DNA methylation in adults and during development of the self‐fertilizing mangrove rivulus,Kryptolebias marmoratus

In addition to genetic variation, epigenetic mechanisms such as DNA methylation might make important contributions to heritable phenotypic diversity in populations. However, it is often difficult to disentangle the contributions of genetic and epigenetic variation to phenotypic diversity. Here, we investigated global DNA methylation and mRNA expression of the methylation‐associated enzymes during embryonic development and in adult tissues of one natural isogenic lineage of mangrove rivulus fish, Kryptolebias marmoratus. Being the best‐known self‐fertilizing hermaphroditic vertebrate affords the opportunity to work with genetically identical individuals to examine, explicitly, the phenotypic effects of epigenetic variance. Using the LUminometric Methylation Assay (LUMA), we described variable global DNA methylation at CpG sites in adult tissues, which differed significantly between hermaphrodite ovotestes and male testes (79.6% and 87.2%, respectively). After fertilization, an immediate decrease in DNA methylation occurred to 15.8% in gastrula followed by re‐establishment to 70.0% by stage 26 (liver formation). Compared to zebrafish, at the same embryonic stages, this reprogramming event seems later, deeper, and longer. Furthermore, genes putatively encoding DNA methyltransferases (DNMTs), Ten‐Eleven Translocation (TET), and MeCP2 proteins showed specific regulation in adult gonad and brain, and also during early embryogenesis. Their conserved domains and expression profiles suggest that these proteins play important roles during reproduction and development. This study raises questions about mangrove rivulus’ peculiar reprogramming period in terms of epigenetic transmission and physiological adaptation of individuals to highly variable environments. In accordance with the general‐purpose genotype model, epigenetic mechanisms might allow for the expression of diverse phenotypes among genetically identical individuals. Such phenotypes might help to overcome environmental challenges, making the mangrove rivulus a valuable vertebrate model for ecological epigenetic studies. The mangrove rivulus, Kryptolebias marmoratus, is the best‐known self‐fertilizing hermaphroditic vertebrate that allows to work with genetically identical individuals to examine, explicitly, the phenotypic effects of epigenetic variance. The reprogramming event is later, more dramatic and longer than in other described vertebrates. High evolutionary conservation and expression patterns of DNMT, TET, and MeCP2 proteins in K. marmoratus suggest biological roles for each member in gametogenesis and development.     

Relative fitness of two hermaphroditic mating types in the androdioecious clam shrimp,Eulimnadia texana

Androdioecy (populations of males and hermaphrodites) is a rare reproductive form, being described from only a handful of plants and animals. One of these is the shrimp Eulimnadia texana, which has populations comprised of three mating types: two hermaphroditic types (monogenics and amphigenics) and males. In a recent study, the amphigenic hermaphrodites were found to be in greater abundance than that predicted from a model of this mating system. Herein, we compare the relative fitness of offspring from amphigenic and monogenic siblings, attempting to understand the greater relative abundance of the former. Populations started with offspring from selfed monogenic hermaphrodites had a net reproductive rate (R) 87% that of offspring from their amphigenic siblings. Additionally, within populations of amphigenic offspring (which included males, monogenics and amphigenics), amphigenics survived longer than monogenics. These differences help to explain the increased relative abundance of amphigenics in natural populations, but amphigenics continue to be more abundant than expected.     

Association behaviour of the self-fertilizing Kryptolebias marmoratus (Poey): the influence of microhabitat use on the potential for a complex mating system

Preliminary observations were conducted to identify conspicuous body postures and movements of males and hermaphrodites in the mangrove killifish Kryptolebias marmoratus . These behaviours were used quantitatively to examine the social interactions for experimental pairings of K. marmoratus of different sexual states ( i.e. simultaneous hermaphrodite and male) in an aquarium with an open-water area and simulated crab burrows. This allowed observation of behaviours that could not be observed in the field. Kryptolebias marmoratus , regardless of sexual state and experimental treatment, spent 40% of the time in the burrow. Hermaphrodites exhibited a preference for associating with males rather than other hermaphrodites. The observed complexity of displayed behaviours and interactions between paired conspecifics indicate that K. marmoratus has a rich repertoire of social behaviour not predicted for a strictly selfing species. Also, land crab burrows play an important role in their social interactions.     

Cutaneous vasoregulation during short- and long-term aerial acclimation in the amphibious mangrove rivulus, Kryptolebias marmoratus

The mangrove rivulus (Kryptolebias marmoratus) is an amphibious fish and evidence suggests that the cutaneous surface is the primary site of gas exchange during emersion. The aim of this study was to determine whether cutaneous blood vessels were regulated in the caudal fin during the initial transition from water to aerial exposure, and after 10 days of aerial acclimation. Acute changes (first 3 min following emersion) in the cutaneous vessels diameter were measured in real-time on live fish using light microscopy. The data show that under control conditions, only arterioles in the caudal fin were vasoactive. During the first 20 s of aerial acclimation the arterioles significantly constricted (− 2.1 ± 0.4 μm), which was followed immediately by a relaxation (from 40 to 180 s). This vasoconstriction was eliminated with the addition of phentolamine (50 μmol l^− 1), which indicates that the vasoconstriction was mediated by α-adrenoreceptors. Longer-term changes in the cutaneous surface vasculature were determined using fluorescent immunohistochemistry and antibodies for the endothelial marker, CD31. Fish aerially acclimated for 10 days exhibited significantly higher levels of endothelial fluorescence in the caudal fin when compared to control fish in water, indicating endothelial cell production (i.e. angiogenesis). These data combined show that for every emersion episode, there is an initial α-adrenergic mediated vasoconstriction, which is most likely, a stress response. This is then followed by a long-term acclimation involving an upregulation in endothelial cell production, which would subsequently enhance blood perfusion to the cutaneous surface and potentially increase the capacity for gas exchange with the external environment.     

Androdioecy in Kryptolebias killifish and the evolution of self‐fertilizing hermaphroditism

Kryptolebias marmoratus is an important experimental fish, and is considered to represent the only vertebrate species comprising self‐fertilizing hermaphroditic individuals. Subsequent to the discovery of this unusual mode of reproduction, approximately 50 years ago, K. marmoratus has been the focus of a series of studies. However, little is known about the evolution of this rare reproduction mode, and data on the biology of closely related species are still unavailable. The present study aimed to histologically analyse the gonads of three K. marmoratus congeners (i.e. Kryptolebias ocellatus, Kryptolebias caudomarginatus, and Kryptolebias brasiliensis) to check the distribution of features related to hermaphroditism that are useful for forming hypotheses about the origin and evolution of the self‐fertilization mode of reproduction through the available phylogenies. The data obtained demonstrate that populations of K. caudomarginatus consist of males and hermaphrodites, which supports the hypothesis that hermaphroditism arose at the base of the clade containing K. caudomarginatus, K. marmoratus, and K. ocellatus as a first step towards a more advanced condition, uniquely shared by both K. marmoratus and K. ocellatus, in which males are rare or absent in natural populations, with the subsequent occurrence of self‐fertilization. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99 , 344–349.     

Strong population structure despite evidence of recent migration in a selfing hermaphroditic vertebrate, the mangrove killifish (Kryptolebias marmoratus)

We employ a battery of 33 polymorphic microsatellite loci to describe geographical population structure of the mangrove killifish (Kryptolebias marmoratus), the only vertebrate species known to have a mixed-mating system of selfing and outcrossing. Significant population genetic structure was detected at spatial scales ranging from tens to hundreds of kilometres in Florida, Belize, and the Bahamas. The wealth of genotypic information, coupled with the highly inbred nature of most killifish lineages due to predominant selfing, also permitted treatments of individual fish as units of analysis. Genetic clustering algorithms, neighbour-joining trees, factorial correspondence, and related methods all earmarked particular killifish specimens as products of recent outcross events that could often be provisionally linked to specific migration events. Although mutation is the ultimate source of genetic diversity in K. marmoratus, our data indicate that interlocality dispersal and outcross-mediated genetic recombination (and probably genetic drift also) play key proximate roles in the local 'clonal' dynamics of this species.     

How does pollination mutualism affect the evolution of prior self‐fertilization? A model

The mode of pollination is often neglected regarding the evolution of selfing. Yet the distribution of mating systems seems to depend on the mode of pollination, and pollinators are likely to interfere with selfing evolution, since they can cause strong selective pressures on floral traits. Most selfing species reduce their investment in reproduction, and display smaller flowers, with less nectar and scents (referred to as selfing syndrome). We model the evolution of prior selfing when it affects both the demography of plants and pollinators and the investment of plants in pollination. Including the selfing syndrome in the model allows to predict several outcomes: plants can evolve either toward complete outcrossing, complete selfing, or to a stable mixed‐mating system, even when inbreeding depression is high. We predict that the evolution to high prior selfing could lead to evolutionary suicides, highlighting the importance of merging demography and evolution in models. The consequence of the selfing syndrome on plant–pollinator interactions could be a widespread mechanism driving the evolution of selfing in animal‐pollinated taxa.     

Effects of reproductive compensation, gamete discounting and reproductive assurance on mating-system diversity in hermaphrodites

Hermaphroditism allows considerable scope for contributing genes to subsequent generations through various mixtures of selfed and outcrossed offspring. The fitness consequences of different family compositions determine the evolutionarily stable mating strategy and depend on the interplay of genetic features, the nature of mating, and factors that govern offspring development. This theoretical article considers the relative contributions of these influences and their interacting effects on mating-system evolution, given a fixed genetic load within a population. Strong inbreeding depression after offspring gain independence selects for exclusive outcrossing, regardless of the intensity of predispersal inbreeding depression, unless insufficient mating limits offspring production. The extent to which selfing evolves under weak postdispersal inbreeding depression depends on predispersal inbreeding depression and the opportunity for resource limitation of offspring production. Mixed selfing and outcrossing is an evolutionarily stable strategy (ESS) if selfed zygotes survive poorly, but selfed offspring survive well, and maternal individuals produce enough "extra" eggs that deaths of unviable outcrossed embryos do not impact offspring production (reproductive compensation). Mixed mating can also be an ESS, despite weak lifetime inbreeding depression, if self-mating reduces the number of male gametes available for outcrossing (male-gamete discounting). Reproductive compensation and male-gamete discounting act largely independently on mating-system evolution. ESS mating systems always involve either complete fertilization or fertilization of enough eggs to induce resource competition among embryos, so although reproductive assurance is adaptive with insufficient mating, it is never an ESS. Our results illustrate the theoretical importance of different constraints on offspring production (availability of male gametes, egg production, and maternal resources) for both the course and outcome of mating-system evolution, whereas unequal competition between selfed and outcrossed embryos has limited effect. These results also underscore the significance of heterogeneity in the nature and intensity of inbreeding depression during the life cycle for the evolution of hermaphrodite mating systems.     

The role of natural enemies in the expression and evolution of mixed mating in hermaphroditic plants and animals

Although a large portion of plant and animal species exhibit intermediate levels of outcrossing, the factors that maintain this wealth of variation are not well understood. Natural enemies are one relatively understudied ecological factor that may influence the evolutionary stability of mixed mating. In this paper, we aim for a conceptual unification of the role of enemies in mating system expression and evolution in both hermaphroditic animals and plants. We review current theory and detail the potential effects of enemies on fundamental mating system parameters. In doing so, we identify situations in which consideration of enemies alters expectations about the stability of mixed mating. Generally, we find that inclusion of the enemy dimension may broaden conditions in which mixed mating systems are evolutionarily stable. Finally, we highlight avenues ripe for future theoretical and empirical work that will advance our understanding of enemies in the expression and evolution of mixed mating in their hosts/victims, including examination of feedback cycles between victims and enemies and quantification of mating system-related parameters in victim populations in the presence and absence of enemies.     

Inbreeding avoidance and the evolution of gender dimorphism in Wurmbea biglandulosa (Colchicaceae)

How females establish in populations of cosexuals is central to understanding the evolution of gender dimorphism in angiosperms. Inbreeding avoidance hypotheses propose that females can establish and be maintained if cosexual fitness is reduced because they self-fertilize, and their progeny express inbreeding depression. Here we assess the role of inbreeding avoidance in maintaining sexual system variation in Wurmbea biglandulosa. We estimated costs of self-pollination, mating patterns, and inbreeding depression in gender monomorphic (cosexuals only) and dimorphic (males and females) populations. Costs of selfing, estimated from seed set of experimentally self- and cross-pollinated flowers, were severe in both males and cosexuals (inbreeding depression, sigma = 0.86). In a field experiment, intact males that could self produced fewer seeds than both emasculated males and females, whereas seed set of intact and emasculated cosexuals did not differ. Thus, pollinator-mediated selfing reduces fitness of males but not cosexuals under natural conditions. Outcrossing rates of males revealed substantial selfing (t = 0.68), whereas females and cosexuals were outcrossed (0.92 and 0.97). For males, progeny inbreeding coefficients exceeded parental coefficients (0.220 vs. 0.009), whereas for females and cosexuals these coefficients did not differ and approached zero. Differences in coefficients between males and their progeny indicate that selfed progeny express severe inbreeding depression (sigma = 0.93). Combined with inbreeding depression for seed set, cumulative sigma = 0.99, indicating that most or all selfed zygotes fail to reach reproductive maturity. We propose that present sexual system variation in W. biglandulosa is maintained by high inbreeding depression coupled with differences in selfing rates among monomorphic and dimorphic populations.     

Rapid loss of self‐incompatibility in experimental populations of the perennial outcrossing plant Linaria cavanillesii

Transitions from self‐incompatibility to self‐compatibility in angiosperms may be frequently driven by selection for reproductive assurance when mates or pollinators are rare, and are often succeeded by loss of inbreeding depression by purging. Here, we use experimental evolution to investigate the spread of self‐compatibility from one such population of the perennial plant Linaria cavanillesii into self‐incompatible (SI) populations that still have high inbreeding depression. We introduced self‐compatible (SC) individuals at different frequencies into replicate experimental populations of L. cavanillesii that varied in access to pollinators. Our experiment revealed a rapid shift to self‐compatibility in all replicates, driven by both greater seed set and greater outcross siring success of SC individuals. We discuss our results in the light of computer simulations that confirm the tendency of self‐compatibility to spread into SI populations under the observed conditions. Our study illustrates the ease with which self‐compatibility can spread among populations, a requisite for species‐wide transitions from self‐incompatibility to self‐compatibility.     

The divergence history of the perennial plant Linaria cavanillesii confirms a recent loss of self‐incompatibility

Many angiosperms prevent inbreeding through a self‐incompatibility (SI) system, but the loss of SI has been frequent in their evolutionary history. The loss of SI may often lead to an increase in the selfing rate, with the purging of inbreeding depression and the ultimate evolution of a selfing syndrome, where plants have smaller flowers with reduced pollen and nectar production. In this study, we used approximate Bayesian computation (ABC) to estimate the timing of divergence between populations of the plant Linaria cavanillesii that differ in SI status and in which SI is associated with low inbreeding depression but not with a transition to full selfing or a selfing syndrome. Our analysis suggests that the mixed‐mating self‐compatible (SC) population may have begun to diverge from the SI populations around 2810 generation ago, a period perhaps too short for the evolution of a selfing syndrome. We conjecture that the SC population of L. cavanillesii is at an intermediate stage of transition between outcrossing and selfing.     

The double edged sword: The demographic consequences of the evolution of self‐fertilization

Phylogenies indicate that the transition from outcrossing to selfing is frequent, with selfing populations being more prone to extinction. The rates of transition to selfing and extinction, acting on different timescales, could explain the observed distributions of extant selfing species among taxa. However, phylogenetic and theoretical studies consider these mechanisms independently, that is transitions do not cause extinction. Here, we theoretically explore the demographic consequences of the evolution of self‐fertilization. Deleterious mutations and mutations modifying the selfing rate are recurrently introduced and the number of offspring depends on individual fitness, allowing for a demographic feedback. We show that mutational meltdowns can be triggered in populations evolving near strict selfing. Populations having survived a demographic crash are more stable than ancestral outcrossing populations once deleterious mutations are purged. The relatively rapid time‐scales at which extinctions occur indicate that during evolutionary transitions the accumulation of deleterious mutations may not be the cause of extinctions observed on longer time scales, but could lead to the underestimation of transition rates from outcrossing to selfing.     

The joint evolution of lifespan and self‐fertilization

In Angiosperms, there exists a strong association between mating system and lifespan. Most self‐fertilizing species are short‐lived, and most predominant or obligate outcrossers are long‐lived. This association is generally explained by the influence of lifespan on the evolution of the mating system, considering lifespan as fixed. Yet, lifespan can itself evolve, and the mating system may as well influence the evolution of lifespan, as is suggested by joint evolutionary shifts of lifespan and mating system between sister species. In this paper, we build modifier models to study the joint evolution of self‐fertilization and lifespan, including both juvenile and adult inbreeding depression. We show that provided that inbreeding depression affects adult survival, self‐fertilization is expected to promote evolution towards shorter lifespan, and that the range of conditions under which selfing can evolve rapidly shrinks as lifespan increases. We study the effects of inbreeding depression affecting various steps in the life cycle and discuss how extrinsic mortality conditions are expected to affect evolutionary associations. In particular, we show that selfers may sometimes remain short‐lived even in a very stable habitat, as a strategy to avoid the deleterious effects of inbreeding.     

Increase in multiple paternity across the reproductive lifespan in a sperm‐storing,hermaphroditic freshwater snail

Polyandry is a common phenomenon and challenges the traditional view of stronger sexual selection in males than in females. In simultaneous hermaphrodites, the physical proximity of both sex functions was long thought to preclude the operation of sexual selection. Laboratory studies suggest that multiple mating and polyandry in hermaphrodites may actually be common, but data from natural populations are sparse. We therefore estimated the rate of multiple paternity and its seasonal variability in the annual, sperm‐storing, simultaneously hermaphroditic freshwater snail Radix balthica for the entire duration of the reproductive lifespan. We also tested whether multiple paternity was associated with clutch size or embryonic development. To obtain these data, we measured and genotyped 60 field‐collected egg clutches using nine highly polymorphic microsatellite markers. Overall, 50% of the clutches had multiple fathers, and both the frequency (20–93% of clutches) and magnitude of multiple paternity (mean 1.3–3.8 fathers per clutch) substantially increased over time, probably because of extensive sperm storage. Most multiply sired clutches (83%) had a dominant father, but neither clutch size nor the proportion of developed embryos per clutch was associated with levels of multiple paternity. Both the evident promiscuity and the frequent skew of paternity shares suggest that sexual selection may be an important evolutionary force in the study population.     

Effects of pollen availability and the mutation bias on the fixation of mutations disabling the male specificity of self‐incompatibility

The evolution of self‐compatibility (SC) by the loss of self‐incompatibility (SI) is regarded as one of the most frequent transitions in flowering plants. SI systems are generally characterized by specific interactions between the male and female specificity genes encoded at the S‐locus. Recent empirical studies have revealed that the evolution of SC is often driven by male SC‐conferring mutations at the S‐locus rather than by female mutations. In this study, using a forward simulation model, we compared the fixation probabilities of male vs. female SC‐conferring mutations at the S‐locus. We explicitly considered the effects of pollen availability in the population and bias in the occurrence of SC‐conferring mutations on the male and female specificity genes. We found that male SC‐conferring mutations were indeed more likely to be fixed than were female SC‐conferring mutations in a wide range of parameters. This pattern was particularly strong when pollen availability was relatively high. Under such a condition, even if the occurrence of mutations was biased strongly towards the female specificity gene, male SC‐conferring mutations were much more often fixed. Our study demonstrates that fixation probabilities of those two types of mutation vary strongly depending on ecological and genetic conditions, although both types result in the same evolutionary consequence—the loss of SI.     

Population genetics, pleiotropy, and the preferential fixation of mutations during adaptive evolution

Ongoing debate centers on whether certain types of mutations are fixed preferentially during adaptive evolution. Although there has been much discussion, no quantitative framework currently exists to test for these biases. Here, we describe a method for distinguishing between the two processes that likely account for biased rates of substitution: variation in mutation rates and variation in the probability that a mutation becomes fixed once it arises. We then use this approach to examine the type and magnitude of these biases during evolutionary transitions across multiple scales: those involving repeated origins of individual traits (flower color change), and transitions involving broad suites of traits (morphological and physiological trait evolution in plants and animals). We show that fixation biases can be strong at both levels of comparison, likely due to differences in the magnitude of deleterious pleiotropy associated with alternative mutation categories. However, we also show that the scale at which these comparisons are made greatly influences the results, as broad comparisons that simultaneously analyze multiple traits obscure heterogeneity in the direction and magnitude of these biases. We conclude that preferential fixation of mutations likely is common in nature, but should be studied on a trait-by-trait basis.     

Feed‐backs among inbreeding,inbreeding depression in sperm traits,and sperm competition can drive evolution of costly polyandry

Ongoing ambitions are to understand the evolution of costly polyandry and its consequences for species ecology and evolution. Emerging patterns could stem from feed‐back dynamics between the evolving mating system and its genetic environment, defined by interactions among kin including inbreeding. However, such feed‐backs are rarely considered in nonselfing systems. We use a genetically explicit model to demonstrate a mechanism by which inbreeding depression can select for polyandry to mitigate the negative consequences of mating with inbred males, rather than to avoid inbreeding, and to elucidate underlying feed‐backs. Specifically, given inbreeding depression in sperm traits, costly polyandry evolved to ensure female fertility, without requiring explicit inbreeding avoidance. Resulting sperm competition caused evolution of sperm traits and further mitigated the negative effect of inbreeding depression on female fertility. The evolving mating system fed back to decrease population‐wide homozygosity, and hence inbreeding. However, the net overall decrease was small due to compound effects on the variances in sex‐specific reproductive success and paternity skew. Purging of deleterious mutations did not eliminate inbreeding depression in sperm traits or hence selection for polyandry. Overall, our model illustrates that polyandry evolution, both directly and through sperm competition, might facilitate evolutionary rescue for populations experiencing sudden increases in inbreeding.