Population genetics and evolution of the mangrove rivulus Kryptolebias marmoratus,the world's only self‐fertilizing hermaphroditic vertebrate

The mangrove rivulus, Kryptolebias marmoratus (Rivulidae, Cyprinodontiformes), is phylogenetically embedded within a large clade of oviparous (egg laying) and otherwise mostly gonochoristic (separate sex) killifish species in the circumtropical suborder Aplocheiloidei. It is unique in its reproductive mode: K. marmoratus is essentially the world's only vertebrate species known to engage routinely in self‐fertilization as part of a mixed‐mating strategy of selfing plus occasional outcrossing with gonochoristic males. This unique form of procreation has profound population‐genetic and evolutionary‐genetic consequences that are the subject of this review.     

Extensive hybridization and past introgression between divergent lineages in a quasi‐clonal hermaphroditic fish: Ramifications for species concepts and taxonomy

Extreme inbreeding is expected to reduce the incidence of hybridization, serving as a prezygotic barrier. Mangrove rivulus is a small killifish that reproduces predominantly by self‐fertilization, producing highly homozygous lines throughout its geographic range. The Bahamas and Caribbean are inhabited by two highly diverged phylogeographic lineages of mangrove rivulus, Kryptolebias marmoratus and a ‘Central clade’ closely related to K. hermaphroditus from Brazil. The two lineages are largely allopatric, but recently were found in syntopy on San Salvador, Bahamas, where a single hybrid was reported. To better characterize the degree of hybridization and the possibility of secondary introgression, here we conducted a detailed genetic analysis of the contact zone on San Salvador. Two mixed populations were identified, one of which contained sexually mature hybrids. The distribution of heterozygosity at diagnostic microsatellite loci in hybrids showed that one of these hybrids was an immediate offspring from the K. marmoratus x Central clade cross, whereas the remaining five hybrids were products of reproduction by self‐fertilization for 1–3 generations following the initial cross. Two hybrids had mitochondrial haplotypes of K. marmoratus and the remaining four hybrids had a haplotype of the Central clade, indicating that crosses go in both directions. In hybrids, alleles of parental lineages were represented in equal proportions suggesting lack of recent backcrossing to either of the parental lineages. However, sympatric populations of two lineages were less diverged than allopatric populations, consistent with introgression. Results are discussed in terms of applicability of the biological species concept for isogenic, effectively clonal, organisms.     

Where do fish go when stranded on land? Terrestrial orientation of the mangrove rivulus Kryptolebias marmoratus

The goal of the present study was to determine which sensory cues the mangrove rivulus Kryptolebias marmoratus, a quasi-amphibious, hermaphroditic fish, uses to orient in an unfamiliar terrestrial environment. In a laboratory setting, K. marmoratus were placed on a terrestrial test arena and were provided the opportunity to move toward reflective surfaces, water, dark colours v. light colours, and orange colouration. Compared with hermaphrodites, males moved more often toward an orange section of the test arena, suggesting that the response may be associated with camouflage or male–male competition, since only males display orange colouration. Younger individuals also moved more often toward the orange quadrant than older individuals, suggesting age-dependent orientation performance or behaviour. Sloped terrain also had a significant effect on orientation, with more movement downhill, suggesting the importance of the otolith-vestibular system in terrestrial orientation of K. marmoratus. By understanding the orientation of extant amphibious fishes, we may be able to infer how sensory biology and behaviour might have evolved to facilitate invasion of land by amphibious vertebrates millions of years ago.     

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.     

Inbred and furious: negative association between aggression and genetic diversity in highly inbred fish

Aggressive behaviour plays an important role in securing resources, defending against predators and shaping social interactions. Although aggression can have positive effects on growth and reproductive success, it is also energetically costly and may increase injury and compromise survival. Individual genetic diversity has been positively associated with aggression, but the cause for such an association is not clear, and it might be related to the ability to recognize kin. To disentangle the relationships between genetic diversity, kinship and aggression, we quantified aggressive behaviour in a wild, self‐fertilizing fish (Kryptolebias marmoratus) with naturally variable degrees of genetic diversity, relatedness and familiarity. We found that in contrast to captive fish, levels of aggression among wild K. marmoratus are positively associated with individual homozygosity, but not with relatedness or familiarity. We suggest that the higher aggression shown by homozygous fish could be related to better kin discrimination and may be facilitated by hermaphrodite competition for scarce males, given the fitness advantages provided by outcrossing in terms of parasite resistance. It seems likely that the relationship between aggression and genetic diversity is largely influenced by both the environment and population history.     

Sex ratio,sex‐specific pattern in vegetative growth and gemma production in an aquatic liverwort,Scapania undulata (Marchantiophyta: Scapaniaceae)

Scapania undulata is an aquatic dioicous liverwort growing in shallow streams in boreal to subtropical zones. We studied the expressed sex ratio, sex‐specific differences in shoot architecture and possible trade‐off between sexual and asexual reproduction in ten populations of S. undulata by surveying 100 plots in ten streams in southern Finland. The expressed sex ratio was male biased, in contrast with the sex ratio in most dioicous bryophytes. It was also highly variable between the streams, but individual plots frequently comprised shoots from only one sex. The overproduction of males might be a strategy to overcome sperm dilution and ensure fertilization over longer distances in water. No size differences between females and males were detected, but they differed in branching patterns. Evidence for a higher cost of sexual reproduction in females than males can be seen from the following: the male‐biased sex ratio; low number of sex‐expressing female shoots in female‐only plots; no co‐occurrence of gemmae and female sex organs on a single branch, and no more than one sexual branch per female shoot. In contrast, high gemma production of male and female sex‐expressing shoots indicates a minimal trade‐off between sexual and asexual reproduction. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 175 , 229–241.     

Changes in reproductive physiology of mangrove rivulus Kryptolebias marmoratus following exposure to environmentally relevant doses of ethinyl oestradiol

Kryptolebias marmoratus exposed to 4 ng l⁻¹ of ethinyl oestradiol (EE2) for 30 days experienced significant changes in endogenous 17β‐oestradiol (E2) and 11‐ketotestosterone (KT) and qualitative changes in gonad morphology. Both hermaphrodites and males showed a significant decrease in E2, whereas only males exhibited a significant decrease in KT. Exposure to EE2 resulted in a decrease in spermatid and spermatocyte density in males and an increase in the number of early stage oocytes in hermaphrodites.     

Ovarian self‐incompatibility in Narcissus papyraceus (Amaryllidaceae) is the result of a pre‐zygotic response

Ovarian self‐incompatibility, including pre‐ and post‐zygotic reactions, is a complex mechanism for which we still lack many details relating to its function and significance. The joint presence of ovarian self‐incompatibility with style polymorphism is a rare combination that is found in the genus Narcissus. Usually, style polymorphic species have heteromorphic (diallelic and linked to style length locus) incompatibility, which prevents fertilization between individuals of the same morph, thereby helping to maintain equal proportions of floral morphs in populations. However, when present, self‐incompatibility in Narcissus is not linked to style polymorphism and cross‐fertilization within each morph is possible. Hence, self‐incompatibility in Narcissus is of particular interest when attempting to unravel the nature of the rejection reaction and aiming to assess possible cryptic differences in the fertilization process in intra‐ and inter‐morph crosses, which might ultimately explain the wide variation of morph‐ratio in the field. We examined the breeding system of Narcissus papyraceus, a style‐dimorphic species that has biased morph ratios in most of its populations. We studied pollen‐tube growth in the pistil and ovule fate after experimentally controlled hand pollinations. The growth of pollen tubes in self‐ and intra‐ and inter‐morph crosses was similar up to the point of micropyle penetration in both morphs but, subsequently, a pre‐zygotic failure appeared to affect male and female gametophytes in selfed pistils. A high proportion of ovules from self‐pollinated flowers showed signs of collapse and self‐pollen tubes were blocked or behaved abnormally before entering the embryo sac. Self‐incompatibility was stronger in the long‐styled morph than in the short‐styled morph. We did not find any conclusive sign of differential functioning between intra‐ and inter‐morph cross‐pollinations in any morph. These results enable us to rule out the possible effects of pollen–pistil interactions in N. papyraceus as a cause of morph‐ratio biases and confirm the exceptional nature of the self‐incompatibility mechanism in this polymorphic species. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 177 , 629–643.     

EVIDENCE THAT AN OUTCROSSING POPULATION IS A DERIVED LINEAGE IN A HERMAPHRODITIC FISH (RIVULUS MARMORATUS)

Rivulus marmoratus is the only known vertebrate with obligate, synchronous hermaphroditic fertilization. Males can be experimentally induced in the laboratory and are rare or absent in most populations, but at the isolated Twin Cays, Belize, locality, males are relatively abundant. At this locality, evidence of outcrossing has been documented in this otherwise automictic cloning species. Phylogenetic analysis of restriction sites and sequence characters revealed that all Florida and Belize western Caribbean populations (including Twin Cays) are phyletically indistinguishable yet divergent from eastern populations in Brazil and the Bahamas. Further, these western lineages shared a common ancestor more recently than all other populations. Therefore, the Twin Cays population is not a remnant ancestral outcrossing population. Outcrossing is suspected to have evolved as a phenotypically plastic character, and its expression in R. marmoratus may be dormant unless triggered by some ecological factor that is not well understood.     

OUTBREEDING DEPRESSION WITH LOW GENETIC VARIATION IN SELFING CAENORHABDITIS NEMATODES

Theory and empirical study produce clear links between mating system evolution and inbreeding depression. The connections between mating systems and outbreeding depression, whereby fitness is reduced in crosses of less related individuals, however, are less well defined. Here we investigate inbreeding and outbreeding depression in self‐fertile androdioecious nematodes, focusing on Caenorhabditis sp. 11. We quantify nucleotide polymorphism for nine nuclear loci for strains throughout its tropical range, and find some evidence of genetic differentiation despite the lowest sequence diversity observed in this genus. Controlled crosses between strains from geographically separated regions show strong outbreeding depression, with reproductive output of F1s reduced by 36% on average. Outbreeding depression is therefore common in self‐fertilizing Caenorhabditis species, each of which evolved androdioecious selfing hermaphroditism independently, but appears strongest in C. sp. 11. Moreover, the poor mating efficiency of androdioecious males extends to C. sp. 11. We propose that self‐fertilization is a key driver of outbreeding depression, but that it need not evolve as a direct result of local adaptation per se. Our verbal model of this process highlights the need for formal theory, and C. sp. 11 provides a convenient system for testing the genetic mechanisms that cause outbreeding depression, negative epistasis, and incipient speciation.     

Reproductive isolating barriers between colour‐differentiated populations of an African annual killifish,Nothobranchius korthausae (Cyprinodontiformes)

Allopatric populations separated by vicariance events are expected to evolve reproductive isolating mechanisms as a result of disparate selection pressures and genetic drift. The appearance of reproductive isolating mechanisms may vary across taxa with differences in the opportunity for mate choice, and may be asymmetrical. In addition, premating barriers may be affected by individual mating experience. We used choice and no‐choice experiments to investigate reproductive isolation between two allopatric (island and mainland) and colour‐differentiated populations of an African annual fish, Nothobranchius korthausae. Assortative mating under experimental conditions was limited and asymmetrical. Preference for sympatric males was only expressed in nonvirgin females from one population. Virgin fish from both populations mated indiscriminately. No difference in the number of eggs laid, fertilization rate and hatching success was detected in no‐choice experiments. All mating combinations produced viable offspring and no postmating barriers were detected in terms of the performance and fertility of F1 hybrids. Overall, we found little evidence for significant reproductive isolation, which is in contrast with the related killifish taxa in which assortative mating can be strong, even among allopatric populations with no colour differentiation. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100 , 62–72.     

Contrasting reproductive strategies of triploid hybrid males in vertebrate mating systems

The scarcity of parthenogenetic vertebrates is often attributed to their ‘inferior’ mode of clonal reproduction, which restricts them to self‐reproduce their own genotype lineage and leaves little evolutionary potential with regard to speciation and evolution of sexual reproduction. Here, we show that for some taxa, such uniformity does not hold. Using hybridogenetic water frogs (Pelophylax esculentus) as a model system, we demonstrate that triploid hybrid males from two geographic regions exhibit very different reproductive modes. With an integrative data set combining field studies, crossing experiments, flow cytometry and microsatellite analyses, we found that triploid hybrids from Central Europe are rare, occur in male sex only and form diploid gametes of a single clonal lineage. In contrast, triploid hybrids from north‐western Europe are widespread, occur in both sexes and produce recombined haploid gametes. These differences translate into contrasting reproductive roles between regions. In Central Europe, triploid hybrid males sexually parasitize diploid hybrids and just perpetuate their own genotype – which is the usual pattern in parthenogens. In north‐western Europe, on the other hand, the triploid males are gamete donors for diploid hybrids, thereby stabilizing the mixed 2n‐3n hybrid populations. By demonstrating these contrasting roles in male reproduction, we draw attention to a new significant evolutionary potential for animals with nonsexual reproduction, namely reproductive plasticity.     

Relationships between ontogenetic changes in prey selection,head shape,sexual maturity,and colour in an Australasian python (Morelia viridis)

We describe a 15‐year study of the loss of reproductive fitness and population decline in Adenostoma sparsifolium, a rosaceous shrub endemic in the fire‐prone chaparral vegetation of southern California (USA) and adjacent northern Baja California, Mexico. Our studies of background extinction concentrated on small relict populations occurring in the eastern Santa Monica Mountains where reproduction is genetically compromised by uniquely high rates of embryonic/endosperm abortion (97–99%) resulting largely from self‐pollination in highly heterozygous populations. Environmental factors further reduce reproductive fitness. The relatively few viable seeds produced are not well adapted to survive wildfires that are a regular (approximately 21 years) occurrence in chaparral. Seedling recruitment after burning is rare and any established seedlings ultimately die from the annual 4–9‐month summer droughts typical of Mediterranean climates. Adult mortality is manifest from wildfire (approximately 6%) and occasional multiple‐year droughts (approximately 15%). Given the virtual absence of new post‐fire reproduction and a low but persistent rate of adult mortality, slow population demise resulting in background extinction is inevitable. We posit that A. sparsifolium is ecologically ‘out of place’ in the present chaparral environment and appears best adapted to a moister climate with summer rains and few wildfires that prevailed before the increasing aridity and warming from mid‐Holocene to the present. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105 , 269–292.     

Resource competition between genetically varied and genetically uniform populations of Daphnia pulex (Leydig): does sexual reproduction confer a short‐term ecological advantage?

Small competitive advantages may suffice to compensate for a large disadvantage in intrinsic growth capacity. This well‐known principle from ecology has recently been applied to the enduring question of how sexual reproduction can persist in the face of invasion by female‐only parthenogens. Small competitive advantages resulting directly from sexual reproduction are predicted to cancel a two‐fold disadvantage in intrinsic growth capacity caused by males (which do not themselves produce offspring) comprising half the sexual population. In this paper we test the principal assumption of this theory, that the genetic variation produced by sexual reproduction confers a competitive advantage over self‐identical asexual invaders. We set up competition between a diverse clonal assembly of Daphnia pulex and genetically uniform populations from single clones. At young ages, the population comprising genetically varied Daphnia had significantly higher birth rates in competition with populations of genetically uniform Daphnia than in competition with itself, indicating competitive release and a Lotka–Volterra competition coefficient α₁₂ < 1. No such difference was apparent under conditions of greater food stress, possibly due to individuals channelling more energy into survival, or for old‐aged populations, possibly as a result of reduced selective pressures for high reproduction in old females. Mean birth rates differed between the clones at all ages in the presence of competition, providing evidence of variation in life history traits between clones. A Lotka–Volterra model predicted empirical estimates of α₁₂ = 0.896 (genetically uniform on varied) and α₂₁ = 1.010 (varied on uniform), which permits immediate coexistence of a sexual population of D. pulex even with an asexual lineage having twice the intrinsic growth capacity. © 2005 The Linnean Society of London, Biological Journal of the Linnean Society, 2005, 85 , 111–123.     

Variation in multiple paternity and sperm utilization patterns in natural populations of a simultaneous hermaphrodite land snail

Mating frequency has important implications for patterns of sexual selection and sexual conflict, and hence for issues such as the maintenance of genetic diversity and speciation. We assessed the level of multiple paternity and sperm utilization patterns in four natural populations of the simultaneous hermaphrodite land snail Arianta arbustorum using four polymorphic microsatellite loci. A total of 1088 offspring from 26 wild‐caught snails were genotyped to determine the number of fathers siring each brood and paternity skew in succeeding clutches. Multiple paternity was detected in the offspring of all 26 mother snails examined with the contribution of two to six fathers. The four populations examined differed in the level of multiple paternity. Snails in the population with the highest density of adults showed the highest level of multiple paternity, whereas snails in the population with the lowest density exhibited the lowest value of multiple paternity. Highly skewed paternity patterns were found in the progeny of 15 (57.7%) of the 26 mother snails. The number and identity of fathers siring the offspring of single mothers also varied among successive clutches. Furthermore, genetic analyses indicate a low level of self‐fertilization in one of the four populations. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99 , 350–361.     

Significant differences in outcrossing rate,self‐incompatibility,and inbreeding depression between two widely hybridizing species of Geum

Geum urbanum and Geum rivale are two widely hybridizing perennial herbs. Estimation of the breeding systems of these taxa using nuclear microsatellite markers scored in mother–progeny arrays demonstrated that, in pure populations, G. urbanum is predominantly selfing (outcrossing rate, t = 0.058 to 0.177), whereas G. rivale is predominantly outcrossing (t = 0.686–0.775). Theory suggests that hybridization between inbreeding and outcrossing species can potentially generate novel inbreeding lineages. However, the establishment of such lineages may be restricted either by self‐incompatibility loci or deleterious recessive alleles derived from the outcrossing parent. To assess the likelihood that hybridization between G. urbanum and G. rivale will generate novel inbreeding lineages, self‐incompatibility and inbreeding depression were investigated in the two taxa. Seed set in the absence of pollinators, and after controlled self‐ and cross‐pollination, was measured to study self‐incompatibility. Inbreeding depression was measured by estimating the relative fitness of offspring from controlled self‐and cross‐pollinations. Geum urbanum was fully self‐compatible [self‐compatibility index (SCI) = 1] and bagged flowers showed full seed set. By contrast, only 3% of bagged flowers set seed in G. rivale and controlled self‐pollinations showed a 60–80% reduction in seed set compared to controlled outcross pollinations (SCI = 0.28). There was no evidence for inbreeding depression in G. urbanum, although significant, albeit low levels of inbreeding depression were detected in one of two G. rivale populations (δ = 0.33). The implication of these results is that if genetic material from G. rivale was incorporated into a hybrid with a selfing morphology, the establishment of this selfing lineage could be compromised by self‐incompatibility and inbreeding depression. The wider implications of these results for evolution in hybrid swarms between G. urbanum and G. rivale are discussed. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 101 , 977–990.     

Variation in male reproductive traits among three bitterling fishes (Acheilognathinae: Cyprinidae) in relation to the mating system

Male traits that correlate with fertilization success include testis size and structure, ejaculate size, ejaculation frequency, and sperm motility. Two hypotheses potentially explain interspecific differences in these traits: sperm competition and sperm limitation. We examined variation in six traits associated with fertilization success in three closely‐related species of bitterling fish; the European bitterling (Rhodeus amarus), the Chinese rose bitterling (Rhodeus ocellatus), and the Chinese bitterling (Rhodeus sinensis). Interspecific differences indicated that the three study species have evolved different sperm allocation strategies. Rhodeus amarus displayed the most developed reproductive apparatus with a number of traits associated with both high levels of sperm production and fertilization efficiency. Rhodeus ocellatus and R. sinensis appear to have more comparable sperm allocation strategies, although relative testis size and spermatozoa head : tail ratio were greater in R. sinensis, suggesting that sperm competition risk may be higher in this species. All three species possessed an unusually well developed sperm duct with evidence of mucin production, which greatly extends the longevity of sperm and, consequently, the period over which fertilization can occur. We discuss these findings in the context of differences in the mating systems of the species examined, and relate the results obtained to differences in the temporal and spatial clustering of fertilizations. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 103 , 622–632.