transmission of gut and cavity symbionts seems to be challenging as they are more vulnerable for replacement by other mircobes. Ants and termites are colony organised insects and transmission of extracellular symbionts could take place horizontally via close contact of different individuals or via feeding of larvae by infected workers. However, reproduction of social insects is accomplished only by few individuals, thus vertical transmission from queens to the offspring is necessary for the foundation of new colonies. Phylogenetic analyses of the gut microbiota of termites indicate symbiont-host coevolution based on vertical transmission in combination with frequent horizontal exchange between congeneric species [47, 48]. Consequently, the special social lifestyle of termites and ants might be one prerequisite for the establishment of stable vertical transmission and cospeciation of extracellular symbionts in these lineages.

Symbioses of nitrogen fixing bacteria with fungi: cyanolichens and symbionts of arbuscular mycorrhizal fungi

In lichen symbioses, a fungal partner (mycobiont) is associated with an extracellular photobiont. The latter are mostly different photosynthetic algae, but cyanobacteria also occur as photobionts in lichens, either alone (bipartite symbiosis) or in combination with algae (tripartite symbiosis) [49]. The benefit to the photobiontic partner is not fully understood, but it might include the provision of water, minerals, protection from predators and UV damage [50]. The advantage for the fungal partner is the provision of photosynthesis-derived carbon metabolites from the photobiont. Cyanobacteria (cyanobionts) provide, in addition to carbon, fixed nitrogen to their hosts. The importance of molecular nitrogen fixation is reflected in the physiological and morphological adaptations of lichen-associated cyanobacteria. These include an increased number of nitrogen-fixing heterocysts in symbiotic Nostoc sp. compared to free-living filaments. A further adaptation is found in tripartite symbioses where the cyanobacteria are concentrated in special areas called cephalodias, where they fix nitrogen and are protected from high oxygen concentrations. In these tripartite symbioses, photosynthesis is restricted to the algal photobionts, and these supply the other partners with fixed carbon compounds [51]. The fact that most cyanobionts are not vertically transmitted and are also found as free-living organisms indicates that they are not obligate symbionts, and thus not dependent on host metabolism. Nevertheless, the morphological characters of lichens suggest a high degree of coevolutionary adaptation of all participants. Although commonly considered a mutualistic interaction, some hypotheses propose that lichen symbioses are a form of parasitism [50]. Even so, the ecological and evolutionary success of lichens suggests mutual benefit is characteristic for the association.

The arbuscular mycorrhizal (AM) symbiosis between fungi and plant roots is the most common of this type of interaction in the rizosphere [52]. The fungus supplies the plant with water and nutrients such as phosphate, while the plant provides the fungus with photosynthetically produced carbohydrates. The AM fungus Gigaspora margarita harbours intracellular bacteria from the genus Burkholderia [53, 54], which supply the fungus with fixed nitrogen. However, the extent of physiological adaptation or reduction of these endosymbionts leading to an obligate status of interaction has yet to be determined.

A further symbiosis, discovered in the Spessart-mountains (Germany), was identified by analysing the fungus Geosiphon pyriformis, related to AM fungi [55]. At the hyphal tips of this fungus, unicellular multinucleated "bladders" develop, which harbour Nostoc punctiforme. It has been shown that these bladders fix CO2, which may be the major contribution of the cyanobacterium to the symbiosis. The symbiont also forms heterocysts, suggesting that nitrogen is fixed as well [56]. However, as these heterocysts are somewhat similar to those of free-living relatives of this Nostoc strain, nitrogen fixation may only serve the needs of the symbiont itself.

Symbioses of nitrogen fixing bacteria with plants

Interactions of bacteria with various groups of plants are the most common symbiotic association for nitrogen assimilation. A multiplicity of bacteria with different physiological backgrounds are involved in these associations, including gram-negative proteobacteria like Rhizobia sp. and Burkholderia sp., gram-positive Frankia sp. [57] and filamentous or unicellular cyanobacteria [58]. The physiological and morphological characteristics of these symbioses range from extracellular communities to highly adapted interfaces within special organs or compartments.

The mutualistic symbioses between various non-photosynthetic proteobacteria of the order Rhizobiales with plants of the orders Fabales, Fagales, Curcurbitales and Rosales are the most extensively studied interactions between bacteria and plants [59]. The rhizobia-legume symbiosis is characterised by typical root-nodule structures of the plant host, which are colonised by the endosymbiotic rhizobia, so-called bacteroids [60]. The nodulated plant roots supply the bacteria with energy-rich carbon compounds and obtain fixed nitrogen by the bacteroids in return. The nodule formation is a highly regulated and complex process driven by both partners. Free-living rhizobia enter the plant root epidermis and induce nodule formation by reprogramming root cortical cells. Of special importance for the establishment of the symbiosis are flavonoids secreted by the plant partner [61] and the subsequent induction of bacterial nodulation (nod) genes [62]. The Nod-factors play a role in the formation of the nodule, a complex structure optimised for the requirements of both partners [63, 64]. Analysis of root epidermal infection and the underlying signal transduction pathways [65–67] indicate that Nod-factors may have evolved following recruitment of pathways, which developed in a phylogenetically more ancient arbuscular mycorrhiza symbiosis [68, 69]. In the nodule, bacteroids reside within parenchym cells, where they are localised in membrane bound vesicles (Figure 3a) [70]. Nitrogenase activity is ensured by the spatial separation of the bacteroids inside the nodule structure and special oxygen-scavenging leghemoglobin that is synthesised in the nodules [71]. An interesting feature of rhizobia is that nitrogen fixation is restricted to symbiotic bacteroids, whereas free-living bacteria do not express nitrogenase [72]. Although the rhizobia-legume symbiosis is a highly adapted and regulated interaction it can not be termed permanent or obligate. Both partners can live and propagate autonomously, and each host generation has to be populated by a new strain of free-living rhizobia.

Rhizobia-legume symbioses are not the only root-nodule forming interactions of bacteria and plants. Actinobacteria of the genus Frankia spp. are known to develop nodules for nitrogen fixation in various families and orders of angiosperms known as actinorhizal plants [73]. Free-living Frankia is characterised by a unique morphology, including three structural forms, hypha, sporangium and vesicle, the latter one being a compartment for nitrogen fixation. Although functionally analogous, Frankia nodules differ from those in rhizobia-legume interactions in development and morphology [74]. In contrast to rhizobia all Frankia strains are also capable of fixing molecular nitrogen as free-living bacteria [75]. The appearance of the Frankia-symbiosis as a nodulation dependent interaction emphasises the adaptation of both partners. Other plants, including important economic crops like Zea mays and Oryza sativa have established associations with different nitrogen-fixing bacteria, including Azospirillum [76] and Azoarcus [77]. However, such symbioses have never been found to result in nodule formation.

Endosymbionts adapted for molecular nitrogen fixation a) A Bradyrhizobium sp. bacteroid in a root-nodule of Glycine max (soybean). b) A Spheroid body of the diatom Rhopalodia gibba. SM: Symbiontophoric membrane SBM: Spheroid body membrane.

In addition, nitrogen fixing cyanobacteria are also often found interacting with plant partners. For example, symbioses of filamentous heterocyst-forming Nostoc sp. have been reported for bryophytes, pteridophytes (Azolla), gymnosperms (cycads) and angiopsperms (Gunnera) [78–81]. In all plant hosts, with the exception of Gunnera, symbiotic Nostoc filaments are localised extracellularly in different locations depending on the host species. In bryophytes, like hornworts, the cyanobacteria are found within cavities of the gametophyte [79], whereas an Azolla sp. harbours the bacterial partners in cavities of the dorsal photosynthetic parts of the leaves [80]. In cycad-cyanobacterial associations the symbionts are limited to specialised coralloid roots where they reside in the cortical cyanobacterial zone [81]. More specialised is the mutualistic intracellular Gunnera-Nostoc symbiosis. Here the process begins with invasion of the petiole glands, followed by intracellular establishment within the meristematic cells of this tissue [60, 78].

The symbioses of cyanobacteria with their plant partners differ remarkably from the rhizobia-legume interactions. First, cyanobacteria show a broad host range and thus differ from rhizobia or Frankia sp., which are limited to legumes or angiosperms, respectively. In addition, cyanobacteria do not induce the formation of highly specialised structures like root-nodules after colonisation of the host but reside in plant structures known as symbiotic cavities [82], which also exist without symbiosis. The lack of nodule-like organs can be explained by the fact that heterocyst forming cyanobacteria also fix nitrogen as free-living cells and therefore do not need a special environment for N2-fixation in symbiosis. This makes them distinct from rhizobia, which only fix nitrogen in the protective environment of the nodule. Although symbiotic cavities do not display the close and highly regulated interface of a legume-nodule they are nevertheless regions that exhibit adaptations for symbiosis. A common specialisation in occupied symbiotic cavities of plant hosts is the elaboration of elongated cells to improve nutrient exchange [83] and the production of mucilage-exopolysaccharides for water storage or as nutrient reserve (e.g. [84, 85]). The infection process is controlled via the production of hormogenium-inducing factors by the host plant, resulting in the development of vegetative cyanobacterial filaments (hormogonia), important for host colonisation [86, 87]. The main adaptations to the symbiotic lifestyle found in the bacterial partners concern changes of morphology and physiology. These include a remarkable increase of heterocysts in symbiotic Nostoc, and higher rates of N2 fixation compared to those of free-living cells. In addition, photosynthesis of symbiotic cyanobacteria is depressed in various associations to avoid competition between symbionts and host for CO2 and light [86].

In conclusion, different adaptations are found in cyanobacterial-plant interactions but they are not as specific and highly regulated as the complex nodule-forming symbioses. A common feature of all bacteria plant symbioses is their non-obligate, non-permanent character, including a lack of vertical transmission of symbionts to the next host generation. An exception might be the Nostoc-Azolla symbiosis, where cyanobacterial homogenia are transmitted via megaspores [88].

Symbioses of nitrogen fixing bacteria with protists

Symbioses of bacteria with unicellular eukaryotes are exceptional as they involve the whole host rather than specialised parts of the host organism. Also these intracellular symbionts require a high degree of regulation and adaptation to maintain the mutualistic relationship. This feature, in conjunction with vertical transmission, suggests that co-evolution and dependence of partners is sufficiently advanced to regard the relationship as unification of two single organisms. The mitochondria and plastids of recent eukaryotes are extreme examples of this kind of association [89, 90]. Cyanobacteria have also been detected in intracellular association with an euglenoid flagellate [91], heterotrophic dinoflagellates [92–94], a filose amoeba [95], diatoms [96, 97] and, extracellularly, with some protists, e.g. diatoms [98]. Only rarely has the nitrogen fixing activity of the prokaryotic partner been demonstrated in these symbioses (e.g. [99]). In the next paragraph the range of symbiotic associations between cyanobacteria and protists is described in a progression of interactions from temporary to permanent. As such, these symbioses provide an opportunity to investigate the cellular changes that may accompany the evolutionary transition from extracellular symbiont to intracellular endosymbiont and cell organelle.

Petalomonas sphagnophila is an apoplastic euglenoid that harbours endosymbiotic Synechocystis species [91]. The cyanobacteria occur inside a perialgal vacuole and remain alive for several weeks, before they are metabolised, so that they must be regarded as temporary endosymbiotic cell inclusions. These intracellular cyanobacteria are thus reminiscent of kleptochloroplasts found in some heterotrophic dinoflagellates, marine snails, foraminifera and ciliates. These associations can be understood as a mechanism for the temporary separation of ingested and digested prey [92–94, 100]. However, in all well-documented cases of kleptochloroplastic interactions, only the plastid or the plastid together with surrounding cell compartments (never the whole cell) is incorporated as a kleptochloroplast by the host. In contrast, the cyanobacteria of P. sphagmophila are not disintegrated during their internalisation by the euglenoid [91]. Symbiont integrity is therefore likely to be a prerequisite for the functioning of the cyanobacterial nitrogen fixing machinery. The enslaved cyanobacteria may also provide energy-rich C-compounds or, as suggested for other symbiotic interactions, vitamin B12 production to it host [101]. These hypotheses are yet to be investigated thoroughly.

Phaeosomes are symbionts found in some representatives of the order Dinophysiales. They exhibit morphological characteristics of Synechocystsis and Synechococcus cells and are located either extracellularly or intracellularly [94]. In the case of intracellular cells, the symbioses seem to be permanent and the benefit of the symbiosis to the host may be efficient nitrogen fixation. However, as in the case of P. sphagnophila, difficulties in cultivating these strains complicate molecular characterisation of the endosymbionts. At present this problem is limiting our understanding of the potential benefits of these prokaryote/eukaryote mergers. Some filamentous cyanobacteria are known to interact with diatoms. Extracellular epibionts, endosymbionts and also symbionts positioned in the periplasmic space between the cell wall and cell membrane of the diatom are known to occur [58, 98]. Electron microscopy scanning of such interactions has demonstrated a dual symbiotic nature of some symbionts. E. g. Richelia intracellularis has been observed to interact either as an epibiont (with Chaetoceros spec.) or as endosymbiont (with Rhizosolenia clevei) [98]. In these examples, nitrogen fixation for the benefit of the host has been demonstrated by the cultivation of the symbiont-diatom association in the absence of an external fixed nitrogen source. Nitrogen fixation is also suggested from morphological features such as the presence of heterocysts. At least in tropical environments, the production of B12 vitamins may also be a further benefit for the host [101].

The cyanobacterial endosymbionts of the diatom Rhopalodia gibba

Some diatoms, including Climacodium frauenfeldianum and Rhopalodia gibba, are known to harbour permanent endosymbionts [96, 97, 102]. As indicated by EM investigations of R. gibba, these endosymbionts are intracellular and are transmitted vertically [102, 103]. The endosymbionts, so-called spheroid bodies [96], are localised in the cytoplasm, and separated by a perialgal vacuole from the cytosol. Each spheroid body is surrounded by a double membrane. As additionally internal membranes are also visible, this morphotype is similar to that of cyanobacteria (Figure 3b). 16S rDNA sequences have been amplified from an environmental sample of C. frauenfeldianum [97] and from isolated spheroid bodies of R. gibba [102]. Phylogenetic analysis groups these sequences together with free-living cyanobacteria of the genus Cyanothece (Figure 2). This robust grouping is also evidenced from phylogenetic analysis of a nitrogenase subunit gene, isolated from R. gibbas's spheroid body [102]. In phylogenetic reconstructions of both genes, the branch lengths separating free-living cyanobacteria and the cell inclusions of C. frauenfeldianum and R. gibba are very short, indicating that origins of the protist symbioses are relatively recent. This is unlike the situation for plastids and extant cyanobacteria, which have an ancient phylogenetic relationship. Cyanothece sp., the closest known free-living relatives of spheroid bodies and the endosymbiont of C. frauenfeldianum, are typical unicellular and diazotrophic cyanobacteria. To protect the nitrogenase from oxygen tension, Cyanothece show a strong physiological periodicity, restricting nitrogen-fixation exclusively to the dark period of growth [104]. During this period, the energy demand for N2 fixation is sustained by large amounts of photosynthetically derived carbohydrates, which are stored as starch particles. Nitrogen fixing activity of R. gibba was first indicated in the 1980s via acetylene reduction assays [99] and confirmed in latter studies [102]. Intracellular localisation of the enzymatic activity has been undertaken by scanning for protein subunits of nitrogenase [102]. Immunogold experiments have shown that the nitrogenase is localised within the diatom spheroid bodies, thereby confirming that the endosymbiont is responsible for the fixation of nitrogen. Furthermore, corresponding genes for the nitrogenase activity have also been isolated from purified spheroid bodies [102]. Interestingly, spheroid body nitrogen fixation in R. gibba is a strictly light dependent process. This might be the result of several adaptations to the endosymbiotic lifestyle. Spheroid bodies lack a characteristic cyanobacterial fluorescence based on photosynthetic pigments, indicating that they have lost photosynthetic activity and that energy for nitrogen fixation is supplied by the host cell. The protection of the nitrogenase enzyme complex is accomplished through the spatial separation of the two pathways, with N2 fixation in spheroid bodies and photosynthesis in the host plastid. The loss of photosynthetic activity of spheroid bodies is also expected to lead to the loss of autonomy resulting in an obligate endosymbiosis. This hypothesis is consistent with the observation that R. gibba cells are never observed without spheroid bodies and that cultivation of the endosymbionts outside the host cells has not been possible [102]. Definitive evidence is still required to determine the exact nature of symbiotic interaction and whether the spheroid body of R. gibba is an obligate endosymbiont, or perhaps even an unrecognised DNA-containing organelle.

HGT may still be underestimated in eukaryotes

Despite potentially frequent HGT in many eukaryotes, identification of acquired genes often is complicated. Although foreign genes may gradually accumulate in recipient genomes, their phylogenetic signal tying them to specific source taxa may be muted or completely erased by substitutions over time. Additionally, HGT from uncultivated or extinct bacterial lineages may not be properly identified 105 . Even if phylogenetic signal is retained, recovery of accurate phylogenies can be complicated. In particular, many gene families are patchily distributed in prokaryotes and eukaryotes, and explanations of such patchiness can be controversial 106-108 .

Interpretations of patchy distributions hinge on underlying assumptions 2 . For researchers who view vertical inheritance as the sole or dominant genetic paradigm, HGT rarely offers a satisfying explanation. In such cases, a patchy distribution is best explained by differential gene losses, misidentification of genes, or simply phylogenetic artifacts. Although these factors can create patchy distributions, indiscriminately resorting to them as the chief explanation not only discounts the obvious existence of HGT in many eukaryotes, but also ignores the gene pool constraints from the common ancestor of eukaryotes and progenitors of organelles. Clearly, some reported cases of HGT turn out to be artifacts 4, 35 , but the existence of some established artifacts does not discount the likelihood of HGT in many other cases.

On the other hand, patchy distributions are easily explained based on current knowledge of HGT. For examples, HGT from prokaryotes, sometimes involving the same genes independently and recurrently 78, 109, 110 , can spread prokaryotic genes among unrelated eukaryotes. Further, the bacterial ancestry of mitochondria and plastids, the widespread distribution of secondary, tertiary, or transient plastids, and the presence of bacterial endosymbionts (e.g. Wolbachia and Rickettsia in animals) in many eukaryotes, are all known to lead to gene transfer and, therefore, bacterial genes in eukaryotic genomes. In such cases, patchy distributions not only are expected, but also clearly reflect the very nature of HGT in eukaryotes 111 .

Given the difficulties and complications discussed above, it is important that putative cases of HGT in eukaryotes be investigated carefully. To do so, independent lines of evidence and alternative scenarios should be considered. Many cases of patchy distribution probably reflect combined effects of duplication, gene loss, HGT and other processes 80, 112, 113 . Nevertheless, as long as vertical inheritance remains the null hypothesis, HGT in eukaryotes will likely be underestimated. Therefore, it is useful to bear in mind that HGT, although difficult to “prove” in every individual case, offers a valid explanation for many of the atypical gene distributions in eukaryotes.

Interactive Dynamics and the Organizational Role of the Eukaryotic Cytoskeleton

The previous two sections have examined the motility of symbionts and organelles, focusing on their different functional contributions to the eukaryotic cell. In both cases the control of the motility of the parts is aimed at satisfying physiological requirements of the eukaryotic cell. However, ongoing endosymbionts and organelles of endosymbiotic origin exhibit a different control of motile capacities which can be understood partly by exploring the evolutionary innovations introduced by the eukaryotic cytoskeleton (compared to the prokaryotic one), partly by analyzing the different roles played by endosymbionts and organelles within the eukaryotic cell.

Despite the discovery of bacterial homologs of actin (Bork et al., 1992), tubulin (de Boer et al., 1992 RayChaudhuri and Park, 1992 Mukherjee et al., 1993) and intermediate filaments (Margolin, 2004) 16 , the eukaryotic cytoskeleton performs new functions, not present in the prokaryotic cell, that allow eukaryotes to move organelles or bacterial pathogens within themselves. Compared to the prokaryotic cytoskeleton, which is involved in the production of cell wall, the maintenance of cell shape and the support for cell division, the eukaryotic one performs several different functions, including intracellular transport of organelles and intracellular signaling. Intracellular transport is unique to the eukaryotic cell 17 , because organelles are enclosed in membranes requiring vesicles for transporting intracellular cargos (Bonifacino and Glick, 2004). Intracellular transport is performed by molecular machines that transport cargoes along actin filaments (myosin) or microtubules (dynein and kinesin) by exploiting ATP hydrolysis (Dawson and Paredez, 2013 Jékely, 2014). The force 18 generated by the eukaryotic cytoskeleton permits a new kind of spatial organization within the eukaryotic cell that cannot be found in the prokaryotic one.

The remodeling of filamentous actin plays a pivotal role both in cell motility (Diez et al., 2005) and is triggered by a variety of cellular signals, including PIP2 19 , Ca 2+ , and small GTPases (Takenawa and Itoh, 2001). The stimulation of purinergic receptors, due to the rise of Ca 2+ , allows actin filaments to accumulate around intracellular organelles in such a way as to slow down their movement through the cytoplasm. The major nucleators of actin polymerization are the Arp2/3 complex and the members of the formin family, which give rise to different actin structures: the Arp 2/3 complex produces branched filaments, whereas formin straight and bundled filaments (Diez et al., 2005).

Since both the endosymbionts (of protists and insects) and organelles are embedded in eukaryotic cells having a eukaryotic cytoskeleton, both should be moved and displaced by molecular motors along actin filaments and microtubules. Nevertheless, the fact that only organelles, and not also endosymbionts, have a cytoskeleton-driven movement is closely connected with the different functional role that organelles and endosymbionts play within the eukaryotic cell.

The movement of organelles permits intracellular communication via vesicle-mediated pathways 20 : the interchange of molecules (e.g., ions, proteins, lipids, etc.) among mitochondria (and plastids), endoplasmic reticulum, Golgi apparatus, lysosomes, and nucleus would not occur if these organelles were not be spatially close (Perico and Sparkes, 2018). In turn, the delivery and the coordinated transfer of molecules enable organelles to perform important physiological tasks that collectively contribute to the self-maintenance of the eukaryotic cell. For example, the spatial proximity between endoplasmic reticulum and Golgi apparatus allows the movement of proteins between them as well as the closeness between mitochondria and other organelles favors the interchange of reducing equivalents and ATP molecules. Since organelle movement plays such a crucial role, the eukaryotic cell modulates the distribution of the organelles with spatiotemporal accuracy by means of changes in network and motor properties (e.g., polarization, signaling, motor mobility, etc.) (Ando et al., 2015 van Bergeijk et al., 2015).

Unlike organelles, endosymbionts do not perform regulatory and homeostatic mechanisms for the host. Accordingly, they require neither displacement nor a fine-tuned dynamic spatiotemporal control from the eukaryotic cell. Indeed, endosymbionts usually provide the host with enzymes necessary for performing catabolic or anabolic pathways (e.g., the enzymes for amino acid anabolism of sap-feeding insects), which are absent or incomplete in the host. The enzymes synthesized by endosymbionts are targeted to the plasma membrane of the host through co-translation or post-translation pathway without the need for spatial proximity to the membrane contact sites of eukaryotic organelles. For these reasons, the host does not need to consume energy to displace endosymbionts and they can be kept in an extremely stable position during the symbiotic association. It is worthy of note that the eukaryotic cytoskeleton can be also employed by bacterial pathogens for performing invasion strategies (Haglund and Welch, 2011 Gouin et al., 2015) by exploiting actin polymerization. Therefore, the fact that (bacterial) endosymbionts are not moved by the cytoskeleton is likely not due to a cytoskeletal limitation, but rather to the uselessness of this displacement within the eukaryotic context.

The eukaryotic cytoskeleton is a fundamental step not only in the transition from prokaryotic to eukaryotic cell but also in the evolution of mitochondria and plastids from long-term stable endosymbionts to organelles. The eukaryotic cytoskeleton has given rise to an extremely dynamic and interconnected network within the eukaryotic cell that has led to complex forms of communication and a fine-tuned spatiotemporal localization of eukaryotic organelles in such a way that the degree of cohesion and mutual dependence among the parts considerably increased. This was a very important innovation during eukaryogenesis because it opened up a more sophisticated form of intracellular communication (vesicular transport instead of simple diffusion) and an effective control over the positioning of organelles. These important biological novelties have made an important contribution to the overall functional integration of the eukaryotic cell.

Special attention should be paid to the major contribution made by the eukaryotic cytoskeleton to the transition from endosymbiotic proto-mitochondria and proto-plastids to organelles. Both mitochondria and plastids have an endosymbiotic origin (α-proteobacteria were likely the ancestors of mitochondria, whereas cyanobacteria of plastids) and they transformed into organelles over millions of years (Martin et al., 2015). It has been stressed that the main events that allowed endosymbionts to become organelles were the massive transfer of genes to the eukaryotic nucleus (endosymbiotic gene transfer) and the appearance of protein import machineries in the membranes of proto-mitochondria and proto-plastids (Theissen and Martin, 2006). We hypothesize that at some point in eukaryogenesis the eukaryotic cytoskeleton must have played a pivotal role in the transformation of proto-mitochondria and proto-plastids into organelles.

Indeed, given that mitochondria and plastids were endosymbionts, they lost most of their genes, including those for cell motility. It is therefore likely that in an initial phase of eukaryogenesis mitochondria and plastids were immobile or, at least, with a very reduced ability to move. Yet, since proto-mitochondria and proto-plastids were progressively performing regulatory and homeostatic mechanisms, it was necessary to provide some mechanisms for displacing and putting them close to other eukaryotic organelles in order to ensure intracellular communication. From this perspective, the eukaryotic cytoskeleton is no longer just a bunch of filaments for controlling cell shape, but an extremely dynamic structure that has allowed mitochondria, plastids, and the other eukaryotic organelles to achieve a high degree of functional integration.

Cell Reproduction

Eukaryotes grow and reproduce through a process called mitosis. In organisms that also reproduce sexually, the reproductive cells are produced by a type of cell division called meiosis. Most prokaryotes reproduce asexually and some through a process called binary fission. During binary fission, the single DNA molecule replicates and the original cell is divided into two identical daughter cells. Some eukaryotic organisms also reproduce asexually through processes such as budding, regeneration, and parthenogenesis.