Is Sexual Conflict an “Engine of Speciation”?

At the end of the last century, sexual conflict was identified as a powerful engine of speciation, potentially even more important than ecological selection. Earlier work that followed—experimental, comparative, and mathematical—provided strong initial support for this assertion. However, as the field matures, both the power of sexual conflict and constraints on the evolution of reproductive isolation as driven by sexual conflict are becoming better understood. From theoretical studies, we now know that speciation is only one of several possible evolutionary outcomes of sexual conflict. In line with these predictions, both experimental evolution studies and comparative analyses of fertilization proteins and of species richness show that sexual conflict leads to, or is associated with, reproductive isolation and speciation in some cases but not in others. Increased genetic variation (especially in females) without reproductive isolation is an underappreciated consequence of sexually antagonistic selection.By the end of 1990s, studies of sexual conflict and sexually antagonistic coevolution moved to the forefront of experimental and theoretical research in evolutionary biology (Rice and Holland 1997; Holland and Rice 1998; Rice 1998). Although the potential evolutionary importance of sexual conflict was anticipated and articulated from a theoretical point of view by Geoff Parker 20 years earlier (Parker 1979), the explosive interest in this topic was a result of groundbreaking experimental work with Drosophila melanogaster by Bill Rice (1993, 1996), which directly showed high potential for sexually antagonistic coevolution.Sexual conflict is a special case of intragenomic conflict (Rice and Holland 1997; Rice 1998; Crespi and Nosil 2013). Sexual conflict occurs if the interests of the sexes with regard to certain aspects of reproduction differ (Parker 1979; Arnqvist and Rowe 2005). Ultimately, sexual conflict arises because of the differences in the roles played by the sexes in the process of reproduction, which in turn lead to the differences between the sexes in the costs and benefits of mating and reproduction (Bateman 1948; Trivers 1972; Parker 1979). Sexual conflict can occur over mating rate (Rice and Holland 1997; Holland and Rice 1998; Rice 1998), offspring size (Haig 2000), parental care (Smith and Härdling 2000; Barta et al. 2002), the use of sperm (Ball and Parker 2003), epigenetic control of development (Rice et al. 2012), etc.Sexual conflict can occur through two genetic routes (Chapman and Partridge 1996; Parker and Partridge 1998). Within-locus conflict occurs when the locus controls a trait expressed in both sexes and the optimum trait values differ between the sexes. As a result, optimizing the trait value in one sex will lead to a fitness reduction in the other sex. Within-locus conflict can be resolved via a number of mechanisms, including the evolution of sex linkage, sex-specific expression of genes, gene duplication, and condition dependence (Bonduriansky and Chenoweth 2009; van Doorn 2009). Between-locus conflict occurs when there are two different (sets of) traits each expressed in one sex only but affecting the fitness of both sexes in opposite directions. In this case, adaptive changes in a trait of one sex cause deleterious fitness consequences for the other sex, which can be negated by the evolution in a trait of the other sex, which in turn will cause deleterious fitness consequences for the first sex. For example, males can evolve adaptations increasing their mating rate, which would be detrimental for females who would then evolve some counteradaptations to decrease the mating rate (Rice 1996).One particularly exciting idea that has emerged from studies of sexual conflict and sexually antagonistic coevolution is that sexual conflict can be an important “engine of speciation” (Rice 1996, 1998; Howard et al. 1998; Parker and Partridge 1998). In standard modern perspective, speciation is a result of genetic divergence between populations accompanied by the evolution of reproductive isolation (Howard and Berlocher 1998; Schluter 2000; Coyne and Orr 2004; Dieckmann et al. 2004; Gavrilets 2004). Genetic divergence can be driven by a variety of evolutionary factors, including mutation, random genetic drift, and natural, sexual, and social selection. Reproductive isolation can follow from a variety of mechanisms, resulting in incompatibilities (including genetic, developmental, morphological, ecological, and behavioral) of males and females from diverging populations or in a reduced fitness of their offspring. As was argued by Rice (1998), Parker and Partridge (1998), and others (e.g., Howard et al. 1998), sexual conflict can contribute to these processes in a number of ways.Below, I briefly summarize several, mostly verbal, theories of biological diversification caused by sexual conflict and then move to discussing some of the more concrete mathematical models and empirical data and patterns.     

Sexual Conflict between Parents: Offspring Desertion and Asymmetrical Parental Care

Parental care is an immensely variable social behavior, and sexual conflict offers a powerful paradigm to understand this diversity. Conflict over care (usually considered as a type of postzygotic sexual conflict) is common, because the evolutionary interests of male and female parents are rarely identical. I investigate how sexual conflict over care may facilitate the emergence and maintenance of diverse parenting strategies and argue that researchers should combine two fundamental concepts in social behavior to understand care patterns: cooperation and conflict. Behavioral evidence of conflict over care is well established, studies have estimated specific fitness implications of conflict for males or females, and experiments have investigated specific components of conflict. However, studies are long overdue to reveal the full implications of conflict for both males and females. Manipulating (or harming) the opposite sex seems less common in postzygotic conflicts than in prezygotic conflicts because by manipulating, coercing, or harming the opposite sex, the reproductive interest of the actor is also reduced. Parental care is a complex trait, although few studies have yet considered the implications of multidimensionality for parental conflict. Future research in parental conflict will benefit from understanding the behavioral interactions between male and female parents (e.g., negotiation, learning, and coercion), the genetic and neurogenomic bases of parental behavior, and the influence of social environment on parental strategies. Empirical studies are needed to put sexual conflict in a population context and reveal feedback between mate choice, pair bonds and parenting strategies, and their demographic consequences for the population such as mortalities and sex ratios. Taken together, sexual conflict offers a fascinating avenue for understanding the causes and consequences of parenting behavior, sex roles, and breeding system evolution.Sexual conflict over care is a type of evolutionary conflict that emerges from the different interests of males and females in regard to parental care (Trivers 1972; Clutton-Brock 1991; Chapman et al. 2003; Arnqvist and Rowe 2005). The conflict arises when the young benefit from the effort of either parent, but each parent pays only the cost of its own effort, so that each parent would have higher fitness if the other parent provides more care (Houston et al. 2005; Lessells 2006; Klug et al. 2012). Conflict refers to the way selection acts on the two sexes that have different optimum values in parental provisioning; between the two optima, sexually antagonistic selection operates (Lessells 2012). Sexual conflict over care can be seen as tug-of-war, because each parent is tempted to pull out of care leaving the other parent to provide more care for the young (Székely et al. 1996; Arnqvist and Rowe 2005; Lessells 2012).Sexual conflict over care seems to be the rule rather than the exception. The conflict may be resolved by one or both parents failing to adopt the optimal parenting for their mate and nonetheless remaining in conflict, or by both parents adopting the optima that suit their mate (i.e., exhibit the maximum provisioning possible). Examples of the latter conflict resolution (whereby the conflict is completely wiped out) are exceedingly rare and seem to be limited to three scenarios. First, conflict over care is not expected in obligate monogamy by both males and females so that the lifetime reproductive successes of both parents are identical. This may occur in semelparous organisms (i.e., both the male and the female put their resources into a single breeding event) or in iteroparous organisms with lifelong exclusive monogamy. Second, males and females might be genetically identical, so even though one or both sexes are polygamous, polygamy would benefit the same genome whether it is in the male or the female phenotype. Third, parental care is cost-free and thus parents provide maximum level of care (P Smiseth, pers. comm.). However, few, if any, organisms fit these restrictive assumptions, and thus conflict-free parenting seems exceedingly rare in nature: (1) some level of polygamy (by males, females, or both sexes) appears to be widespread; (2) the reproduction by genetically identical individuals (clones) as separate sexes (males and females) seems unlikely although not impossible if sex is determined environmentally; and (3) care provisioning, as far as we are aware, does have costs that discourage parents from providing their absolute maxima for a given batch of offspring.Parents may have conflicting interest over caring or deserting the young, the amount of care provided for each young, the number of simultaneous mates, the size and sex ratio of their brood, and the synchronization of birth for a clutch or litter of young (Westneat and Sargent 1996; Houston et al. 2005; Klug et al. 2012; Lessells 2012). Conflict between parents over care is usually labeled as a postzygotic conflict although resources had been already allocated into the gametes before fertilization as part of parental provisioning (Clutton-Brock 1991); other examples of postzygotic conflicts include infanticide and genomic imprinting (Chapman et al. 2003; Tregenza et al. 2006; Lessells 2012; see Palombit 2014).Studies of conflict over care are fascinating for at least four major reasons. First, parental care is diverse. There is great variation both between and within species in the types of care provided, duration of care, and the sex of the care-providing parent (Wilson 1975; Clutton-Brock 1991; McGraw et al. 2010; Royle et al. 2012), and sexual conflict is thought to be one of the main drivers of this diversity. Second, parental care is one of the core themes in breeding systems and sex role evolution, and it is increasingly evident that parental care can only be understood by dissecting the entangled relationships between ecological and life-history settings, and the variety of mating and parenting behavior (Székely et al. 2000; Webb et al. 2002; Wedell et al. 2006; Jennions and Kokko 2010; Klug et al. 2012). Third, parental care was (and is) one of the test beds of evolutionary game theory. Numerous models have been developed to understand how parents interact with each other and with their offspring (Trivers 1972; Maynard Smith 1977; Houston and Davies 1985; Balshine-Earn and Earn 1998; McNamara et al. 1999, 2000; Webb et al. 1999; Johnstone and Hinde 2006; Johnstone et al. 2014). Parental care research is one field in which empiricists are extensively testing the predictions of evolutionary game theoretic models in both the laboratory and wild populations (Székely et al. 1996; Balshine-Earn and Earn 1998; Harrison et al. 2009; Klug et al. 2012; Lessells 2012; van Dijk et al. 2012), although the congruence between theoretical and empirical work is not as tight as often assumed (Houston et al. 2013). Finally, parental care—wherever it occurs—is often a major component of fitness, because whether the offspring are cared for or abandoned has a large impact on their survival, maturation, and reproduction (Smiseth et al. 2012). Therefore, parental care (or the lack of it) may have an impact on population productivity and population growth and influences the resilience of populations to various threats (Bessa-Gomes et al. 2004; Veran and Beissinger 2009; Blumstein 2010). Thus, understanding the behavioral interactions between parents and the fitness implications of these interactions is highly relevant for population dynamics and biodiversity conservation (Alonzo and Sheldon 2010; Blumstein 2010).Sexual conflict over care has been reviewed recently (van Dijk and Székely 2008; Lessells 2012; Houston et al. 2013). Here, I focus on three issues that have not been extensively covered by previous reviews: (1) why sexual conflict over care occurs in some environments, whereas in others parental cooperation appears to dominate; (2) how can one detect sexual conflict over care; and (3) what are the implications of sexual conflict over care for macroevolution. I view causes and implications of parental care primarily from empirical perspectives; there are excellent reviews on the rich theoretical literature (Lessells 2006, 2012; Klug et al. 2012; Houston et al. 2013). My intention is not to be comprehensive; instead, I use selected examples to illustrate salient features of conflict over care. I focus on ecological and evolutionary aspects; for a discussion of the genetic and neuroendocrine bases of parental care, see Adkins-Regan (2005), McGraw et al. (2010), and Champagne and Curley (2012). I prefer to use the term “parental care” instead of “parental investment,” because the latter, as admitted by Trivers (1985), is extremely difficult to estimate empirically and thus may have a limited use in empirical studies (Mock and Parker 1997; McGraw et al. 2010). The term “parental investment” can be deceptive, if used without directly demonstrating the full costs of care. The term “parental care” is less restrictive, because it refers to any form of parental behavior that appears to increase the fitness of an offspring and is likely to have evolved for this function (Clutton-Brock 1991; Smiseth et al. 2012). In this review, I focus on families in the narrow sense (i.e., two parents and their offspring), although in numerous organisms the families are more extensive and may include several generations of offspring living together and/or unrelated individuals that assist the parents rearing the young.     

Sexual Conflict and Seminal Fluid Proteins: A Dynamic Landscape of Sexual Interactions

Sexual reproduction requires coordinated contributions from both sexes to proceed efficiently. However, the reproductive strategies that the sexes adopt often have the potential to give rise to sexual conflict because they can result in divergent, sex-specific costs and benefits. These conflicts can occur at many levels, from molecular to behavioral. Here, we consider sexual conflict mediated through the actions of seminal fluid proteins. These proteins provide many excellent examples in which to trace the operation of sexual conflict from molecules through to behavior. Seminal fluid proteins are made by males and provided to females during mating. As agents that can modulate egg production at several steps, as well as reproductive behavior, sperm “management,” and female feeding, activity, and longevity, the actions of seminal proteins are prime targets for sexual conflict. We review these actions in the context of sexual conflict. We discuss genomic signatures in seminal protein (and related) genes that are consistent with current or previous sexual conflict. Finally, we note promising areas for future study and highlight real-world practical situations that will benefit from understanding the nature of sexual conflicts mediated by seminal proteins.Both sexes benefit from successful reproduction, but the different reproductive strategies adopted by males and females may result in differential costs and benefits. This can result in sexual conflict before, during, and after mating. Conflict in the more familiar form of competition can also occur between females and between males, with the latter situation including interejaculate competition. Of the many “weapons” in these conflicts and competitions, this article focuses on the seminal fluid proteins (SFPs) that are made by males and transferred to females during mating. These proteins represent a crucial interface of functional activity between male and female. Transfer of SFPs can affect physiology and, in some animals, the behavior and life span of mated females (reviewed in Chapman 2001; Gillott 2003; Poiani 2006; Avila et al. 2011; Rodríguez-Martínez et al. 2011). Because SFPs have important effects on the most intimate of interactions between the sexes, they are prime candidates to become subject to sexually antagonistic selection (Arnqvist and Rowe 2005). With increasing knowledge of the functions of SFPs, their roles in inter- and intrasexual conflict and their evolutionary responses to conflict are becoming ever more apparent. Here, we explore the roles, evolution, and significance of these male-derived players in sexual conflict. We refer the reader to previous reviews for much of the detailed functional information on SFPs (e.g., Chapman 2001; Gillott 2003; Kubli 2003; Arnqvist and Rowe 2005; Poiani 2006; Sirot et al. 2009; Avila et al. 2011; Rodríguez-Martínez et al. 2011) and focus here instead on selected examples, drawn largely from the study of insects.     

Paradox of Mother's Curse and the Maternally Provisioned Offspring Microbiome

Strict maternal transmission creates an “asymmetric sieve” favoring the spread of mutations in organelle genomes that increase female fitness, but diminish male fitness. This phenomenon, called “Mother''s Curse,” can be viewed as an asymmetrical case of intralocus sexual conflict. The evolutionary logic of Mother''s Curse applies to each member of the offspring microbiome, the community of maternally provisioned microbes, believed to number in the hundreds, if not thousands, of species for host vertebrates, including humans. Taken together, these observations pose a compelling evolutionary paradox: How has maternal transmission of an offspring microbiome become a near universal characteristic of the animal kingdom when the genome of each member of that community poses a potential evolutionary threat to the fitness of host males? I review features that limit or reverse Mother''s Curse and contribute to resolving this paradox. I suggest that the evolution of vertical symbiont transmission requires conditions that mitigate the evolutionary threat to host males.The genomes of mitochondria, chloroplasts, and many symbiotic microbes are transmitted maternally by host females to their offspring. Maternal transmission can be transovariole (intracellular, within the egg) or contagious, during gestation, birth, or feeding (Sonneborn 1950; Smith and Dunn 1991; Gillham 1994; O’Neill et al. 1997). Vertically transmitted (VT) symbiont lineages tend to be genetically homogeneous within hosts (Birky et al. 1983, 1989; Funk et al. 2000). Maternal uniparental transmission creates an “asymmetric sieve” wherein mutations advantageous for females, but harmful for males, can spread through a population (Cosmides and Tooby 1981; Frank and Hurst 1996; Zeh and Zeh 2005; Burt and Trivers 2006). Such mutations spread because deleterious male-specific fitness effects do not affect the response to natural selection of the maternally transmitted entities. This adaptive process favoring the transmitting sex is called Mother''s Curse (MC) (Gemmell et al. 2004) and it has been referred to as an irreconcilable instance of intralocus conflict: “… exclusively maternal transmission of cytoplasmic genes (e.g., in mitochondria) can result in suboptimal mitochondrial function in males … a form of [intralocus sexual conflict] that apparently cannot be resolved, because selection on mitochondria in males cannot produce a response” (Bonduriansky and Chenoweth 2009, p. 285).Mitochondria are ubiquitous in animals and despite the indisputable evolutionary logic of MC (Frank and Hurst 1996) there are no reported cases of sperm-killing or son-killing mitochondria (Burt and Trivers 2006). Moreover, many species of animals possess an offspring microbiome, a community of microbes transmitted uniparentally from mother to offspring at some point in development, whether prefertilization, postfertilization, or postnatal (Funkhouser and Bordenstein 2013). In some vertebrates, including humans, this community is believed to number in the hundreds of species (Funkhouser and Bordenstein 2013). Prolonged periods of maternal care, as in mammals and birds, as well as kin-structured sociality, afford many opportunities for maternal provisioning of microbes to developing offspring. The social insects, in particular, show obligate mutualisms with a microbiome that confers important nutritional benefits for its host (Baumann 2005; Engel and Moran 2013), the termites being a classic example (Ikeda-Ohtsubo and Brune 2009).Together, the evolutionary logic of MC and the widespread existence of maternally transmitted hereditary symbioses pose a paradox for evolutionary biology. The maternally provisioned microbiome (MC) consists of tens to hundreds of genomes affording ample opportunity, along with mitochondrial and organelle genomes, for the occurrence of mutations that benefit females while harming host males. Assembling a VT community as a host nutritional or defensive adaptation requires evading MC not once, but from a continuous siege over evolutionary time. This is the Mother''s Curse–microbiome (MC–MB) paradox. It conceptually affiliated with the “paradox of mutualism,” the persistence of interspecific mutualisms despite the advantages of cheating by one or the other member of the mutualism (Heath and Stinchcombe 2014). Symbiont “cheating” on only half the members of a host species, the males, might offer marginal benefits relative to wholesale cheating on both host sexes. Nevertheless, the MC–MB paradox deserves research attention.In this review, I discuss inbreeding, kin selection, compensatory evolution, and defensive advantages against more virulent pathogens (or predators and herbivores) as means for resolving the MC–MB paradox. First, I review the simple population genetics of MC. I discuss how host inbreeding and kin selection (Unckless and Herren 2009; Wade and Brandvain 2009), alone or in concert, allow for a response to selection on male fertility and viability fitness effects of maternally transmitted genomes. As a result, inbreeding and kin selection can limit or prevent the spread of mutations in a hereditary symbiosis (Cowles 1915) that are harmful to males. I will show that, for both inbreeding and kin selection, there exist conditions that “favor the spread of maternally transmitted mutations harmful to females”; a situation that is the reverse of MC. However, many outbreeding, asocial species harbor maternally provisioned microbiomes and these solutions cannot be applied to them.I also consider the evolution of compensatory nuclear mutations that mitigate or eliminate the harm to males of organelles or symbionts, spreading via MC dynamics. However, I find that the relative rate of compensatory evolution is only 1/4 the rate of evolution of male-harming symbionts. Thus, an evolutionary rescue of host males via compensatory host nuclear mutations requires that there be fourfold or more opportunities for compensation offered by a larger host nuclear genome. The larger the number of species in a host microbiome, the more difficult it is to entertain host nuclear compensatory mutations as a resolution of the MC–MB paradox.Next, I consider the situation in which a deleterious, VT symbiont harms its host but prevents host infection by a more severely deleterious contagiously transmitted pathogen (Lively et al. 2005; see also Clay 1988). This is a case in which absolute harm to a host by a maternally provisioned symbiont becomes a “relative” fitness advantage. This is a scenario that may be common in hosts with speciose microbial communities, especially if each microbial species increases host resistance or outright immunity to infectious, virulent pathogens.Finally, I discuss models of symbiont domestication and capture via the evolution of vertical transmission from an ancestral state of horizontal transmission (Drown et al. 2013). I show that the evolution of vertical transmission requires conditions that tend to restrict the capacity for male harming by symbionts. Each of these scenarios significantly expands the range of evolutionary possibilities permitted for the coevolution of host–symbiont assemblages, especially those microbial communities that are maternally, uniparentally transmitted across host generations. Unfortunately, current data do not permit discriminating among these various evolutionary responses to MC, so none can be definitively considered a resolution of the MC–MB paradox.     

Biology of the TAM Receptors

The TAM receptors—Tyro3, Axl, and Mer—comprise a unique family of receptor tyrosine kinases, in that as a group they play no essential role in embryonic development. Instead, they function as homeostatic regulators in adult tissues and organ systems that are subject to continuous challenge and renewal throughout life. Their regulatory roles are prominent in the mature immune, reproductive, hematopoietic, vascular, and nervous systems. The TAMs and their ligands—Gas6 and Protein S—are essential for the efficient phagocytosis of apoptotic cells and membranes in these tissues; and in the immune system, they act as pleiotropic inhibitors of the innate inflammatory response to pathogens. Deficiencies in TAM signaling are thought to contribute to chronic inflammatory and autoimmune disease in humans, and aberrantly elevated TAM signaling is strongly associated with cancer progression, metastasis, and resistance to targeted therapies.The name of the TAM family is derived from the first letter of its three constituents—Tyro3, Axl, and Mer (Prasad et al. 2006). As detailed in Figure 1, members of this receptor tyrosine kinase (RTK) family were independently identified by several different groups and appear in the early literature under multiple alternative names. However, Tyro3, Axl, and Mer (officially c-Mer or MerTK for the protein, Mertk for the gene) have now been adopted as the NCBI designations. The TAMs were first grouped into a distinct RTK family (the Tyro3/7/12 cluster) in 1991, through PCR cloning of their kinase domains (Lai and Lemke 1991). The isolation of full-length cDNAs for Axl (O''Bryan et al. 1991), Mer (Graham et al. 1994), and Tyro3 (Lai et al. 1994) confirmed their segregation into a structurally distinctive family of orphan RTKs (Manning et al. 2002b). The two ligands that bind and activate the TAMs—Gas6 and Protein S (Pros1)—were identified shortly thereafter (Ohashi et al. 1995; Stitt et al. 1995; Mark et al. 1996; Nagata et al. 1996).Open in a separate windowFigure 1.TAM receptors and ligands. The TAM receptors (red) are Tyro3 (Lai and Lemke 1991; Lai et al. 1994)—also designated Brt (Fujimoto and Yamamoto 1994), Dtk (Crosier et al. 1994), Rse (Mark et al. 1994), Sky (Ohashi et al. 1994), and Tif (Dai et al. 1994); Axl (O''Bryan et al. 1991)—also designated Ark (Rescigno et al. 1991), Tyro7 (Lai and Lemke 1991), and Ufo (Janssen et al. 1991); and Mer (Graham et al. 1994)—also designated Eyk (Jia and Hanafusa 1994), Nyk (Ling and Kung 1995), and Tyro12 (Lai and Lemke 1991). The TAMs are widely expressed by cells of the mature immune, nervous, vascular, and reproductive systems. The TAM ligands (blue) are Gas6 and Protein S (Pros1). The carboxy-terminal SHBG domains of the ligands bind to the immunoglobulin (Ig) domains of the receptors, induce dimerization, and activate the TAM tyrosine kinases. When γ-carboxylated in a vitamin-K-dependent reaction, the amino-terminal Gla domains of the dimeric ligands bind to the phospholipid phosphatidylserine expressed on the surface on an apposed apoptotic cell or enveloped virus. See text for details. (From Lemke and Burstyn-Cohen 2010; adapted, with permission, from the authors.)Subsequent progress on elucidating the biological roles of the TAM receptors was considerably slower and ultimately required the derivation of mouse loss-of-function mutants (Camenisch et al. 1999; Lu et al. 1999). The fact that Tyro3^−/−, Axl^−/−, and Mer^−/− mice are all viable and fertile permitted the generation of a complete TAM mutant series that included all possible double mutants and even triple mutants that lack all three receptors (Lu et al. 1999). Remarkably, these Tyro3^−/−Axl^−/−Mer^−/− triple knockouts (TAM TKOs) are viable, and for the first 2–3 wk after birth, superficially indistinguishable from their wild-type counterparts (Lu et al. 1999). Because many RTKs play essential roles in embryonic development, even single loss-of-function mutations in RTK genes often result in an embryonic-lethal phenotype (Gassmann et al. 1995; Lee et al. 1995; Soriano 1997; Arman et al. 1998). The postnatal viability of mice in which an entire RTK family is ablated completely—the TAM TKOs can survive for more than a year (Lu et al. 1999)—is therefore highly unusual. Their viability notwithstanding, the TAM mutants go on to develop a plethora of phenotypes, some of them debilitating (Camenisch et al. 1999; Lu et al. 1999; Lu and Lemke 2001; Scott et al. 2001; Duncan et al. 2003; Prasad et al. 2006). Almost without exception, these phenotypes are degenerative in nature and reflect the loss of TAM signaling activities in adult tissues that are subject to regular challenge, renewal, and remodeling. These activities are the subject of this review.     

Sexual Cannibalism as a Manifestation of Sexual Conflict

Sexual cannibalism is a well-known example for sexual conflict and has many facets that determine the costs and benefits for the cannibal and the victim. Here, I focus on species in which sexual cannibalism is a general component of a mating system in which males invest maximally in mating with a single (monogyny) or two (bigyny) females. Sexual cannibalism can be a male strategy to maximize paternity and a female strategy to prevent paternity monopolization by any or a particular male. Considerable variation exists between species (1) in the potential of males to monopolize females, and (2) in the success of females in preventing monopolization by males. This opens up exciting future possibilities to investigate sexually antagonistic coevolution in a largely unstudied mating system.Sexual cannibalism, the killing and consumption of potential or actual mating partners in a mating context, has been termed a “pinnacle of sexual conflict” because of the dramatic ending of the act for one mating partner, mostly the male (Elgar and Schneider 2004). This contradiction of traditional sex roles may be one reason why the phenomenon of sexual cannibalism has intrigued naturalists for a long time. In the context of sexual conflict, sexually cannibalistic behavior of females is a harmful trait, and antagonistic traits are expected to evolve in males, which can be considered the reverse of most other examples in which females respond to male harm (see Perry and Rowe 2014). I will discuss potential antagonistic traits to sexual cannibalism in males but will also show that the above view is too simplistic when it comes to spider mating systems characterized by very low male mating rates.It is important to note that there are different kinds of sexual cannibalism based on very different evolutionary scenarios (Elgar and Schneider 2004; Prenter et al. 2006; Wilder et al. 2009). The most extreme divide exists between cannibalism before sperm transfer, which can only benefit the cannibal, and sexual cannibalism during or after sperm transfer (from here on termed postinsemination sexual cannibalism), which can benefit the cannibal and the victim (Elgar and Schneider 2004). Despite a longer history of research on preinsemination sexual cannibalism, the evolutionary causes and consequences of postinsemination sexual cannibalism are generally less debated.There are reports (often anecdotal) on the occurrence of sexual cannibalism from diverse invertebrate taxa (Elgar 1992) and it may well occur in all predatory invertebrates that are potentially cannibalistic (Polis 1981). It is beyond the scope of this brief review to list and evaluate all reported occurrences. Rather, I will start with a brief account of the generally discussed causes and consequences of sexual cannibalism and will then concentrate on the conflicting interests of the sexes regarding postinsemination sexual cannibalism in mating systems that are characterized by very low male mating rates.Studies that investigate sexual cannibalism experimentally are mostly concerned with (1) nutritional aspects, (2) the importance of sexual size dimorphism and sexual selection, and, increasingly, (3) behavioral syndromes. The aggressive spillover hypothesis suggests that preinsemination sexual cannibalism is part of a behavioral syndrome in which aggression against mating partners spills over from a foraging context (Arnqvist and Henriksson 1997). There is mixed support for this idea in the few species that have been looked at. In several spider species, females consistently differ in their aggressiveness and these differences affect sexual cannibalism (for a recent debate about the evidence for this hypothesis, see Johnson 2013; Kralj-Fišer et al. 2013b; Pruitt and Keiser 2013).A majority of studies have taken a unilateral view and have been concerned with the “motivation” of the cannibal; because sexual cannibalism generally occurs in predators, hunger is a well-supported motivation (Wilder et al. 2009). Many predators are food-limited, and, assuming a trade-off between foraging and mating, the balance may tilt toward foraging under particular circumstances (modeled by Newman and Elgar 1991). Food and mate availability will influence the costs and benefits of sexual cannibalism for females and have been one focus of a recent review on sexual cannibalism (Wilder et al. 2009).In all predatory and cannibalistic animals, mating partners impose selection on each other’s abilities to avoid or resist aggression. This selection pressure is asymmetrical if one sex is physically dominant. Indeed, the differences in size between females and males often determine the frequency of sexual cannibalism, perhaps because the potential to resist a cannibalistic attack is size-dependent (Elgar 1992; Wilder and Rypstra 2008). Usually, males are the victims and females are the cannibals. Yet, reversed sexual cannibalism has also been reported and appears to be associated with the reversed pattern in sexual size dimorphism. Examples are the water spider, Arygoneta aquatica (Schutz and Taborsky 2005, 2011) and role-reversed wolf spiders (Aisenberg et al. 2011). In the gnaphosid spider, Micaria sociabilis, large, young males cannibalize old and relatively smaller females (Sentenska and Pekar 2013). These examples further support the notion that the relative size differences of a mating pair play a part in determining the likelihood of sexual cannibalism. Patterns can be found both on a between-species comparative scale and on a within-species scale (Wilder and Rypstra 2008; Wilder et al. 2009), and they are also reported as an underlying pattern in cannibalism outside a mating context (Bleakley et al. 2013). Furthermore, there is anecdotal evidence for the same pattern in hermaphrodites (e.g., Goto and Yoshida 1985; Michiels et al. 2003), which may constitute a particularly interesting case to study, as the power asymmetries are less obviously related to the male or female role.In asymmetric encounters, the costs and risks of aggressive behavior toward potential mating partners are low for the dominant partner. Toward smaller males, females could use aggressiveness as a means of partner choice. Indeed, many studies suggest that sexual selection in addition to gaining a meal may be the adaptive value of sexual cannibalism (Prenter et al. 2006). From the female perspective, aggressive behavior directed toward males may serve as a general screening of partner quality, a mechanism often described as indirect mate choice (Elgar and Nash 1988; Prenter et al. 2006; Kralj-Fišer et al. 2012). A screening method implies that females attack every male, and suitors that cannot withstand and persist an attack will be killed and consumed; alternatively, females may differentiate between males and attack and consume only those males that do not meet certain quality criteria (reviewed in Prenter et al. 2006). The latter has been found in wolf spiders (Wilgers and Hebets 2012). The latter mechanism of direct choice is more complex than the indirect one as it requires perception and assessment of quality cues, and large enough benefits of choosiness are expected to match the costs. Mate rejection via sexual cannibalism is considered a particularly extreme case of sexual conflict mostly because rejection can lead to death. Although this may be true for the individual male that loses all future reproductive success, frequencies of preinsemination sexual cannibalism might be rather low (Kralj-Fišer et al. 2013b). Please note that in almost every species, a certain proportion of individuals will be excluded from the mating market and will have no mating success. The claim that prevention of mating success via sexual cannibalism results in more intense sexual conflict than exclusion from mating with less drastic measures has, to my knowledge, never been tested. Because of the scarcity of data on natural frequencies of preinsemination cannibalism, a meta-analysis would not reveal a realistic picture at this stage. Hence, to date, it is not feasible to compare the relative strength of selection imposed by a cannibalistic mate choice strategy against a strategy with less drastic consequences of mate rejection. More studies are needed to unravel the exact nature of sexual selection under the threat of ending as a meal. Below, I will briefly sketch possible responses to selection imposed by sexually cannibalistic females before or during insemination.     

Origin and Evolution of the Self-Organizing Cytoskeleton in the Network of Eukaryotic Organelles

The eukaryotic cytoskeleton evolved from prokaryotic cytomotive filaments. Prokaryotic filament systems show bewildering structural and dynamic complexity and, in many aspects, prefigure the self-organizing properties of the eukaryotic cytoskeleton. Here, the dynamic properties of the prokaryotic and eukaryotic cytoskeleton are compared, and how these relate to function and evolution of organellar networks is discussed. The evolution of new aspects of filament dynamics in eukaryotes, including severing and branching, and the advent of molecular motors converted the eukaryotic cytoskeleton into a self-organizing “active gel,” the dynamics of which can only be described with computational models. Advances in modeling and comparative genomics hold promise of a better understanding of the evolution of the self-organizing cytoskeleton in early eukaryotes, and its role in the evolution of novel eukaryotic functions, such as amoeboid motility, mitosis, and ciliary swimming.The eukaryotic cytoskeleton organizes space on the cellular scale and this organization influences almost every process in the cell. Organization depends on the mechanochemical properties of the cytoskeleton that dynamically maintain cell shape, position organelles, and macromolecules by trafficking, and drive locomotion via actin-rich cellular protrusions, ciliary beating, or ciliary gliding. The eukaryotic cytoskeleton is best described as an “active gel,” a cross-linked network of polymers (gel) in which many of the links are active motors that can move the polymers relative to each other (Karsenti et al. 2006). Because prokaryotes have only cytoskeletal polymers but lack motor proteins, this “active gel” property clearly sets the eukaryotic cytoskeleton apart from prokaryotic filament systems.Prokaryotes contain elaborate systems of several cytomotive filaments (Löwe and Amos 2009) that share many structural and dynamic features with eukaryotic actin filaments and microtubules (Löwe and Amos 1998; van den Ent et al. 2001). Prokaryotic cytoskeletal filaments may trace back to the first cells and may have originated as higher-order assemblies of enzymes (Noree et al. 2010; Barry and Gitai 2011). These cytomotive filaments are required for the segregation of low copy number plasmids, cell rigidity and cell-wall synthesis, cell division, and occasionally the organization of membranous organelles (Komeili et al. 2006; Thanbichler and Shapiro 2008; Löwe and Amos 2009). These functions are performed by dynamic filament-forming systems that harness the energy from nucleotide hydrolysis to generate forces either via bending or polymerization (Löwe and Amos 2009; Pilhofer and Jensen 2013). Although the identification of actin and tubulin homologs in prokaryotes is a major breakthrough, we are far from understanding the origin of the structural and dynamic complexity of the eukaryotic cytoskeleton.Advances in genome sequencing and comparative genomics now allow a detailed reconstruction of the cytoskeletal components present in the last common ancestor of eukaryotes. These studies all point to an ancestrally complex cytoskeleton, with several families of motors (Wickstead and Gull 2007; Wickstead et al. 2010) and filament-associated proteins and other regulators in place (Jékely 2003; Richards and Cavalier-Smith 2005; Rivero and Cvrcková 2007; Chalkia et al. 2008; Eme et al. 2009; Fritz-Laylin et al. 2010; Eckert et al. 2011; Hammesfahr and Kollmar 2012). Genomic reconstructions and comparative cell biology of single-celled eukaryotes (Raikov 1994; Cavalier-Smith 2013) allow us to infer the cellular features of the ancestral eukaryote. These analyses indicate that amoeboid motility (Fritz-Laylin et al. 2010; although, see Cavalier-Smith 2013), cilia (Cavalier-Smith 2002; Mitchell 2004; Jékely and Arendt 2006; Satir et al. 2008), centrioles (Carvalho-Santos et al. 2010), phagocytosis (Cavalier-Smith 2002; Jékely 2007; Yutin et al. 2009), a midbody during cell division (Eme et al. 2009), mitosis (Raikov 1994), and meiosis (Ramesh et al. 2005) were all ancestral eukaryotic cellular features. The availability of functional information from organisms other than animals and yeasts (e.g., Chlamydomonas, Tetrahymena, Trypanosoma) also allow more reliable inferences about the ancestral functions of cytoskeletal components (i.e., not only their ancestral presence or absence) and their regulation (Demonchy et al. 2009; Lechtreck et al. 2009; Suryavanshi et al. 2010).The ancestral complexity of the cytoskeleton in eukaryotes leaves a huge gap between prokaryotes and the earliest eukaryote we can reconstruct (provided that our rooting of the tree is correct) (Cavalier-Smith 2013). Nevertheless, we can attempt to infer the series of events that happened along the stem lineage, leading to the last common ancestor of eukaryotes. Meaningful answers will require the use of a combination of gene family history reconstructions (Wickstead and Gull 2007; Wickstead et al. 2010), transition analyses (Cavalier-Smith 2002), and computer simulations relevant to cell evolution (Jékely 2008).     

Wnt Signaling in Vertebrate Axis Specification

The Wnt pathway is a major embryonic signaling pathway that controls cell proliferation, cell fate, and body-axis determination in vertebrate embryos. Soon after egg fertilization, Wnt pathway components play a role in microtubule-dependent dorsoventral axis specification. Later in embryogenesis, another conserved function of the pathway is to specify the anteroposterior axis. The dual role of Wnt signaling in Xenopus and zebrafish embryos is regulated at different developmental stages by distinct sets of Wnt target genes. This review highlights recent progress in the discrimination of different signaling branches and the identification of specific pathway targets during vertebrate axial development.Wnt pathways play major roles in cell-fate specification, proliferation and differentiation, cell polarity, and morphogenesis (Clevers 2006; van Amerongen and Nusse 2009). Signaling is initiated in the responding cell by the interaction of Wnt ligands with different receptors and coreceptors, including Frizzled, LRP5/6, ROR1/2, RYK, PTK7, and proteoglycans (Angers and Moon 2009; Kikuchi et al. 2009; MacDonald et al. 2009). Receptor activation is accompanied by the phosphorylation of Dishev-elled (Yanagawa et al. 1995), which appears to transduce the signal to both the cell membrane and the nucleus (Cliffe et al. 2003; Itoh et al. 2005; Bilic et al. 2007). Another common pathway component is β-catenin, an abundant component of adherens junctions (Nelson and Nusse 2004; Grigoryan et al. 2008). In response to signaling, β-catenin associates with T-cell factors (TCFs) and translocates to the nucleus to stimulate Wnt target gene expression (Behrens et al. 1996; Huber et al. 1996; Molenaar et al. 1996).This β-catenin-dependent activation of specific genes is often referred to as the “canonical” pathway. In the absence of Wnt signaling, β-catenin is destroyed by the protein complex that includes Axin, GSK3, and the tumor suppressor APC (Clevers 2006; MacDonald et al. 2009). Wnt proteins, such as Wnt1, Wnt3, and Wnt8, stimulate Frizzled and LRP5/6 receptors to inactivate this β-catenin destruction complex, and, at the same time, trigger the phosphorylation of TCF proteins by homeodomain-interacting protein kinase 2 (HIPK2) (Hikasa et al. 2010; Hikasa and Sokol 2011). Both β-catenin stabilization and the regulation of TCF protein function by phosphorylation appear to represent general strategies that are conserved in multiple systems (Sokol 2011). Thus, the signaling pathway consists of two branches that together regulate target gene expression (Fig. 1).Open in a separate windowFigure 1.Conserved Wnt pathway branches and components. In the absence of Wnt signals, glycogen synthase kinase 3 (GSK3) binds Axin and APC to form the β-catenin destruction complex. Some Wnt proteins, such as Wnt8 and Wnt3a, stimulate Frizzled and LRP5/6 receptors to inhibit GSK3 activity and stabilize β-catenin (β-cat). Stabilized β-cat forms a complex with T-cell factors (e.g., TCF1/LEF1) to activate target genes. Moreover, GSK3 inhibition leads to target gene derepression by promoting TCF3 phosphorylation by homeodomain-interacting protein kinase 2 (HIPK2) through an unknown mechanism, for which β-catenin is required as a scaffold. This phosphorylation results in TCF3 removal from target promoters and gene activation. Other Wnt proteins, such as Wnt5a and Wnt11, use distinct receptors such as ROR2 and RYK, in addition to Frizzled, to control the the cytoskeletal organization through core planar cell polarity (PCP) proteins, small GTPases (Rho/Rac/Cdc42), and c-Jun amino-terminal kinase (JNK).Other Wnt proteins, such as Wnt5a or Wnt11, strongly affect the cytoskeletal organization and morphogenesis without stabilizing β-catenin (Torres et al. 1996; Angers and Moon 2009; Wu and Mlodzik 2009). These “noncanonical” ligands do not influence TCF3 phosphorylation (Hikasa and Sokol 2011), but may use distinct receptors such as ROR1/2 and RYK instead of or in addition to Frizzled (Hikasa et al. 2002; Lu et al. 2004; Mikels and Nusse 2006; Nishita et al. 2006, 2010; Schambony and Wedlich 2007; Grumolato et al. 2010; Lin et al. 2010; Gao et al. 2011). In such cases, signaling mechanisms are likely to include planar cell polarity (PCP) components, such as Vangl2, Flamingo, Prickle, Diversin, Rho GTPases, and c-Jun amino-terminal kinases (JNKs), which do not directly affect β-catenin stability (Fig. 1) (Sokol 2000; Schwarz-Romond et al. 2002; Schambony and Wedlich 2007; Komiya and Habas 2008; Axelrod 2009; Itoh et al. 2009; Tada and Kai 2009; Sato et al. 2010; Gao et al. 2011). This simplistic dichotomy of the Wnt pathway does not preclude some Wnt ligands from using both β-catenin-dependent and -independent routes in a context-specific manner.Despite the existence of many pathway branches, only the β-catenin-dependent branch has been implicated in body-axis specification. Recent experiments in lower vertebrates have identified additional pathway components and targets and provided new insights into the underlying mechanisms.     

Autophagy and Neuronal Cell Death in Neurological Disorders

Autophagy is implicated in the pathogenesis of major neurodegenerative disorders although concepts about how it influences these diseases are still evolving. Once proposed to be mainly an alternative cell death pathway, autophagy is now widely viewed as both a vital homeostatic mechanism in healthy cells and as an important cytoprotective response mobilized in the face of aging- and disease-related metabolic challenges. In Alzheimer’s, Parkinson’s, Huntington’s, amyotrophic lateral sclerosis, and other diseases, impairment at different stages of autophagy leads to the buildup of pathogenic proteins and damaged organelles, while defeating autophagy’s crucial prosurvival and antiapoptotic effects on neurons. The differences in the location of defects within the autophagy pathway and their molecular basis influence the pattern and pace of neuronal cell death in the various neurological disorders. Future therapeutic strategies for these disorders will be guided in part by understanding the manifold impact of autophagy disruption on neurodegenerative diseases.Soon after the discovery of lysosomes by de Duve in the 1950s, electron microscopists recognized the presence of cytoplasmic organelles within membrane-limited vacuoles (Clark 1957) and observed what appeared to be the progressive breakdown of these contents (Ashford and Porter 1962). Proposing that “prelysosomes” containing sequestered cytoplasm matured to autolysosomes by fusion with primary lysosomes, de Duve and colleagues (de Duve 1963; de Duve and Wattiaux 1966) named this process “autophagy” (self-eating). Neurons, as cells particularly rich in acid phosphatase-positive lysosomes, were a preferred model in the initial investigations of autophagy. Early studies of pathologic states such as neuronal chromatolysis (Holtzman and Novikoff 1965; Holtzman et al. 1967) linked neurodegenerative phenomena to robust proliferation of autophagic vacuoles (AVs) and lysosomes. Although de Duve appreciated the importance of lysosomes for maintaining cell homeostasis, he was especially intrigued with their potential as “suicide bags” capable of triggering cell death by releasing proteases into the cytoplasm. Despite some support for this notion (Brunk and Brun 1972), the concept was not significantly embraced until many decades later. Instead, for many years, lysosomes and autophagy were mainly considered to perform cellular housekeeping and to scavenge and clean up debris during neurodegeneration in preparation for regenerative processes. The connection between autophagy and neuronal cell death reemerged in the 1970s from observations of Clarke and colleagues, who presented evidence that the developing brain deployed autophagy as a form of programmed neuronal cell death during which autophagy was massively up-regulated to eliminate cytoplasmic components, at once killing the neuron and reducing its cell mass for easy removal. Self-degradation was suggested as a more efficient elimination mechanism than apoptosis, which requires a large population of phagocytic cells and access of these cells to the dying region (Baehrecke 2005). Indeed, the best evidence for this process is in the context of massive cell death, as in metamorphosis and involutional states (Das et al. 2012).Clarke proposed that autophagic cell death (ACD)—type 2 programmed cell death (PCD)—could be a relatively common alternative route to death distinct from apoptosis—type 1 PCD (Clarke 1990)—or caspase-independent cell death—type 3 PCD (Fig. 1). The distinguishing features of ACD are marked proliferation of AVs and progressive disappearance of organelles but relative preservation of cytoskeletal and nuclear integrity until late in the process (Schweichel and Merker 1973; Hornung et al. 1989). In this original concept of ACD or type 2 PCD, death is achieved by autophagic digestion of organelles and essential regulatory molecules and elimination of death inhibitory factors (Baehrecke 2005). With the advent of the molecular era of autophagy research in the 1990s, it became possible to verify the most important implication of ACD, namely, that the death could be prevented by inhibiting autophagy genetically or pharmacologically. Meanwhile, reports of prominent lysosomal/autophagic pathology in Alzheimer’s disease (AD) (Cataldo et al. 1997; Nixon et al. 2000, 2005) and other neuropathic states (Anglade et al. 1997; Rubinsztein et al. 2005) raised important questions about whether autophagy pathology signifies a prodeath program or an attempt to maintain survival—a critical question for any potential therapy based on autophagy modulation. In this article, we will examine evidence for the various neuroprotective roles of autophagy and review our current understanding of how specific stages of autophagy may become disrupted and influence the neurodegenerative pattern seen in major adult-onset neurological diseases. We will particularly focus on how neurons regulate the balance between prosurvival autophagy and well-established cell death mechanisms in making life or death decisions.Open in a separate windowFigure 1.Neuronal cell death: three general morphological types of dying cells in the developing nervous system, as initially classified by Schweichel and Merker (1973) and later Clarke (1990). (A,B) Type 1 (“apoptotic”) cell death: (A) A neuron, from the brain of a postnatal day 6 mouse pup, in the middle of apoptotic degeneration showing cell shrinkage, cytoplasmic condensation, ruffled plasma membrane, and a highly electron-dense nucleus. Endoplasmic reticulum (ER) is still recognizable and some are dilated. A small number of autophagic vacuoles (AVs) can be seen (arrows). (B) A late-stage apoptotic neuron displaying electron-dense chromatin balls (CB), each surrounded by a small amount of highly condensed cytoplasm. (Panel from Yang et al. 2008; reprinted, with permission, from the American Association of Pathologists and Bacteriologists.) (C) Type 2 (“autophagic”) cell death: a deafferented isthmo-optic neuron in developing chick brain after uptake of horseradish peroxidase to highlight (electron dense) endocytic and autophagic compartments. The cell death pattern features pyknosis, abundant AVs, and sometimes dilated ER and mitochondria. (Panel from Hornung et al. 1989; reproduced, with permission, from John Wiley & Sons) (D) Type 3 (“cytoplasmic, nonlysosomal”) cell death: a motoneuron displaying markedly dilated rough ER, Golgi, and nuclear envelope, late vacuolization, and increased chromatin granularity. (Panel from Chu-Wang and Oppenheim 1978; reproduced, with permission, from John Wiley & Sons) Scale bars, 1 µm (A,B); 2 µm (C,D).     

Origins of Eukaryotic Sexual Reproduction

Sexual reproduction is a nearly universal feature of eukaryotic organisms. Given its ubiquity and shared core features, sex is thought to have arisen once in the last common ancestor to all eukaryotes. Using the perspectives of molecular genetics and cell biology, we consider documented and hypothetical scenarios for the instantiation and evolution of meiosis, fertilization, sex determination, uniparental inheritance of organelle genomes, and speciation.The transition from prokaryote to protoeukaryote to the last eukaryotic common ancestor (LECA) entailed conservation, modification, and reconfiguration of preexisting genetic circuits via mutation, horizontal gene transfer (HGT), endosymbiosis, and selection, as detailed in previous articles of this collection. During the course of this evolutionary trajectory, the LECA became sexual, reassorting and recombining chromosomes in a process that entails regulated fusions of haploid gametes and diploid → haploid reductions via meiosis. That the LECA was sexual is no longer a matter of speculation/debate as evidence of sex, and of genes exclusively involved in meiosis, has been found in all of the major eukaryotic radiations (Brawley and Johnson 1992; Ramesh et al. 2005; Kobiyama et al. 2007; Malik et al. 2008; Phadke and Zufall 2009; Fritz-Laylin et al. 2010; Lahr et al. 2011; Peacock et al. 2011; Vanstechelman et al. 2013).We propose that the transition to a sexual LECA entailed four innovations: (1) alternation of ploidy via cell–cell fusion and meiosis; (2) mating-type regulation of cell–cell fusion via differentiation of complementary haploid gametes (isogametic and then anisogametic), a prelude to species-isolation mechanisms; (3) mating-type-regulated coupling of the diploid/meiotic state to the formation of adaptive diploid resting spores; and (4) mating-type-regulated transmission of organelle genomes. Our working assumption is that the protoeukaryote → LECA era featured numerous sexual experiments, most of which failed but some of which were incorporated, integrated, and modified. Therefore, this list is not intended to suggest a sequence of events; rather, the four innovations most likely coevolved in a parallel and disjointed fashion.Once these core sexual-cycle themes were in place, the evolution of eukaryotic sex has featured countless prezygotic and postzygotic variations, the outcome being the segregation of panmictic populations into distinct species with distinctive adaptations.For additional reviews on the evolution of sex, the interested reader is referred to Goodenough (1985), Dacks and Roger (1999), Schurko et al. (2009), Wilkins and Holliday (2009), Gross and Bhattacharya (2010), Lee et al. (2010), Perrin (2012), and Calo et al. (2013).     

Bioenergetic Constraints on the Evolution of Complex Life

All morphologically complex life on Earth, beyond the level of cyanobacteria, is eukaryotic. All eukaryotes share a common ancestor that was already a complex cell. Despite their biochemical virtuosity, prokaryotes show little tendency to evolve eukaryotic traits or large genomes. Here I argue that prokaryotes are constrained by their membrane bioenergetics, for fundamental reasons relating to the origin of life. Eukaryotes arose in a rare endosymbiosis between two prokaryotes, which broke the energetic constraints on prokaryotes and gave rise to mitochondria. Loss of almost all mitochondrial genes produced an extreme genomic asymmetry, in which tiny mitochondrial genomes support, energetically, a massive nuclear genome, giving eukaryotes three to five orders of magnitude more energy per gene than prokaryotes. The requirement for endosymbiosis radically altered selection on eukaryotes, potentially explaining the evolution of unique traits, including the nucleus, sex, two sexes, speciation, and aging.Evolutionary theory has enormous explanatory power and is understood in detail at the molecular genetic level, yet it cannot easily predict even the past. The history of life on Earth is troubling. Life apparently arose very early, perhaps 4 billion years ago, but then remained essentially bacterial for probably some 2–3 billion years. Bacteria and archaea explored almost every conceivable metabolic niche and still dominate in terms of biomass. Yet, in morphological diversity and genomic complexity, bacteria barely begin to compare with eukaryotes, even at the level of cells, let alone multicellular plants and animals. Eukaryotes are monophyletic and share a common ancestor that by definition arose only once, probably between 1.5 and 2 billion years ago, although the dates are poorly constrained (Knoll et al. 2006; Parfrey et al. 2011). The eukaryotic common ancestor already had a nucleus, nuclear pore complexes, introns and exons, straight chromosomes, mitosis and meiotic sex, a dynamic cytoskeleton, an endoplasmic reticulum, and mitochondria, making it difficult to trace the evolution of these traits from a prokaryotic state (Koonin 2010). The “eukaryotic niche”—limited metabolic diversity but enormous morphological complexity—was never invaded by prokaryotes. In short, life arose early, stagnated in morphological complexity for several billion years, and then rather abruptly gave rise to a single group—the eukaryotes—which explored the morphological realm of life in ways never seen in bacteria or archaea.Consider the possibility of life evolving on other planets. Would it follow a similar trajectory? If not, why not? Evolutionary theory gives little insight. The perplexing history of life on Earth conceals a paradox relating to natural selection. If basal eukaryotic traits such as the nucleus, meiotic sex, and phagocytosis arose by selection, starting with a prokaryotic ancestor, and each step offered some small advantage over the last, then why don’t the same traits arise repeatedly in prokaryotes too? Prokaryotes made many a start. There are examples of bacteria or archaea with nucleus-like structures (Lindsay et al. 2001), recombination (Smith et al. 1993), linear chromosomes (Bentley et al. 2002), internal membranes (Pinevich 1997), multiple replicons (Robinson and Bell 2007), giant size (Schulz and Jorgensen 2001), extreme polyploidy (Mendell et al. 2008), a dynamic cytoskeleton (Vats and Rothfield 2009), predation (Davidov and Jurkevitch 2009), parasitism (Moran 2007), introns and exons (Simon and Zimmerly 2008), intercellular signaling (Waters and Bassler 2005), endocytosis-like processes (Lonhienne et al. 2010), and even endosymbionts (Wujek 1979; von Dohlen et al. 2001). Yet, for each of these traits, bacteria and archaea stopped well short of the baroque complexity of eukaryotes. Compare this with the evolution of eyes. From a simple, light-sensitive spot in an early metazoan, morphologically disparate eyes arose on scores of occasions (Vopalensky and Kozmic 2009). This is exactly what evolutionary theory predicts. Each step offers an advantage in its own ecological setting, so morphologically different eyes arise on multiple occasions. Why is this not the case for traits such as the nucleus, meiotic sex, and phagocytosis? To suggest that lateral gene transfer (LGT) or bacterial conjugation is equivalent to meiotic sex will not do: Neither involves a systematic and reciprocal exchange of alleles across the entire genome.The simplest explanation is a bottleneck. The “big bang” radiation of major eukaryotic supergroups, combined with the apparent absence of surviving evolutionary intermediates between prokaryotes and the last eukaryotic common ancestor, does indeed hint at a bottleneck at the origin of eukaryotes. There is no shortage of environmental possibilities, from snowball glaciations to rising atmospheric oxygen. The most widely held explanation contends that when oxygen levels rose after the great oxidation event, some proto-eukaryotic cells acquired mitochondria, which protected them against oxygen toxicity (Andersson and Kurland 1999) and enabled them to exploit oxygen as a terminal electron acceptor in respiration (Sagan 1967), giving the first eukaryotes an enormous competitive advantage. They swiftly occupied new niches made available by oxygen, outcompeting to extinction any other prokaryotes that tried subsequently to invade this niche (de Duve 2007; Gross and Bhattacharya 2010). But this is an evolutionary “just-so story” and has no evidence to support it. The idea that mitochondria might protect against oxygen toxicity is nonsense: The single-electron donors of respiratory chains are among the most potent free-radical generators known. And what was to stop facultatively aerobic bacteria—from which the mitochondria evolved, hence already present—from occupying the aerobic niche first?In fact, the limited evidence available suggests that oxygen had little to do with it (Müller et al. 2012; van der Giezen and Lenton 2012). A large, diverse group of morphologically simple protists dubbed archezoa are the key here. The archezoa appear to lack mitochondria; and three decades ago, looked to branch deeply in the eukaryotic tree. Cavalier-Smith postulated that some archezoa might be primitively amitochondriate: surviving evolutionary intermediates between prokaryotes and eukaryotes (Cavalier-Smith 1987, 1989). But 20 years of careful molecular biology and phylogenetics have shown that all known archezoa possess specialized organelles that derive from mitochondria, namely hydrogenosomes or mitosomes (Keeling 1998; Embley and Martin 2006; van der Giezen 2009; Archibald 2011). The archezoa are obviously not real evolutionary intermediates, and radical developments in phylogenomics have transformed the eukaryotic tree to a “big-bang” radiation with no early branching archezoa (Koonin 2010). The archezoa remain significant not because they are genuine evolutionary intermediates, but because they are true ecological intermediates. Critically, they were not outcompeted to extinction by more sophisticated aerobic eukaryotes. On the contrary, they lost their capacity for aerobic respiration and depend instead on anaerobic fermentations, yet remain, morphologically, more complex than bacteria or archaea.The fact that the archezoa are a phylogenetically disparate group that arose on multiple occasions is equally significant. The “intermediate” niche is viable and was invaded many times, without the new arrivals being outcompeted to extinction by existing cells, or vice versa. Yet each time the invader was an anaerobic eukaryote, which adapted by reductive evolution to the niche—not bacteria or archaea evolving slightly greater complexity. What is the likelihood of this bias? Given at least 20 independent origins of archezoa (van der Giezen 2009; Müller et al. 2012), the probability of these ecological intermediates arising each time from the eukaryotes rather than prokaryotes is less than one in a million. It is far more parsimonious to assume that there was something about the structure of eukaryotes that facilitated their invasion of this intermediate niche; and, conversely, something about the structure of prokaryotes that tended to preclude their evolution of greater morphological complexity. But this quite reasonable statement is loaded because it implies that prokaryotes existed for nearly 4 billion years, and throughout that time showed no tendency to evolve greater morphological complexity. In stark contrast, eukaryotes arose just once, a seemingly improbable event.Here I argue that the constraint on prokaryotes was bioenergetic. There was, indeed, a bottleneck at the origin of eukaryotes, but it was biological (restrictive), not environmental (selective). It related to the physical structure of prokaryotic cells: Both bacteria and archaea respire across their plasma membrane. I make three key points, which arguably apply to life elsewhere in the universe, and are therefore proposed as biological principles that could guide our understanding of life generally: (1) chemiosmotic coupling is as universal as the genetic code, for fundamental reasons relating to the origin of life; (2) prokaryotes are constrained by chemiosmotic coupling across their plasma membrane, but eukaryotes escaped this constraint through a rare and stochastic endosymbiosis between two prokaryotes, giving them orders of magnitude more energy per gene; and (3) this endosymbiosis, in turn, produced a unique genomic asymmetry, transforming the selection pressures acting on eukaryotes and driving the evolution of unique eukaryotic traits.     

The Persistent Contributions of RNA to Eukaryotic Gen(om)e Architecture and Cellular Function

Currently, the best scenario for earliest forms of life is based on RNA molecules as they have the proven ability to catalyze enzymatic reactions and harbor genetic information. Evolutionary principles valid today become apparent in such models already. Furthermore, many features of eukaryotic genome architecture might have their origins in an RNA or RNA/protein (RNP) world, including the onset of a further transition, when DNA replaced RNA as the genetic bookkeeper of the cell. Chromosome maintenance, splicing, and regulatory function via RNA may be deeply rooted in the RNA/RNP worlds. Mostly in eukaryotes, conversion from RNA to DNA is still ongoing, which greatly impacts the plasticity of extant genomes. Raw material for novel genes encoding protein or RNA, or parts of genes including regulatory elements that selection can act on, continues to enter the evolutionary lottery.

Everything has been said already, but not yet by everyone.—Karl Valentin

Sturgeon''s Revelation: Ninety percent of science fiction is crud, but then, ninety percent of everything is crud.—Theodore Sturgeon

They think that intelligence is about noticing things that are relevant (detecting patterns); in a complex world, intelligence consists in ignoring things that are irrelevant (avoiding false patterns).—Nassim Nicholas Taleb (Taleb 2010)

Of all extant cellular macromolecules, RNA is the most ancient, persisting as much as 4 × 10⁹ years in our planet’s life-forms. The ability to combine genotype with phenotype such as catalytic activity (Noller and Chaires 1972; Kruger et al. 1982; Guerrier-Takada et al. 1983; Noller et al. 1992) leveled a major hurdle in understanding the origin of life. The salient discoveries eliminated the virtually impossible prerequisite for two to three different classes of macromolecules to converge as an evolving unit. At the same time, RNA provides a required continuity in the path of evolution (Yarus 2011) during various genetic takeovers or evolutionary transitions (Cairns-Smith 1982; Szathmáry and Smith 1995). In a remarkably insightful article dating back half a century, Alex Rich foresaw much of what now is becoming mainstream, for example, that RNA was ancestral to protein and DNA (Rich 1962). This landmark publication received little attention over the years; even early proponents of an RNA world did not refer to this article (Woese 1967; Crick 1968; Orgel 1968; Gilbert 1986), although at least one of the investigators must have had knowledge about the article, as it was cited in a different context concerning the stereochemical possibility of six distinct base pairs (Crick 1968). The origin of the DNA genome from RNA and that “DNA may be regarded as a derivative molecule which has evolved in the form that it only carries out part of the primitive nucleic acid function” is another correct prediction (Rich 1962). Furthermore, the investigator presaged mechanisms such as antisense RNA control of gene expression, short interfering RNAs (siRNAs), and perhaps microRNAs (miRNAs): “If both strands are active, then the DNA would produce two RNA strands which are complementary to each other. Only one of these might be active in protein synthesis, and the other strand might be a component of the control or regulatory signal” (Rich 1962).In this article, I shall present the rise and persistence of RNA from the dawn of an RNA world and discuss current evolutionary principles already apparent in an RNA world. In comparison to Archaea and Bacteria, the eukaryotic genome is a better vantage point, as archaeal and bacterial genomes are more derived and, thus, lost many of the RNA signatures that eukaryotes still show. It is likely that eukaryotic DNA genomes not only kept much more of their RNA/RNP world heritage than previously anticipated, but also continue to evolve novel RNAs in various functional roles.     

Architecture of the Mammalian Golgi

Since its first visualization in 1898, the Golgi has been a topic of intense morphological research. A typical mammalian Golgi consists of a pile of stapled cisternae, the Golgi stack, which is a key station for modification of newly synthesized proteins and lipids. Distinct stacks are interconnected by tubules to form the Golgi ribbon. At the entrance site of the Golgi, the cis-Golgi, vesicular tubular clusters (VTCs) form the intermediate between the endoplasmic reticulum and the Golgi stack. At the exit site of the Golgi, the trans-Golgi, the trans-Golgi network (TGN) is the major site of sorting proteins to distinct cellular locations. Golgi functioning can only be understood in light of its complex architecture, as was revealed by a range of distinct electron microscopy (EM) approaches. In this article, a general concept of mammalian Golgi architecture, including VTCs and the TGN, is described.In 1898 Camillo Golgi was the first to visualize, describe, and ultimately name the Golgi complex. Using a histochemical impregnation method causing the reduction and deposition of silver, he defined the Golgi in neuronal cells as a reticular apparatus stained by the “black reaction” (Golgi 1898). In the 1950s, the first ultrastructural images of the Golgi were revealed using the then newly developed electron microscope (EM) (Dalton 1954; Farquhar and Rinehart 1954; Sjostrand and Hanzon 1954; Dalton and Felix 1956), reviewed by Farquhar and Palade (1981). In 1961, the thiamine pyrophosphatase reaction developed by Novikoff and Goldfischer allowed cytochemical labeling of Golgi membranes, which revealed the ubiquitous cellular distribution of this organelle (Novikoff and Goldfischer 1961). In the many years of ultrastructural research that have followed, the visualization of the Golgi has gone hand-in-hand with the developing EM techniques.The intriguing structural complexity of the Golgi has made it one of the most photographed organelles in the cell. However, a full understanding of Golgi architecture is hard to deduce from the ultrathin (70–100 nm) sections used in standard transmission EM preparations. Rambourg and Clermont (1974) were the first to investigate the Golgi in three dimensions (3D), using stereoscopy (Rambourg 1974). In this approach a “thick” (150–200 nm), EM section is photographed at two distinct angles, after which the pairs of photographs are viewed with a stereoscope. Over the years, stereoscopy was applied to a variety of cells and has greatly contributed to our current understanding of Golgi architecture (Lindsey and Ellisman 1985; Rambourg and Clermont 1990; Clermont et al. 1994; Clermont et al. 1995). An alternative approach to study 3D structure is serial sectioning, by which a series of adjacent (serial) thin sections are collected. The Golgi can be followed throughout these sections and be constructed into a 3D model (Beams and Kessel 1968; Dylewski et al. 1984; Rambourg and Clermont 1990). In the nineties, 3D-EM was boosted by the introduction of high-voltage, dual axis 3D electron tomography (Ladinsky et al. 1999; Koster and Klumperman 2003; Marsh 2005; Marsh 2007; Noske et al. 2008), which allows the analysis of sections of up to 3–4 µm with a 4–6 nm resolution in the z-axis. The sections are photographed in a tilt series of different angles, which are reconstructed into a 3D tomogram that allows one to “look beyond” a given structure and reveals how it relates to other cellular compartments.Membranes with a similar appearance can differ in protein content and function. These differences are revealed by protein localization techniques. Therefore, in addition to the “classical” EM techniques providing ultrastructural details, EM methods that determine protein localization within the context of the cellular morphology have been crucial to further our understanding on the functional organization of the Golgi. For example, by enzyme-activity-based cytochemical staining the cis-to-trans-polarity in the distribution of Golgi glycosylation enzymes was discovered, reviewed by Farquhar and Palade (1981), which was key to understanding the functional organization of the Golgi stack in protein and lipid glycosylation. With the development of immunoEM methods, using antibodies, the need for enzyme activity for protein localization was overcome. This paved the way for the localization of a wide variety of proteins, such as the cytoplasmic coat complexes associated with the Golgi (Rabouille and Klumperman 2005).A logical next step in EM-based imaging of the Golgi would be to combine protein localization with 3D imaging, but this is technically challenging. A number of protocols enabling protein localization in 3D have recently been described (Trucco et al. 2004; Grabenbauer et al. 2005; Gaietta et al. 2006; Zeuschner et al. 2006; Meiblitzer-Ruppitsch et al. 2008), but these have only been applied in a limited manner to Golgi studies. Another approach that holds great potential for Golgi research is correlative microscopy (CLEM). Live cell imaging of fluorescent proteins has revolutionized cell biology by the real time visualization of dynamic events. However, live cell imaging does not reveal membrane complexity. By CLEM, live cells are first viewed by light microscopy and then prepared for EM (Mironov et al. 2008; van Rijnsoever et al. 2008). When coupled with the recent introduction of super resolution light microscopy techniques for real time imaging, the combination with EM for direct correlation with ultrastructural resolution has great potential (Hell 2009; Lippincott-Schwartz and Manley 2009).The 100th anniversary of the discovery of the Golgi, in 1998, triggered a wave of reviews on this organelle, including those focusing on Golgi architecture (Rambourg 1997; Farquhar and Palade 1998). More recent reviews that describe Golgi structure in great detail are provided by Marsh (2005) and Hua (2009). In this article, the most recent insights in mammalian Golgi architecture as revealed by distinct EM approaches are integrated into a general concept.     

Protein Solubility and Protein Homeostasis: A Generic View of Protein Misfolding Disorders

According to the “generic view” of protein aggregation, the ability to self-assemble into stable and highly organized structures such as amyloid fibrils is not an unusual feature exhibited by a small group of peptides and proteins with special sequence or structural properties, but rather a property shared by most proteins. At the same time, through a wide variety of techniques, many of which were originally devised for applications in other disciplines, it has also been established that the maintenance of proteins in a soluble state is a fundamental aspect of protein homeostasis. Taken together, these advances offer a unified framework for understanding the molecular basis of protein aggregation and for the rational development of therapeutic strategies based on the biological and chemical regulation of protein solubility.Virtually every complex biochemical process taking place in living cells depends on the ability of the molecules involved to self-assemble into functional structures (Dobson 2003; Robinson et al. 2007; Russel et al. 2009), and a sophisticated quality control system is responsible for regulating the reactions leading to this organization within the cellular environment (Dobson 2003; Balch et al. 2008; Hartl and Hayer-Hartl 2009; Powers et al. 2009; Vendruscolo and Dobson 2009). Proteins are the molecules that are essential for enabling, regulating, and controlling almost all the tasks necessary to maintain such a balance. To function, the majority of our proteins need to fold into specific three-dimensional structures following their biosynthesis in the ribosome (Hartl and Hayer-Hartl 2002). The wide variety of highly specific structures that results from protein folding, and which serve to bring key functional groups into close proximity, has enabled living systems to develop an astonishing diversity and selectivity in their underlying chemical processes by using a common set of just 20 basic molecular components, the amino acids (Dobson 2003). Given the central importance of protein folding, it is not surprising that the failure of proteins to fold correctly, or to remain correctly folded, is at the origin of a wide variety of pathological conditions, including late-onset diabetes, cystic fibrosis, and Alzheimer’s and Parkinson’s diseases (Dobson 2003; Chiti and Dobson 2006; Haass and Selkoe 2007). In many of these disorders proteins self-assemble in an aberrant manner into large molecular aggregates, notably amyloid fibrils (Chiti and Dobson 2006; Ramirez-Alvarado et al. 2010).     

Endocytosis of Viruses and Bacteria

Of the many pathogens that infect humans and animals, a large number use cells of the host organism as protected sites for replication. To reach the relevant intracellular compartments, they take advantage of the endocytosis machinery and exploit the network of endocytic organelles for penetration into the cytosol or as sites of replication. In this review, we discuss the endocytic entry processes used by viruses and bacteria and compare the strategies used by these dissimilar classes of pathogens.Many of the most widespread and devastating diseases in humans and livestock are caused by viruses and bacteria that enter cells for replication. Being obligate intracellular parasites, viruses have no choice. They must transport their genome to the cytosol or nucleus of infected cells to multiply and generate progeny. Bacteria and eukaryotic parasites do have other options; most of them can replicate on their own. However, some have evolved to take advantage of the protected environment in the cytosol or in cytoplasmic vacuoles of animal cells as a niche favorable for growth and multiplication. In both cases (viruses and intracellular bacteria), the outcome is often destructive for the host cell and host organism. The mortality and morbidity caused by infectious diseases worldwide provide a strong rationale for research into pathogen–host cell interactions and for pursuing the detailed mechanisms of transmission and dissemination. The study of viruses and bacteria can, moreover, provide invaluable insights into fundamental aspects of cell biology.Here, we focus on the mechanisms by which viral and bacterial pathogens exploit the endocytosis machinery for host cell entry and replication. Among recent reviews on this topic, dedicated uniquely to either mammalian viruses or bacterial pathogens, we recommend the following: Cossart and Sansonetti (2004); Pizarro-Cerda and Cossart (2006); Kumar and Valdivia (2009); Cossart and Roy (2010); Mercer et al. (2010b); Grove and Marsh (2011); Kubo et al. (2012); Vazquez-Calvo et al. (2012a); Sun et al. (2013).The term “endocytosis” is used herein in its widest sense, that is, to cover all processes whereby fluid, solutes, ligands, and components of the plasma membrane as well as particles (including pathogenic agents) are internalized by cells through the invagination of the plasma membrane and the scission of membrane vesicles or vacuoles. This differs from current practice in the bacterial pathogenesis field, where the term “endocytosis” is generally reserved for the internalization of molecules or small objects, whereas the uptake of bacteria into nonprofessional phagocytes is called “internalization” or “bacterial-induced phagocytosis.” In addition, the term “phagocytosis” is reserved for internalization of bacteria by professional phagocytes (macrophages, polymorphonuclear leucocytes, dendritic cells, and amoebae), a process that generally but not always leads to the destruction of the ingested bacteria (Swanson et al. 1999; May and Machesky 2001; Henry et al. 2004; Zhang et al. 2010). With a few exceptions, we will not discuss phagocytosis of bacteria or the endocytosis of protozoan parasites such as Toxoplasma and Plasmodium (Robibaro et al. 2001).