Carapace epithelia are rich in large filamentous actin bundles in Daphnia magna,Daphnia pulex,and Sida crystallina (Crustacea: Cladocera)

Cladocerans (water fleas) are planktonic crustaceans that typically have a bivalved carapace. Each valve of the carapace consists of two cuticle‐secreting epithelial layers that are separated by a hemolymphatic chamber and joined by pillar structures. Ultrastructural analyses in several species of Cladocera have shown that the carapace epithelia and pillars contain filamentous structures of unknown composition. In the present study we used a fluorescent phalloidin conjugate to show that the carapaces of three cladocerans, Daphnia magna, D. pulex, and Sida crystallina, are rich in large bundles of filamentous actin (F‐actin). In D. magna we employed confocal microscopy and orthogonal views of three‐dimensional reconstructions to show that these bundles extend radially from foci in the pillars towards the integument surfaces, and their structure is consistent with that of contractile stress fibers. Using a fluorescent lipophilic stain, DiOC₆(3), we show that the F‐actin bundles are distributed in membrane‐rich regions within the carapace epithelia, and that, in the superficial epithelium, these may be large membrane‐bound organelles. In D. magna, the F‐actin bundles are present in embryonic, juvenile instar, and adult, developmental stages, and through development the bundles become larger, contain more F‐actin, and become more widely spaced. We present an alignment of the deduced amino acid sequences of six putative D. pulex actin genes, and discuss the implications that their respective sequences have on the likelihood of their inclusion into the F‐actin bundles of the carapace. Our identification of these large F‐actin bundles within the pillars of three cladocerans provides new insight into the role these structures play in influencing carapace dynamics within this order.     

Evolutionary history of alpine and subalpine Daphnia in western North America

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Sperm number assessed by flow cytometry in species of Daphnia (Crustacea,Cladocera)

Daphnia magna and Daphnia pulex are two important model species in ecotoxicology. In daphniids, studies of the effects of contaminants have mostly focused on female life history traits, yet it would also be important to examine male reproductive traits, particularly in relation to endocrine disruptors. In this study, we developed a protocol that uses flow cytometry to measure sperm number in individual males of different species of Daphnia. We tested our protocol on 114 males from several clones of three common species of Daphnia. Sperm count varied widely among individuals and reached high numbers (up to 1.45 × 10⁵). Positive relationships between male length and sperm number were observed in D. pulex and Daphnia pulicaria, but not in D. magna. Important inter‐clonal differences in sperm production were observed in all species, with some clones producing very little sperm. Duplicated sperm samples showed on average only 6% difference in sperm counts. Sperm counts were stable at least over a 2‐hr period and up to 5 hr for most samples. This sperm isolation protocol and flow cytometric enumeration approach will be of major interest to ecotoxicologists.     

Variation in chronic radiation exposure does not drive life history divergence among Daphnia populations across the Chernobyl Exclusion Zone

Ionizing radiation is a mutagen with known negative impacts on individual fitness. However, much less is known about how these individual fitness effects translate into population‐level variation in natural environments that have experienced varying levels of radiation exposure. In this study, we sampled genotypes of the freshwater crustacean, Daphnia pulex, from the eight inhabited lakes across the Chernobyl Exclusion Zone (CEZ). Each lake has experienced very different levels of chronic radiation exposure since a nuclear power reactor exploded there over thirty years ago. The sampled Daphnia genotypes represent genetic snapshots of current populations and allowed us to examine fitness‐related traits under controlled laboratory conditions at UK background dose rates. We found that whilst there was variation in survival and schedules of reproduction among populations, there was no compelling evidence that this was driven by variation in exposure to radiation. Previous studies have shown that controlled exposure to radiation at dose rates included in the range measured in the current study reduce survival, or fecundity, or both. One limitation of this study is the lack of available sites at high dose rates, and future work could test life history variation in various organisms at other high radiation areas. Our results are nevertheless consistent with the idea that other ecological factors, for example competition, predation or parasitism, are likely to play a much bigger role in driving variation among populations than exposure to the high radiation dose rates found in the CEZ. These findings clearly demonstrate that it is important to examine the potential negative effects of radiation across wild populations that are subject to many and varied selection pressures as a result of complex ecological interactions.     

Evolutionary history of contagious asexuality in Daphnia pulex

Asexual taxa are short-lived, suggesting that transitions to asexuality represent evolutionary dead-ends. However, with high rates of clonal origin and coexistence of asexuals and sexuals via selective asymmetries, asexuality may persist in the long term as a result of a dynamic equilibrium between clonal origin and extinction. Few such systems have been studied in detail. Here, we investigate the evolutionary history of asexual lineages of Daphnia pulex, which are derived from sexual relatives via the inheritance of a dominant female-limited meiosis-suppressing locus and inhabit ponds throughout northeastern North America (NA). Our extensive sampling and subsequent phylogenetic analysis using mitochondrial sequence data reveals a young and genetically diverse asexual assemblage, reflecting high rates of clonal origin due to the contagious nature of asexuality. Yet, asexuality is restricted to two phylogroups (B and C) with historical and/or present associations with northeastern NA and is absent from a northwestern phylogroup (A), supporting a recent northeastern origin of asexuality in this species. Furthermore, macrogeographic patterns of genetic variability indicate that phylogroups B and C recolonized northeastern NA from opposite directions, yet their presently overlapping geographic distributions are similarly divided into an eastern asexual and a western sexual region. We attribute these patterns to a recent contagious spread of asexuality from a northeastern source. If environment-mediated selective asymmetries play no significant role in determining the outcome of competitive interactions between sexuals and asexuals, regions of contact may be setting the stage for continued asexual conquests.     

Hybrid asexuality as a primary postzygotic barrier between nascent species: On the interconnection between asexuality,hybridization and speciation

Although sexual reproduction is ubiquitous throughout nature, the molecular machinery behind it has been repeatedly disrupted during evolution, leading to the emergence of asexual lineages in all eukaryotic phyla. Despite intensive research, little is known about what causes the switch from sexual reproduction to asexuality. Interspecific hybridization is one of the candidate explanations, but the reasons for the apparent association between hybridization and asexuality remain unclear. In this study, we combined cross‐breeding experiments with population genetic and phylogenomic approaches to reveal the history of speciation and asexuality evolution in European spined loaches (Cobitis). Contemporary species readily hybridize in hybrid zones, but produce infertile males and fertile but clonally reproducing females that cannot mediate introgressions. However, our analysis of exome data indicates that intensive gene flow between species has occurred in the past. Crossings among species with various genetic distances showed that, while distantly related species produced asexual females and sterile males, closely related species produce sexually reproducing hybrids of both sexes. Our results suggest that hybridization leads to sexual hybrids at the initial stages of speciation, but as the species diverge further, the gradual accumulation of reproductive incompatibilities between species could distort their gametogenesis towards asexuality. Interestingly, comparative analysis of published data revealed that hybrid asexuality generally evolves at lower genetic divergences than hybrid sterility or inviability. Given that hybrid asexuality effectively restricts gene flow, it may establish a primary reproductive barrier earlier during diversification than other “classical” forms of postzygotic incompatibilities. Hybrid asexuality may thus indirectly contribute to the speciation process.     

On the fate of sexual traits under asexuality

Environmental shifts and life‐history changes may result in formerly adaptive traits becoming non‐functional or maladaptive. In the absence of pleiotropy and other constraints, such traits may decay as a consequence of neutral mutation accumulation or selective processes, highlighting the importance of natural selection for adaptations. A suite of traits are expected to lose their adaptive function in asexual organisms derived from sexual ancestors, and the many independent transitions to asexuality allow for comparative studies of parallel trait maintenance versus decay. In addition, because certain traits, notably male‐specific traits, are usually not exposed to selection under asexuality, their decay would have to occur as a consequence of drift. Selective processes could drive the decay of traits associated with costs, which may be the case for the majority of sexual traits expressed in females. We review the fate of male and female sexual traits in 93 animal lineages characterized by asexual reproduction, covering a broad taxon range including molluscs, arachnids, diplopods, crustaceans and eleven different hexapod orders. Many asexual lineages are still able occasionally to produce males. These asexually produced males are often largely or even fully functional, revealing that major developmental pathways can remain quiescent and functional over extended time periods. By contrast, for asexual females, there is a parallel and rapid decay of sexual traits, especially of traits related to mate attraction and location, as expected given the considerable costs often associated with the expression of these traits. The level of decay of female sexual traits, in addition to asexual females being unable to fertilize their eggs, would severely impede reversals to sexual reproduction, even in recently derived asexual lineages. More generally, the parallel maintenance versus decay of different trait types across diverse asexual lineages suggests that neutral traits display little or no decay even after extended periods under relaxed selection, while extensive decay for selected traits occurs extremely quickly. These patterns also highlight that adaptations can fix rapidly in natural populations of asexual organisms, in spite of their mode of reproduction.     

Functional rare males in diploid parthenogenetic Artemia

Functional males that are produced occasionally in some asexual taxa – called ‘rare males’ – raise considerable evolutionary interest, as they might be involved in the origin of new parthenogenetic lineages. Diploid parthenogenetic Artemia produce rare males, which may retain the ability to mate with females of related sexual lineages. Here, we (i) describe the frequency of male progeny in populations of diploid parthenogenetic Artemia, (ii) characterize rare males morphologically, (iii) assess their reproductive role, using cross‐mating experiments with sexual females of related species from Central Asia and characterize the F1 hybrid offspring viability and (iv) confirm genetically both the identity and functionality of rare males using DNA barcoding and microsatellite loci. Our result suggests that these males may have an evolutionary role through genetic exchange with related sexual species and that diploid parthenogenetic Artemia is a good model system to investigate the evolutionary transitions between sexual species and parthenogenetic strains.     

Epidemiology of asexuality induced by the endosymbiotic Wolbachia across phytophagous wasp species: host plant specialization matters

Among eukaryotes, sexual reproduction is by far the most predominant mode of reproduction. However, some systems maintaining sexuality appear particularly labile and raise intriguing questions on the evolutionary routes to asexuality. Thelytokous parthenogenesis is a form of spontaneous loss of sexuality leading to strong distortion of sex ratio towards females and resulting from mutation, hybridization or infection by bacterial endosymbionts. We investigated whether ecological specialization is a likely mechanism of spread of thelytoky within insect communities. Focusing on the highly specialized genus Megastigmus (Hymenoptera: Torymidae), we first performed a large literature survey to examine the distribution of thelytoky in these wasps across their respective obligate host plant families. Second, we tested for thelytoky caused by endosymbionts by screening in 15 arrhenotokous and 10 thelytokous species for Wolbachia, Cardinium, Arsenophonus and Rickettsia endosymbionts and by performing antibiotic treatments. Finally, we performed phylogenetic reconstructions using multilocus sequence typing (MLST) to examine the evolution of endosymbiont‐mediated thelytoky in Megastigmus and its possible connections to host plant specialization. We demonstrate that thelytoky evolved from ancestral arrhenotoky through the horizontal transmission and the fixation of the parthenogenesis‐inducing Wolbachia. We find that ecological specialization in Wolbachia's hosts was probably a critical driving force for Wolbachia infection and spread of thelytoky, but also a constraint. Our work further reinforces the hypothesis that community structure of insects is a major driver of the epidemiology of endosymbionts and that competitive interactions among closely related species may facilitate their horizontal transmission.     

Daphnia comes of age: an ecological model in the genomic era

Scientists in various subdisciplines of biology have long relied on model organisms to push the frontiers of knowledge forward as far as possible in their specific field. Today, interdisciplinary science requires model organisms that can push our understanding on multiple frontiers and help us formulate and address more complex questions. Members of the genus Daphnia represent just such an interdisciplinary model. Daphnia are aquatic microcrustaceans (also known as waterfleas) that have long been central to the study of ecology and toxicology and have recently been developed as a genomic model. A recent survey of both nuclear and mitochondrial markers in populations of the Daphnia pulex complex from high-altitude lakes in South America ( Mergeay et al . 2008, this issue ) provides an excellent example of how genetic data and ecological information can be used to push the boundaries of our understanding in molecular ecology. In this species complex, extensive hybridization has occurred resulting in polyploidization and, consequently, asexuality. Their data reveal high levels of genetic diversity, incongruence in phylogenetic signal among genomes (nuclear and mitochondrial), cryptic species in the complex, and a new model for the historical spread of the species throughout the Americas. Their data indicate that genome-level changes have occurred in this species which have profound consequences in an ecological context, the implications of which can be more fully appreciated because of our extensive knowledge of the ecology and natural history of the genus Daphnia .     

Approximate Bayesian estimation of extinction rate in the Finnish Daphnia magna metapopulation

Approximate Bayesian computation (ABC) is useful for parameterizing complex models in population genetics. In this study, ABC was applied to simultaneously estimate parameter values for a model of metapopulation coalescence and test two alternatives to a strict metapopulation model in the well‐studied network of Daphnia magna populations in Finland. The models shared four free parameters: the subpopulation genetic diversity (θ_S), the rate of gene flow among patches (4Nm), the founding population size (N₀) and the metapopulation extinction rate (e) but differed in the distribution of extinction rates across habitat patches in the system. The three models had either a constant extinction rate in all populations (strict metapopulation), one population that was protected from local extinction (i.e. a persistent source), or habitat‐specific extinction rates drawn from a distribution with specified mean and variance. Our model selection analysis favoured the model including a persistent source population over the two alternative models. Of the closest 750 000 data sets in Euclidean space, 78% were simulated under the persistent source model (estimated posterior probability = 0.769). This fraction increased to more than 85% when only the closest 150 000 data sets were considered (estimated posterior probability = 0.774). Approximate Bayesian computation was then used to estimate parameter values that might produce the observed set of summary statistics. Our analysis provided posterior distributions for e that included the point estimate obtained from previous data from the Finnish D. magna metapopulation. Our results support the use of ABC and population genetic data for testing the strict metapopulation model and parameterizing complex models of demography.     

All-male asexuality: origin and maintenance of androgenesis in the Asian clam Corbicula

Androgenesis is a rare form of asexual male reproduction found in disparate taxa across the Tree of Life. Phylogenetic analyses of mitochondrial genes suggest that androgenesis has arisen repeatedly in the Asian clam genus Corbicula. Two of these androgenetic species have been introduced to North America. Multiple lines of genetic evidence suggest that although nuclear recombination between these two species is rare, mitochondrial genome capture is a frequent consequence of androgenetic parasitism of heterospecific eggs. Egg parasitism may also rarely result in partial nuclear genome capture between closely related species of Corbicula, which provides a mechanism for the otherwise clonal species to avoid the deleterious effects of asexuality. Egg parasitism among congeners may explain why androgenesis has been maintained in Corbicula after fixation and has not yet led to population extinction. This mechanism also provides an explanation for the apparent multiple origins of androgenesis in Corbicula as seen on the mitochondrial DNA phylogeny. We suggest that a single androgenetic lineage may have repeatedly captured mitochondrial genomes (as well as portions of nuclear genomes) from various sexual species, resulting in several distinct androgenetic species with distantly related mtDNA genomes and divergent morphologies.     

Extinction hazards in experimental Daphnia magna populations: effects of genotype diversity and environmental variation

Extinction is ubiquitous in natural systems and the ultimate fate of all biological populations. However, the factors that contribute to population extinction are still poorly understood, particularly genetic diversity and composition. A laboratory experiment was conducted to examine the influences of environmental variation and genotype diversity on persistence in experimental Daphnia magna populations. Populations were initiated in two blocks with one, two, three, or six randomly selected and equally represented genotypes, fed and checked for extinction daily, and censused twice weekly over a period of 170 days. Our results show no evidence for an effect of the number of genotypes in a population on extinction hazard. Environmental variation had a strong effect on hazards in both experimental blocks, but the direction of the effect differed between blocks. In the first block, variable environments hastened extinction, while in the second block, hazards were reduced under variable food input. This occurred despite greater fluctuations in population size in variable environments in the second block of our experiment. Our results conflict with previous studies, where environmental variation consistently increased extinction risk. They are also at odds with previous studies in other systems that documented significant effects of genetic diversity on population persistence. We speculate that the lack of sexual reproduction, or the phenotypic similarity among our experimental lines, might underlie the lack of a significant effect of genotype diversity in our study.     

Quantitative evaluation of hybridization and the impact on biodiversity conservation

Anthropogenic hybridization is an increasing conservation threat worldwide. In South Africa, recent hybridization is threatening numerous ungulate taxa. For example, the genetic integrity of the near‐threatened bontebok (Damaliscus pygargus pygargus) is threatened by hybridization with the more common blesbok (D. p. phillipsi). Identifying nonadmixed parental and admixed individuals is challenging based on the morphological traits alone; however, molecular analyses may allow for accurate detection. Once hybrids are identified, population simulation software may assist in determining the optimal conservation management strategy, although quantitative evaluation of hybrid management is rarely performed. In this study, our objectives were to describe species‐wide and localized rates of hybridization in nearly 3,000 individuals based on 12 microsatellite loci, quantify the accuracy of hybrid assignment software (STRUCTURE and NEWHYBRIDS), and determine an optimal threshold of bontebok ancestry for management purposes. According to multiple methods, we identified 2,051 bontebok, 657 hybrids, and 29 blesbok. More than two‐thirds of locations contained at least some hybrid individuals, with populations varying in the degree of introgression. HYBRIDLAB was used to simulate four generations of coexistence between bontebok and blesbok, and to optimize a threshold of ancestry, where most hybrids will be detected and removed, and the fewest nonadmixed bontebok individuals misclassified as hybrids. Overall, a threshold Q‐value (admixture coefficient) of 0.90 would remove 94% of hybrid animals, while a threshold of 0.95 would remove 98% of hybrid animals but also 8% of nonadmixed bontebok. To this end, a threshold of 0.90 was identified as optimal and has since been implemented in formal policy by a provincial nature conservation agency. Due to widespread hybridization, effective conservation plans should be established and enforced to conserve native populations that are genetically unique.     

Effects of algal food quality on sexual reproduction of Daphnia magna

The objective of our study was to investigate sexual reproduction of Daphnia magna associated with mating behaviors and hatching rates, according to different algal food sources. Since a diatom is known to contain more abundant long‐chain poly unsaturated fatty acids (PUFAs), we hypothesized that the diatom‐consuming D. magna would exhibit more successful reproduction rates. Upon the hypothesis, we designed three experiments using two algal species, a green alga (Chlorella vulgaris) and a diatom (Stephanodiscus hantzschii). From the results, we found that the mating frequency and copulation duration increased in the treatment with S. hantzschii, resulting in a significant increase of hatching rates of resting eggs. In the other two repetitive mating strategies (e.g., one female vs. multiple males, and one male vs. multiple females), we found that the hatching rates of resting eggs were greater in the S. hantzschii treatment. In addition to the mating strategy, male body size significantly increased in the diatom treatment, hence average diameter of penis was also statistically different among the treatments (greater diameter in the S. hantzschii treatment). To examine the effect of algal food quality, we estimated quantity of fatty acids in the two algal species. Our result showed that S. hantzschii had a higher proportion of long‐chain PUFAs than C. vulgaris. Furthermore, a stable isotope analysis revealed that carbon and nitrogen originated from S. hantzschii were more assimilated to D. magna. In summary, our study manifested that diatom consumption of D. magna leads to more successful sexual reproduction. We then discussed how the diatom consumption of zooplankton influences food web dynamics in a freshwater ecosystem.     

Patterns of hybridization among cutthroat trout and rainbow trout in northern Rocky Mountain streams

Introgressive hybridization between native and introduced species is a growing conservation concern. For native cutthroat trout and introduced rainbow trout in western North America, this process is thought to lead to the formation of hybrid swarms and the loss of monophyletic evolutionary lineages. Previous studies of this phenomenon, however, indicated that hybrid swarms were rare except when native and introduced forms of cutthroat trout co‐occurred. We used a panel of 86 diagnostic, single nucleotide polymorphisms to evaluate the genetic composition of 3865 fish captured in 188 locations on 129 streams distributed across western Montana and northern Idaho. Although introgression was common and only 37% of the sites were occupied solely by parental westslope cutthroat trout, levels of hybridization were generally low. Of the 188 sites sampled, 73% contained ≤5% rainbow trout alleles and 58% had ≤1% rainbow trout alleles. Overall, 72% of specimens were nonadmixed westslope cutthroat trout, and an additional 3.5% were nonadmixed rainbow trout. Samples from seven sites met our criteria for hybrid swarms, that is, an absence of nonadmixed individuals and a random distribution of alleles within the sample; most (6/7) were associated with introgression by Yellowstone cutthroat trout. In streams with multiple sites, upstream locations exhibited less introgression than downstream locations. We conclude that although the widespread introduction of nonnative trout within the historical range of westslope cutthroat trout has increased the incidence of introgression, sites containing nonadmixed populations of this taxon are common and broadly distributed.     

Sexual reproduction of Daphnia pulex in a temporary habitat

Species of Daphnia (Crustacea: Cladocera) typically reproduce by cyclical parthenogenesis, in which a period of all-female parthenogenetic reproduction is followed by sexual reproduction. Sex in Daphnia is determined by the environment, with factors such as temperature, photoperiod and crowding stimulating the production of males and sexual females. Previous studies on Daphnia pulex from temporary pond habitats demonstrated the coexistence of male-producing and non-male-producing (NMP) females, as determined under crowding in the laboratory. A strong genetic component to this sex allocation variation suggested that sex expression in D. pulex is better described as a result of genotype-environment interaction. The present study examined the switch from parthenogenetic to sexual reproduction in two temporary-pond populations of D. pulex. Both populations showed a very early investment in sexual reproduction, independent of population density, by producing males very soon after the populations were reestablished from resting eggs in the early spring. Approximately 40% of the initial broods were male. Additional evidence for gender specialization was obtained by observing the sex of two or three successive broods for 85 individual females. Fifty-eight females produced successive broods of females, 13 females produced successive broods of males and 14 females produced successive broods which included both male and female broods. Females that produced successive female broods under natural conditions included a higher frequency of NMP females compared to a random sample of females, confirming the existence of NMP females. Sexual females were observed in both populations after the first appearence of males, suggesting that the presence of males may stimulate the production of sexual females. For D. pulex populations in a temporary environment, there appears to be an increased emphasis on sexual reproduction and a decreased influence of the environment on sex determination, compared to Daphnia populations in more permanent habitats. Received: 19 February 1996 / Accepted: 20 January 1997     

Selection on incremental variation of eye size in a wild population of Daphnia

Several studies of eye morphology have analysed macroevolutionary patterns in the diversity of eyes, and although these studies are often linked to environment or behaviour, they provide only indirect evidence of selection. Specific data to show the microevolutionary potential for adaptation by natural selection in eye morphology have been lacking. We document directional selection on eye size, an important determinant of visual capabilities, in a wild population of the freshwater microcrustacean Daphnia. We show that even slight changes in eye size may have major consequences for fitness. An increase in eye diameter of 19.9 μm – slightly more than one standard deviation – is associated with an increase in clutch size of one egg, or an increase of nearly 20% of the mean clutch size. Furthermore, relative eye size is genetically variable and thus could evolve in response to the observed selective pressure. We conclude that selection on incremental variation in eye size may have led to differences observed on broader taxonomic scales.