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Symbiogenesis: Beyond the endosymbiosis theory?
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Symbiogenesis: beyond the endosymbiosis theory? Duur K. Aanen , Paul Eggleton PII: DOI: Reference:
S0022-5193(17)30361-2 10.1016/j.jtbi.2017.08.001 YJTBI 9165
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Journal of Theoretical Biology
Received date: Revised date: Accepted date:
1 February 2017 23 June 2017 2 August 2017
Please cite this article as: Duur K. Aanen , Paul Eggleton , Symbiogenesis: beyond the endosymbiosis theory?, Journal of Theoretical Biology (2017), doi: 10.1016/j.jtbi.2017.08.001
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ACCEPTED MANUSCRIPT Symbiogenesis: beyond the endosymbiosis theory?
Duur K. Aanen Department of Plant Sciences, Laboratory of Genetics, Wageningen University, 6708 PB Wageningen, Netherlands.
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E-mail: [email protected]
Paul Eggleton
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Life Sciences Department, the Natural History Museum, London, SW7 5BD, UK
Abstract
Symbiogenesis, literally ‘becoming by living together’, refers to the crucial role of
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symbiosis in major evolutionary innovations. The term usually is reserved for the major transition to eukaryotes and to photosynthesising eukaryotic algae and plants by
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endosymbiosis. However, in some eukaryote lineages endosymbionts have been lost secondarily, showing that symbiosis can trigger a major evolutionary innovation, even if
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symbionts were lost secondarily. This leads to the intriguing possibility that symbiosis has played a role in other major evolutionary innovations as well, even if not all extant
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representatives of such groups still have the symbiotic association. We evaluate this hypothesis for two innovations in termites (Termitoidae, also known informally as
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“Isoptera”): i) the role of flagellate gut protist symbionts in the transition to eusociality from cockroach-like ancestors, and ii) the role of non-gut associated symbionts in the transition to „higher‟ termites, characterized by the absence of flagellate gut protists. In both cases we identify a crucial role for symbionts, even though in both cases, subsequently, symbionts were lost again in some lineages. We also briefly discuss additional possible examples of symbiogenesis. We conclude that symbiogenesis is more broadly applicable than just for the endosymbiotic origin of eukaryotes and photosynthetic eukaryotes, and may be a useful concept to acknowledge the important role of symbiosis for evolutionary innovation. However, we do not accept Lynn Margulis‟s view that symbiogenesis will lead to a paradigm
ACCEPTED MANUSCRIPT shift from neo-Darwinism, as the role of symbiosis in evolutionary change can be integrated with existing theory perfectly. Key words: cockroaches; endosymbiosis theory; eusociality; flagellates; gut symbionts; insects; termites Introduction
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Even though the significance of symbiosis for ecosystem functioning and evolutionary change is widely appreciated, the term symbiogenesis goes further. According to Lynn Margulis, „Symbioses are ecological relationships that, over a long period of time, may become
symbiogenesis. In cases when new behaviors, structures or taxa , i.e. new tissues, new organs, new species, new genera, or even new phyla emerge, new relationships at many different
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levels can be identified as the consequence of symbiosis, then symbiogenesis has been demonstrated‟ (Margulis, 2010). Margulis was not the first to express the idea of symbiogenesis. The term was first introduced by the Russian Konstantin Sergeivich Mereschkovsky (Mereschkowsky, 1910) and is equivalent to symbionticism or
microsymbiotic complexes, both of which terms were independently coined by the Swedish-
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American Ivan Emanuel Wallin (Wallin, 1927).
Even though the term symbiogenesis in principle is widely applicable, it is usually reserved
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for the origin of eukaryotes by endosymbiosis (https://en.wikipedia.org/wiki/Symbiogenesis; but see (Guerrero et al., 2013)). It holds that the organelles distinguishing eukaryotic from
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prokaryotic cells evolved from symbiosis of individual single-celled prokaryotes (Bacteria and Archaea). The mitochondria and plastids (for example chloroplasts), and possibly other
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organelles of eukaryote cells are thought to represent formerly free-living prokaryotes taken one inside the other in endosymbiosis around 1.5 billion years ago (Cavalier-Smith, 2013).
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The endosymbiosis theory is strongly supported by various lines of evidence. Most importantly, mitochondria and chloroplasts have retained part of their ancestral genome separate from that of the nuclear genome, which testifies to their endosymbiotic origin (Gray and Doolittle, 1982). However, there has been a long debate on the scenario of eukaryogenesis. According to the archezoan hypothesis, a primitive eukaryote, but one without mitochondria, had evolved before the origin of mitochondria. This primitive eukaryote took up an endosymbiont, which developed into mitochondria (Cavalier-Smith,
ACCEPTED MANUSCRIPT 1993). According to the rival hypothesis, symbiogenesis, an Archaeon became a eukaryote as the result of endosymbiosis (Koonin, 2015; Speijer et al., 2015). Recent research provides compelling support for the symbiogenesis hypothesis, as amitochondriate eukaryotes are highly derived within eukaryotes and not their sister group (Hirt et al., 1999). Eukaryotes arose from a lineage of archaebacteria (Lokiarchaeota) that had elements of a cytoskeleton, and thus presumably the ability to phagocytose (Spang et al.,
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2015; Zaremba-Niedzwiedzka et al., 2017). Apart from this characteristic, however, most eukaryotic features, such as components of the nuclear pore and the spliceosome as well as spliceosomal introns, first arose after the acquisition of mitochondria.
The crucial distinction between the two scenarios is the role of symbiosis for the major
transition to eukaryotes, either just a contributing factor (archezoan hypothesis) or a crucial
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factor sine qua non (symbiogenesis). To test the generality of the symbiogenesis hypothesis, we evaluate the role of symbiosis for other evolutionary innovations: the transition to eusociality from cockroaches to termites, and subsequently, the transition to the so-called “higher” termites (Termitidae). We also briefly discuss the role of symbiosis in some other evolutionary innovations, such as the colonisation of land by higher plants, and algae. We
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finish by evaluating whether the concept symbiogensis will lead to a paradigm shift in evolutionary theory, especially for the origin of species, as predicted by Lynn Margulis
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(Margulis and Sagan, 2002).
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1. The transition to eusociality in termites Termites are eusocial cockroaches. Recent studies indicate that subsocial woodroaches of the
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genus Cryptocercus are their sister group (Inward et al., 2007a). The most decisive characteristic of eusociality is reproductive division of labour. Most individuals altruistically
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forego reproduction, at least temporarily, to help the colony raise more offspring. There is no consensus on how this condition could evolve in the ancestor of termites, but several contributing factors have been identified. First, all termites are strictly monogamous for life. This lifetime monogamy implies that relatedness between reproductives and their own offspring is the same as relatedness among siblings (i.e. 0.5) (Boomsma, 2009). This high relatedness among siblings may be the ultimate explanation for the evolution of eusociality: any marginal increase in the total number of individuals produced by becoming a helper could have sufficed to tip the balance away from independent, solitary reproduction. Second, ecological factors contributed to this balance between the benefits of staying solitary and
ACCEPTED MANUSCRIPT becoming eusocial (Bourke, 1999). Termites., at least ancestrally, depend upon dead, decaying wood, which has a patchy distribution throughout the environment. This means there is a high cost to dispersing to find new patches, and these resources must be defended for the group to survive. These requirements may make a high social order beneficial for the survival of the group. Third, it has been proposed that cooperative breeding led to the loss of flagellates at molt and interdependence of colony members for the re-acquisition of
1.1.
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specialized gut symbionts (Nalepa, 1994; Nalepa, 2015; Nalepa et al., 2001). Symbiont acquisition/cycling
Next to their eusocial organisation in colonies, termites are perhaps best known for their
symbiotic associations with microorganisms from the three domains of life that help them digest lignocellulose (Brune, 2014). Six early diverging lineages all have flagellate gut
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protists, which enable the insects to digest cellulose (Figure 1). The only family that lacks flagellate protist gut symbionts are the Termitidae, the most diverse and derived group of termites (see par. 1.3). It has been proposed by several authors that the evolution of eusociality is linked to the loss of flagellates at molt (Lin and Michener, 1972; Starr, 1979; Wilson, 1971). During ecdysis, or rather just prior to ecdysis (Nalepa, 1994), termites lose
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their flagellates and reacquire symbionts from nest mates via feeding on hindgut fluids of a nestmate, a feeding mode called proctodeal (anal) trophallaxis. This makes group living
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mandatory (Andrew, 1930; Cleveland, 1925).
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Next to facilitating the reacquisition of symbionts from nest mates, proctodeal trophallaxis has some additional consequences for group living, affecting the social, nutritional and microbial
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environment (Nalepa, 2015). First, unlike coprophagy, proctodeal trophallaxis requires physical contact and behavioural interaction. Second, proctodeal trophallaxis facilitates the transfer between individuals of material that would otherwise degrade in the outside
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environment, such as hormones, enzymes, metabolites, which may serve as physiological or behavioural signals (Nalepa, 2015). Third, the symbionts transferred may also partially serve as nutritional sources, as they may be harmed during transfer, for example due to gut passage. Proctodeal trophallaxis probably also has been crucial to establish reliable vertical transmission of hindgut protists, and thereby has contributed to the obligate symbiotic relationship with them in termites (Bell et al., 2007). It has been argued that gut protists in the ancestor of termites and Cryptocercus were passed on to other individuals of a group via coprophagy of encysted protists (Nalepa et al., 2001). However, flagellates only form cysts
ACCEPTED MANUSCRIPT during molting of their host, and cysts are never found in the feces of adults (Bell et al., 2007; Cleveland et al., 1934). Since Cryptocercus is subsocial and semelparous, adults spend their entire lives nurturing one set of offspring, so that there are no older nymphs when adults reproduce. Therefore, vertical transmission of flagellate gut symbionts could not occur via coprophagy of encysted flagellates, but only via proctodeal trophallaxis. Nalepa (Nalepa, 2015) argues that in the common ancestor of termites and Cryptocercus an
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obligate relationship with gut symbionts, vertical transmission via proctodeal trophallaxis, and subsociality were a co-evolved set of characters. Proctodeal feeding in young families assured passage of the entire complex of microorganisms present in the hindgut fluids, facilitating coevolution not only between host and microbiome, but also between microorganisms. The result was a growing dependence of the host upon its microbiome, which reinforced selection
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for assured vertical transmission of the gut microbiome via subsociality and trophallactic behaviour. The switch from horizontal to vertical gut symbiont transmission thus was of key influence in the transition from gregarious to subsocial behaviour in the ancestor of Cryptocercus and termites. It also preconditioned the transition to eusociality by establishing the behavioural basis of trophallactic exchanges (Nalepa et al., 2001). The transition to eusociality
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1.2.
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The defining distinction between subsociality seen in Cryptocercus woodroaches and eusociality seen in termites is that multiple overlapping generations of offspring are produced
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by a single pair. The crucial first step to set this transition in motion was that older nymphs assumed responsibility for feeding and maintaining younger siblings (Bell et al., 2007;
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Nalepa, 2015). This relieved their parents of the cost of brood care and allowed them to invest in additional offspring, which led to overlapping generations of brood. A second consequence of this step, assuming that feeding of siblings takes away some resources from reproduction,
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was that it delayed maturation and thus reproduction of the offspring. Nalepa (Nalepa, 2015) has described the development of an incipient colony of termites as a recapitulation of this evolutionary transition, with reproductives initially participating in raising the offspring, in the absence of workers, and gradually specialising in reproductive tasks only, when the first workers appear in the colony. 1.3.
The non-flagellate termites
ACCEPTED MANUSCRIPT The Termitidae are the most derived of the six termite families. They constitute roughly 70% of all described species and contain the highest diversity of lifestyles, including soil-feeding and fungus-growing and have the highest diversity in social organisation. A defining characteristic of termitids is the absence of flagellate protist gut symbionts. A key question to understand the evolution of termitids is to explain the loss of protist gut symbionts.
A scenario proposed by Eggleton and co-workers (e.g. (Inward et al., 2007b)) for the
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transition to higher termites is a shift from digestion mainly in the gut by flagellate protist symbionts to mainly external digestion by fungal and/or bacterial symbionts. According to this model, the first step was the acquisition of external, i.e. non-gut associated, symbionts, which allowed the loss of internal flagellate gut symbionts (“externalising the gut”). The extant clade Macrotermitinae, the fungus-growing termites, still show this feature, having
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externalised the gut so that a significant part of the degradation of organic matter occurs in the fungus-comb rather than in the gut. Many details of substrate degradation in this group and variation therein remain to be discovered (Bignell, 2016). A recent study in the species Macrotermes natalensis, however. shows that wood is masticated and passes through the
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worker gut with only minor cellulose degradation before being defecated on to the fungus comb (rather than being turned into nest material; (Nobre and Aanen, 2012; Poulsen et al., 2014); we define the faecal material here as “carton” – partially digested dead plant naterial.
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Much of the digestive burden carried originally by the flagellates is therefore passed on to the
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fungus (Rouland-Lefevre, 2000) and the flagellates are redundant. After the adoption of carton material as an „external rumen‟ (rather than as nest material), two
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routes were followed: i) elaboration of the external rumen into an ectosymbiosis with fungi (as seen in the extant subfamily Macrotermitinae); ii) replacing flagellate functions in
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cellulose breakdown by bacteria, and termite functions associated with elaboration of the gut. The recent finding that the species Sphaerotermes sphaerothorax, uniquely characterized by bacterium rather than fungus combs (Garniersillam et al., 1989), is the sister group of all nonfungus growing higher termites, supports this scenario (Figure 1). However, the genera Labritermes and Foraminitermes, previously found to be sister group to Sphaerotermes (Inward et al., 2007b), were not included in the analysis found in Figure1. Labritermes and Foraminitermes also represent the first attempt at soil-feeding, and have very different guts from the other soil-feeders, so their phylogenetic placement is crucial to understand the
ACCEPTED MANUSCRIPT evolution of combs and soil feeding. In addition, more work is required to establish the details of bacterial digestion in combs in Sphaerotermes.
Soil-feeding termites and diverse lifestyles
fungus-growing termites Fungus comb
Adoption of fungi as external symbiont Adoption of bacteria as external symbionts
cockroaches
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‘Complex gut’, loss of external rumen
‘lower termites’ (flagellate protozoan gut symbionts; few species; simple colony structures; generally nesting inside food)
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Termitidae (‘higher termites’; no flagellate protist gut symbionts; highly speciose; highly diverse lifestyles; most derived feeding groups, including various forms of soil feeding; nesting outside food source)
Loss of flagellates Adoption of carton material to build combs as an ‘external rumen’
Transition to eusociality Transition to family-group subsociality
Flagellate protozoan gut symbionts
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Figure 1. A reconstruction of the key events leading to termites and Termitidae („higher
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termites‟), on a schematic representation of a recent phylogenetic reconstruction of termites (Bourguignon et al., 2015). All termite families (except the termitids), and the woodroach sistergroup of termites, Cryptocercus, have flagellate protist gut symbionts, responsible for
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cellulose breakdown. The acquisition of flagellate gut symbionts is associated with a transition to family-group subsociality. In the next transition, to higher termites, flagellate gut
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symbionts were secondarily lost. The adoption of carton material to build combs as an external rumen removed the need for gut protist symbionts. The basal clade within the higher
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termites is formed by the fungus-growing termites (extant subfamily Macrotermitinae), which specialized on fungal symbionts. The basal clade within the clade consisting of all nonfungus-farming higher termites is the species Sphaerotermes sphaerothorax, uniquely characterized by bacterial combs. According to this phylogenetic position, and the possession of combs in all Macrotermitinae, one of the two most parsimonious reconstructions in the evolution of an external rumen in higher termites, is „external rumen first‟, subsequently followed by loss in the sister group of S. sphaerothorax and the evolution of more complex gut morphologies facilitating cellulose degradation with bacterial gut symbionts (the other,
ACCEPTED MANUSCRIPT equally parsimonious reconstruction is two independent gains of comb, one in the ancestor of fungus-growing termites, and one in the ancestor of S. sphaerothorax; however, assuming a higher probability to lose the complex comb structure, this is less likely).
2. Potential other examples of symbiogenesis The colonization of land by phototrophs depended on symbiotic associations with fungi.
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Lichens are symbioses between a cyanobacterium or a green alga inside a vegetative stroma of an ascomycete or, less frequently, a basidiomycete. There is evidence that some nonlichenised ascomycete fungi descend from lichenized ancestors, implying secondary loss of the photosynthetic symbiont (Lutzoni et al., 2001).
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The colonisation of land by multicellular phototrophic organisms involved the radiation of the Plantae some 400 million years ago (Selosse and Le Tacon, 1998). It is generally believed that this colonization also depended on a symbiotic association with fungi, arbuscular mycorrhizal fungi (Lewis, 1987; Selosse and Le Tacon, 1998). Those non-septate fungi form mutualistic
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associations with the roots of some 90% of higher plant species including ferns and some mosses (Selosse and Le Tacon, 1998). The fungus grows intercellularly and forms vesicles
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and intracellular arbuscules, and is involved in the uptake of minerals in particular phosphorus, and also provides protection against plant pathogens.
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Arbuscular mycorrhizal fungi (AM fungi) belong to a small group, about 200 species, of zygomycetes, the Glomales. They are all obligatorily symbiotic, probably asexual, and only
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distantly related to other zygomycetes. The small number of species and their distant relationship with other zygomycetes suggest a slow rate of evolutionary change in a stable
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environment (Law and Lewis, 1983). There are strong arguments for an ancient association with land plants. AM fungi are widespread among extant land plants, covering representatives of all major groups. Furthermore, fossil Glomales have been reported from 460 million years ago, an age confirmed by molecular clock estimations (Redecker et al., 2000; Simon et al., 1993).
The early land plants probably lacked roots, so they relied on associating with fungi for the uptake of water and minerals. The plant genus Aglaophyton is among the first plants known to have had a mycorrhizal relationship with fungi (Remy et al., 1994). It lacked roots, and like
ACCEPTED MANUSCRIPT other rootless land plants of the Silurian and early Devonian may have relied on mycorrhizal fungi for acquisition of water and nutrients from the soil.
Subsequently, however, in some lineages of land plants, this symbiosis was lost or substituted by other symbionts or adaptations, such as nitrogen-fixing bacteria, ectomycorrhizal fungi or by advanced root morphologies. Therefore, the major innovation of land colonization by plants may represent another example of symbiogenesis. This innovation was made possible
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thanks to the acquisition of symbionts. However, upon acquiring this innovation, subsequently, the symbionts were secondarily lost in some derived lineages.
3. Discussion.
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In her seminal paper On the origin of mitosing cells, Lynn Margulis proposed that eukaryotic cells were the result of endosymbiosis (Sagan, 1967). Subsequent research conclusively has confirmed this prediction. In her later work, Margulis went on to argue that symbiosis not only had contributed to evolution, but that it was the source of evolutionary innovation, through a process that had already been called symbiogenesis (Mereschkowsky, 1910). For
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eukaryotes, a symbiogenetic origin was demonstrated in subsequent years through various lines of evidence. However, the usefulness of symbiogenesis as a general unifying principle
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remains to be determined.
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We analysed several cases where symbiosis has facilitated major evolutionary innovations. We provided arguments that flagellate gut protists played a crucial role in the transition to
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eusociality in termites, even though subsequently, in one derived lineage this symbiosis was lost. The loss of those gut symbionts led to the clade Termitidae, which contains the highest number of species, the highest diversity of lifestyles, including soil-feeding and fungus-
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growing and the highest diversity in social organisation. As such, the loss of those symbionts represents a second major innovation in termites. We have provided arguments that this major innovation also depended on symbiosis, viz. an ectosymbiosis with fungi. Furthermore, our analysis on the colonization of land by higher plants demonstrates that this major innovation also depended on symbiosis, as did the colonisation of land by algae through lichens.
In all those cases, a similar pattern emerges: symbiosis facilitates an evolutionary innovation, even though subsequently, the symbiotic association is lost in some lineages, substituted by
ACCEPTED MANUSCRIPT other symbionts or by host adaptations. This shows that the concept symbiogenesis is more widely applicable than to the origin of eukaryotes and photosynthetic eukaryotes. However, does evolutionary theory need an additional category next to mutation, selection and drift? In other words: does the concept symbiogenesis belong in the same conceptual rank as the currently recognized main categories?
Margulis emphasised the role of symbiogenesis to explain discontinuities in evolution and
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speciation: “Evolution is not gradual as Darwin insisted. Species do not change by accumulation of random mutations as NeoDarwinists insist.” (Margulis, 2010). However, this representation of neo-Darwinian evolutionary theory is a caricature. On a similar basis, one could reject evolution when the evolution of altruism via kin selection was described (Hamilton, 1964a; Hamilton, 1964b), which seemed to be diametrically opposed to
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individual-level selection. This has not happened. Inclusive fitness theory has turned out to be a very fruitful addition to evolutionary theory. The development of evolutionary theory did not stop with the neo-Darwinian synthesis, but has continued to be modified and adapted since. In that sense, the history of evolutionary thinking is like biological evolution itself,
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change by adaptive and random processes, usually gradual but sometimes saltatory.
Acknowledgements
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D.K.A. was supported by The Netherlands Organisation for Scientific Research (VICI; NWO
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Symbiogenesis: beyond the endosymbiosis theory? Duur K. Aanen , Paul Eggleton PII: DOI: Reference:
S0022-5193(17)30361-2 10.1016/j.jtbi.2017.08.001 YJTBI 9165
To appear in:
Journal of Theoretical Biology
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1 February 2017 23 June 2017 2 August 2017
Please cite this article as: Duur K. Aanen , Paul Eggleton , Symbiogenesis: beyond the endosymbiosis theory?, Journal of Theoretical Biology (2017), doi: 10.1016/j.jtbi.2017.08.001
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ACCEPTED MANUSCRIPT Symbiogenesis: beyond the endosymbiosis theory?
Duur K. Aanen Department of Plant Sciences, Laboratory of Genetics, Wageningen University, 6708 PB Wageningen, Netherlands.
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E-mail: [email protected]
Paul Eggleton
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Life Sciences Department, the Natural History Museum, London, SW7 5BD, UK
Abstract
Symbiogenesis, literally ‘becoming by living together’, refers to the crucial role of
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symbiosis in major evolutionary innovations. The term usually is reserved for the major transition to eukaryotes and to photosynthesising eukaryotic algae and plants by
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endosymbiosis. However, in some eukaryote lineages endosymbionts have been lost secondarily, showing that symbiosis can trigger a major evolutionary innovation, even if
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symbionts were lost secondarily. This leads to the intriguing possibility that symbiosis has played a role in other major evolutionary innovations as well, even if not all extant
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representatives of such groups still have the symbiotic association. We evaluate this hypothesis for two innovations in termites (Termitoidae, also known informally as
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“Isoptera”): i) the role of flagellate gut protist symbionts in the transition to eusociality from cockroach-like ancestors, and ii) the role of non-gut associated symbionts in the transition to „higher‟ termites, characterized by the absence of flagellate gut protists. In both cases we identify a crucial role for symbionts, even though in both cases, subsequently, symbionts were lost again in some lineages. We also briefly discuss additional possible examples of symbiogenesis. We conclude that symbiogenesis is more broadly applicable than just for the endosymbiotic origin of eukaryotes and photosynthetic eukaryotes, and may be a useful concept to acknowledge the important role of symbiosis for evolutionary innovation. However, we do not accept Lynn Margulis‟s view that symbiogenesis will lead to a paradigm
ACCEPTED MANUSCRIPT shift from neo-Darwinism, as the role of symbiosis in evolutionary change can be integrated with existing theory perfectly. Key words: cockroaches; endosymbiosis theory; eusociality; flagellates; gut symbionts; insects; termites Introduction
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Even though the significance of symbiosis for ecosystem functioning and evolutionary change is widely appreciated, the term symbiogenesis goes further. According to Lynn Margulis, „Symbioses are ecological relationships that, over a long period of time, may become
symbiogenesis. In cases when new behaviors, structures or taxa , i.e. new tissues, new organs, new species, new genera, or even new phyla emerge, new relationships at many different
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levels can be identified as the consequence of symbiosis, then symbiogenesis has been demonstrated‟ (Margulis, 2010). Margulis was not the first to express the idea of symbiogenesis. The term was first introduced by the Russian Konstantin Sergeivich Mereschkovsky (Mereschkowsky, 1910) and is equivalent to symbionticism or
microsymbiotic complexes, both of which terms were independently coined by the Swedish-
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American Ivan Emanuel Wallin (Wallin, 1927).
Even though the term symbiogenesis in principle is widely applicable, it is usually reserved
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for the origin of eukaryotes by endosymbiosis (https://en.wikipedia.org/wiki/Symbiogenesis; but see (Guerrero et al., 2013)). It holds that the organelles distinguishing eukaryotic from
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prokaryotic cells evolved from symbiosis of individual single-celled prokaryotes (Bacteria and Archaea). The mitochondria and plastids (for example chloroplasts), and possibly other
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organelles of eukaryote cells are thought to represent formerly free-living prokaryotes taken one inside the other in endosymbiosis around 1.5 billion years ago (Cavalier-Smith, 2013).
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The endosymbiosis theory is strongly supported by various lines of evidence. Most importantly, mitochondria and chloroplasts have retained part of their ancestral genome separate from that of the nuclear genome, which testifies to their endosymbiotic origin (Gray and Doolittle, 1982). However, there has been a long debate on the scenario of eukaryogenesis. According to the archezoan hypothesis, a primitive eukaryote, but one without mitochondria, had evolved before the origin of mitochondria. This primitive eukaryote took up an endosymbiont, which developed into mitochondria (Cavalier-Smith,
ACCEPTED MANUSCRIPT 1993). According to the rival hypothesis, symbiogenesis, an Archaeon became a eukaryote as the result of endosymbiosis (Koonin, 2015; Speijer et al., 2015). Recent research provides compelling support for the symbiogenesis hypothesis, as amitochondriate eukaryotes are highly derived within eukaryotes and not their sister group (Hirt et al., 1999). Eukaryotes arose from a lineage of archaebacteria (Lokiarchaeota) that had elements of a cytoskeleton, and thus presumably the ability to phagocytose (Spang et al.,
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2015; Zaremba-Niedzwiedzka et al., 2017). Apart from this characteristic, however, most eukaryotic features, such as components of the nuclear pore and the spliceosome as well as spliceosomal introns, first arose after the acquisition of mitochondria.
The crucial distinction between the two scenarios is the role of symbiosis for the major
transition to eukaryotes, either just a contributing factor (archezoan hypothesis) or a crucial
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factor sine qua non (symbiogenesis). To test the generality of the symbiogenesis hypothesis, we evaluate the role of symbiosis for other evolutionary innovations: the transition to eusociality from cockroaches to termites, and subsequently, the transition to the so-called “higher” termites (Termitidae). We also briefly discuss the role of symbiosis in some other evolutionary innovations, such as the colonisation of land by higher plants, and algae. We
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finish by evaluating whether the concept symbiogensis will lead to a paradigm shift in evolutionary theory, especially for the origin of species, as predicted by Lynn Margulis
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(Margulis and Sagan, 2002).
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1. The transition to eusociality in termites Termites are eusocial cockroaches. Recent studies indicate that subsocial woodroaches of the
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genus Cryptocercus are their sister group (Inward et al., 2007a). The most decisive characteristic of eusociality is reproductive division of labour. Most individuals altruistically
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forego reproduction, at least temporarily, to help the colony raise more offspring. There is no consensus on how this condition could evolve in the ancestor of termites, but several contributing factors have been identified. First, all termites are strictly monogamous for life. This lifetime monogamy implies that relatedness between reproductives and their own offspring is the same as relatedness among siblings (i.e. 0.5) (Boomsma, 2009). This high relatedness among siblings may be the ultimate explanation for the evolution of eusociality: any marginal increase in the total number of individuals produced by becoming a helper could have sufficed to tip the balance away from independent, solitary reproduction. Second, ecological factors contributed to this balance between the benefits of staying solitary and
ACCEPTED MANUSCRIPT becoming eusocial (Bourke, 1999). Termites., at least ancestrally, depend upon dead, decaying wood, which has a patchy distribution throughout the environment. This means there is a high cost to dispersing to find new patches, and these resources must be defended for the group to survive. These requirements may make a high social order beneficial for the survival of the group. Third, it has been proposed that cooperative breeding led to the loss of flagellates at molt and interdependence of colony members for the re-acquisition of
1.1.
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specialized gut symbionts (Nalepa, 1994; Nalepa, 2015; Nalepa et al., 2001). Symbiont acquisition/cycling
Next to their eusocial organisation in colonies, termites are perhaps best known for their
symbiotic associations with microorganisms from the three domains of life that help them digest lignocellulose (Brune, 2014). Six early diverging lineages all have flagellate gut
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protists, which enable the insects to digest cellulose (Figure 1). The only family that lacks flagellate protist gut symbionts are the Termitidae, the most diverse and derived group of termites (see par. 1.3). It has been proposed by several authors that the evolution of eusociality is linked to the loss of flagellates at molt (Lin and Michener, 1972; Starr, 1979; Wilson, 1971). During ecdysis, or rather just prior to ecdysis (Nalepa, 1994), termites lose
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their flagellates and reacquire symbionts from nest mates via feeding on hindgut fluids of a nestmate, a feeding mode called proctodeal (anal) trophallaxis. This makes group living
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mandatory (Andrew, 1930; Cleveland, 1925).
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Next to facilitating the reacquisition of symbionts from nest mates, proctodeal trophallaxis has some additional consequences for group living, affecting the social, nutritional and microbial
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environment (Nalepa, 2015). First, unlike coprophagy, proctodeal trophallaxis requires physical contact and behavioural interaction. Second, proctodeal trophallaxis facilitates the transfer between individuals of material that would otherwise degrade in the outside
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environment, such as hormones, enzymes, metabolites, which may serve as physiological or behavioural signals (Nalepa, 2015). Third, the symbionts transferred may also partially serve as nutritional sources, as they may be harmed during transfer, for example due to gut passage. Proctodeal trophallaxis probably also has been crucial to establish reliable vertical transmission of hindgut protists, and thereby has contributed to the obligate symbiotic relationship with them in termites (Bell et al., 2007). It has been argued that gut protists in the ancestor of termites and Cryptocercus were passed on to other individuals of a group via coprophagy of encysted protists (Nalepa et al., 2001). However, flagellates only form cysts
ACCEPTED MANUSCRIPT during molting of their host, and cysts are never found in the feces of adults (Bell et al., 2007; Cleveland et al., 1934). Since Cryptocercus is subsocial and semelparous, adults spend their entire lives nurturing one set of offspring, so that there are no older nymphs when adults reproduce. Therefore, vertical transmission of flagellate gut symbionts could not occur via coprophagy of encysted flagellates, but only via proctodeal trophallaxis. Nalepa (Nalepa, 2015) argues that in the common ancestor of termites and Cryptocercus an
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obligate relationship with gut symbionts, vertical transmission via proctodeal trophallaxis, and subsociality were a co-evolved set of characters. Proctodeal feeding in young families assured passage of the entire complex of microorganisms present in the hindgut fluids, facilitating coevolution not only between host and microbiome, but also between microorganisms. The result was a growing dependence of the host upon its microbiome, which reinforced selection
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for assured vertical transmission of the gut microbiome via subsociality and trophallactic behaviour. The switch from horizontal to vertical gut symbiont transmission thus was of key influence in the transition from gregarious to subsocial behaviour in the ancestor of Cryptocercus and termites. It also preconditioned the transition to eusociality by establishing the behavioural basis of trophallactic exchanges (Nalepa et al., 2001). The transition to eusociality
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1.2.
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The defining distinction between subsociality seen in Cryptocercus woodroaches and eusociality seen in termites is that multiple overlapping generations of offspring are produced
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by a single pair. The crucial first step to set this transition in motion was that older nymphs assumed responsibility for feeding and maintaining younger siblings (Bell et al., 2007;
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Nalepa, 2015). This relieved their parents of the cost of brood care and allowed them to invest in additional offspring, which led to overlapping generations of brood. A second consequence of this step, assuming that feeding of siblings takes away some resources from reproduction,
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was that it delayed maturation and thus reproduction of the offspring. Nalepa (Nalepa, 2015) has described the development of an incipient colony of termites as a recapitulation of this evolutionary transition, with reproductives initially participating in raising the offspring, in the absence of workers, and gradually specialising in reproductive tasks only, when the first workers appear in the colony. 1.3.
The non-flagellate termites
ACCEPTED MANUSCRIPT The Termitidae are the most derived of the six termite families. They constitute roughly 70% of all described species and contain the highest diversity of lifestyles, including soil-feeding and fungus-growing and have the highest diversity in social organisation. A defining characteristic of termitids is the absence of flagellate protist gut symbionts. A key question to understand the evolution of termitids is to explain the loss of protist gut symbionts.
A scenario proposed by Eggleton and co-workers (e.g. (Inward et al., 2007b)) for the
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transition to higher termites is a shift from digestion mainly in the gut by flagellate protist symbionts to mainly external digestion by fungal and/or bacterial symbionts. According to this model, the first step was the acquisition of external, i.e. non-gut associated, symbionts, which allowed the loss of internal flagellate gut symbionts (“externalising the gut”). The extant clade Macrotermitinae, the fungus-growing termites, still show this feature, having
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externalised the gut so that a significant part of the degradation of organic matter occurs in the fungus-comb rather than in the gut. Many details of substrate degradation in this group and variation therein remain to be discovered (Bignell, 2016). A recent study in the species Macrotermes natalensis, however. shows that wood is masticated and passes through the
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worker gut with only minor cellulose degradation before being defecated on to the fungus comb (rather than being turned into nest material; (Nobre and Aanen, 2012; Poulsen et al., 2014); we define the faecal material here as “carton” – partially digested dead plant naterial.
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Much of the digestive burden carried originally by the flagellates is therefore passed on to the
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fungus (Rouland-Lefevre, 2000) and the flagellates are redundant. After the adoption of carton material as an „external rumen‟ (rather than as nest material), two
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routes were followed: i) elaboration of the external rumen into an ectosymbiosis with fungi (as seen in the extant subfamily Macrotermitinae); ii) replacing flagellate functions in
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cellulose breakdown by bacteria, and termite functions associated with elaboration of the gut. The recent finding that the species Sphaerotermes sphaerothorax, uniquely characterized by bacterium rather than fungus combs (Garniersillam et al., 1989), is the sister group of all nonfungus growing higher termites, supports this scenario (Figure 1). However, the genera Labritermes and Foraminitermes, previously found to be sister group to Sphaerotermes (Inward et al., 2007b), were not included in the analysis found in Figure1. Labritermes and Foraminitermes also represent the first attempt at soil-feeding, and have very different guts from the other soil-feeders, so their phylogenetic placement is crucial to understand the
ACCEPTED MANUSCRIPT evolution of combs and soil feeding. In addition, more work is required to establish the details of bacterial digestion in combs in Sphaerotermes.
Soil-feeding termites and diverse lifestyles
fungus-growing termites Fungus comb
Adoption of fungi as external symbiont Adoption of bacteria as external symbionts
cockroaches
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‘Complex gut’, loss of external rumen
‘lower termites’ (flagellate protozoan gut symbionts; few species; simple colony structures; generally nesting inside food)
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Termitidae (‘higher termites’; no flagellate protist gut symbionts; highly speciose; highly diverse lifestyles; most derived feeding groups, including various forms of soil feeding; nesting outside food source)
Loss of flagellates Adoption of carton material to build combs as an ‘external rumen’
Transition to eusociality Transition to family-group subsociality
Flagellate protozoan gut symbionts
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Figure 1. A reconstruction of the key events leading to termites and Termitidae („higher
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termites‟), on a schematic representation of a recent phylogenetic reconstruction of termites (Bourguignon et al., 2015). All termite families (except the termitids), and the woodroach sistergroup of termites, Cryptocercus, have flagellate protist gut symbionts, responsible for
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cellulose breakdown. The acquisition of flagellate gut symbionts is associated with a transition to family-group subsociality. In the next transition, to higher termites, flagellate gut
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symbionts were secondarily lost. The adoption of carton material to build combs as an external rumen removed the need for gut protist symbionts. The basal clade within the higher
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termites is formed by the fungus-growing termites (extant subfamily Macrotermitinae), which specialized on fungal symbionts. The basal clade within the clade consisting of all nonfungus-farming higher termites is the species Sphaerotermes sphaerothorax, uniquely characterized by bacterial combs. According to this phylogenetic position, and the possession of combs in all Macrotermitinae, one of the two most parsimonious reconstructions in the evolution of an external rumen in higher termites, is „external rumen first‟, subsequently followed by loss in the sister group of S. sphaerothorax and the evolution of more complex gut morphologies facilitating cellulose degradation with bacterial gut symbionts (the other,
ACCEPTED MANUSCRIPT equally parsimonious reconstruction is two independent gains of comb, one in the ancestor of fungus-growing termites, and one in the ancestor of S. sphaerothorax; however, assuming a higher probability to lose the complex comb structure, this is less likely).
2. Potential other examples of symbiogenesis The colonization of land by phototrophs depended on symbiotic associations with fungi.
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Lichens are symbioses between a cyanobacterium or a green alga inside a vegetative stroma of an ascomycete or, less frequently, a basidiomycete. There is evidence that some nonlichenised ascomycete fungi descend from lichenized ancestors, implying secondary loss of the photosynthetic symbiont (Lutzoni et al., 2001).
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The colonisation of land by multicellular phototrophic organisms involved the radiation of the Plantae some 400 million years ago (Selosse and Le Tacon, 1998). It is generally believed that this colonization also depended on a symbiotic association with fungi, arbuscular mycorrhizal fungi (Lewis, 1987; Selosse and Le Tacon, 1998). Those non-septate fungi form mutualistic
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associations with the roots of some 90% of higher plant species including ferns and some mosses (Selosse and Le Tacon, 1998). The fungus grows intercellularly and forms vesicles
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and intracellular arbuscules, and is involved in the uptake of minerals in particular phosphorus, and also provides protection against plant pathogens.
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Arbuscular mycorrhizal fungi (AM fungi) belong to a small group, about 200 species, of zygomycetes, the Glomales. They are all obligatorily symbiotic, probably asexual, and only
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distantly related to other zygomycetes. The small number of species and their distant relationship with other zygomycetes suggest a slow rate of evolutionary change in a stable
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environment (Law and Lewis, 1983). There are strong arguments for an ancient association with land plants. AM fungi are widespread among extant land plants, covering representatives of all major groups. Furthermore, fossil Glomales have been reported from 460 million years ago, an age confirmed by molecular clock estimations (Redecker et al., 2000; Simon et al., 1993).
The early land plants probably lacked roots, so they relied on associating with fungi for the uptake of water and minerals. The plant genus Aglaophyton is among the first plants known to have had a mycorrhizal relationship with fungi (Remy et al., 1994). It lacked roots, and like
ACCEPTED MANUSCRIPT other rootless land plants of the Silurian and early Devonian may have relied on mycorrhizal fungi for acquisition of water and nutrients from the soil.
Subsequently, however, in some lineages of land plants, this symbiosis was lost or substituted by other symbionts or adaptations, such as nitrogen-fixing bacteria, ectomycorrhizal fungi or by advanced root morphologies. Therefore, the major innovation of land colonization by plants may represent another example of symbiogenesis. This innovation was made possible
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thanks to the acquisition of symbionts. However, upon acquiring this innovation, subsequently, the symbionts were secondarily lost in some derived lineages.
3. Discussion.
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In her seminal paper On the origin of mitosing cells, Lynn Margulis proposed that eukaryotic cells were the result of endosymbiosis (Sagan, 1967). Subsequent research conclusively has confirmed this prediction. In her later work, Margulis went on to argue that symbiosis not only had contributed to evolution, but that it was the source of evolutionary innovation, through a process that had already been called symbiogenesis (Mereschkowsky, 1910). For
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eukaryotes, a symbiogenetic origin was demonstrated in subsequent years through various lines of evidence. However, the usefulness of symbiogenesis as a general unifying principle
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remains to be determined.
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We analysed several cases where symbiosis has facilitated major evolutionary innovations. We provided arguments that flagellate gut protists played a crucial role in the transition to
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eusociality in termites, even though subsequently, in one derived lineage this symbiosis was lost. The loss of those gut symbionts led to the clade Termitidae, which contains the highest number of species, the highest diversity of lifestyles, including soil-feeding and fungus-
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growing and the highest diversity in social organisation. As such, the loss of those symbionts represents a second major innovation in termites. We have provided arguments that this major innovation also depended on symbiosis, viz. an ectosymbiosis with fungi. Furthermore, our analysis on the colonization of land by higher plants demonstrates that this major innovation also depended on symbiosis, as did the colonisation of land by algae through lichens.
In all those cases, a similar pattern emerges: symbiosis facilitates an evolutionary innovation, even though subsequently, the symbiotic association is lost in some lineages, substituted by
ACCEPTED MANUSCRIPT other symbionts or by host adaptations. This shows that the concept symbiogenesis is more widely applicable than to the origin of eukaryotes and photosynthetic eukaryotes. However, does evolutionary theory need an additional category next to mutation, selection and drift? In other words: does the concept symbiogenesis belong in the same conceptual rank as the currently recognized main categories?
Margulis emphasised the role of symbiogenesis to explain discontinuities in evolution and
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speciation: “Evolution is not gradual as Darwin insisted. Species do not change by accumulation of random mutations as NeoDarwinists insist.” (Margulis, 2010). However, this representation of neo-Darwinian evolutionary theory is a caricature. On a similar basis, one could reject evolution when the evolution of altruism via kin selection was described (Hamilton, 1964a; Hamilton, 1964b), which seemed to be diametrically opposed to
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individual-level selection. This has not happened. Inclusive fitness theory has turned out to be a very fruitful addition to evolutionary theory. The development of evolutionary theory did not stop with the neo-Darwinian synthesis, but has continued to be modified and adapted since. In that sense, the history of evolutionary thinking is like biological evolution itself,
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change by adaptive and random processes, usually gradual but sometimes saltatory.
Acknowledgements
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D.K.A. was supported by The Netherlands Organisation for Scientific Research (VICI; NWO
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