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Genome size in the rotifer Brachionus asplanchnoidis, which belongs to the B. plicatilis species complex, is greatly enlarged and extremely variable (205–407 Mbp). Such variation raises the question whether large genome size differences among individuals might cause reproductive barriers, which could trigger speciation within this group by restricting gene flow across populations. To test this hypothesis, we used B. asplanchnoidis clones from three geographic populations and conducted assays to quantify reproductive isolation among clones differing in genome size, and we examined the population structure of all three populations using amplified fragment length polymorphisms (AFLPs). AFLPs indicated that these populations were genetically separated, but we also found hints of natural gene flow. Clones from different populations with genome size differences of up to 1.7-fold could interbred successfully in the laboratory and give rise to viable, fertile ‘hybrid’ offspring. Genome sizes of these ‘hybrids’ were intermediate between those of their parents, and fitness in terms of male production, population growth, and egg development time was not negatively affected. Thus, we found no evidence for reproductive isolation or nascent speciation within B. asplanchnoidis. Instead, our results suggest that gene flow within this species can occur despite a remarkably large range of genome sizes.

Eukaryotic genomes vary greatly in size due to variation in the proportion of non-coding DNA, a pattern that emerges both in comparisons at a larger taxonomic scale and at the level of individuals within a species. The rotifer Brachionus asplanchnoidis represents one of the most extreme cases of intraspecific genome size variation among Eukaryotes, displaying almost 2-fold variation within a geographic population. Here we used a whole-genome sequencing approach to identify the underlying DNA sequence differences by assembling a high-quality reference genome draft for one individual of the population and aligning short-reads of 15 individuals from the same geographic population. We identified large, contiguous copy number variable regions (CNVs), which exhibited significant coverage differences among individuals, and whose coverage overall scaled with genome size. CNVs were mainly composed of tandemly repeated satellite DNA, with only few interspersed genes or other sequences, and were characterized by an elevated GC-content. Judging from their distributions across contigs, some CNVs are fragments of accessory (B-)chromosomes while others resemble large insertions to normal chromosomes. CNV patterns in offspring of two parents with divergent genome size, and CNV patterns in several individuals from an inbred line differing in genome size demonstrated inheritance and accumulation of CNVs across generations. Our study provides unprecedented insights into genome size evolution at microevolutionary time scales and thus paves the way for studying genome size evolution in contemporary populations rather than inferring patterns and processes a posteriori from species comparisons.

Many organisms display oscillations in population size. Theory predicts that these fluctuations can be generated by predator–prey interactions, and empirical studies using life model systems, such as a rotifer‐algae community consisting of Brachionus calyciflorus as predator and Chlorella vulgaris as prey, have been successfully used for studying such dynamics. B. calyciflorus is a cyclical parthenogen (CP) and clones often differ in their sexual propensity, that is, the degree to which they engage into sexual or asexual (clonal) reproduction. Since sexual propensities can affect growth rates and population sizes, we hypothesized that this might also affect population oscillations. Here, we studied the dynamical behaviour of B. calyciflorus clones representing either CPs (regularly inducing sex) or obligate parthenogens (OPs). We found that the amplitudes of population cycles to be increased in OPs at low nutrient levels. Several other population dynamic parameters seemed unaffected. This suggests that reproductive mode might be an important additional variable to be considered in future studies of population oscillations., Many organisms find themselves in predator‐prey relationships where growth rates of prey and attack rates of predators determine the dynamics. Since sexual reproduction can affect growth rates and population sizes compared to asexual reproduction, we hypothesized that this might also affect population oscillations. Using a predator‐prey system with Brachionus calyciflorus rotifer clones representing either cyclical parthenogens (regularly inducing sex) or obligate parthenogens, feeding on Chlorella vulgaris algal populations, we found that the amplitudes of predator population cycles are increased in obligate parthenogens at low nutrient levels.

Within-species variation in genome size has been documented in many animals and plants. Despite its importance for understanding eukaryotic genome diversity, there is only sparse knowledge about how individual-level processes mediate genome size variation in populations. Here we study a natural population of the rotifer Brachionus asplanchnoidis whose members differ up to 1.9-fold in genome size, but were still able to interbreed and produce viable offspring. We show that genome size is highly heritable and can be artificially selected up or down, but not beyond a minimum diploid genome size. Analyses of segregation patterns in haploid males reveal that large genomic elements (several megabases in size) provide the substrate of genome size variation. These elements, and their segregation patterns, explain the generation of new genome size variants, the short-term evolutionary potential of genome size change in populations, and some seemingly paradoxical patterns, like an increase in genome size variation among highly inbred lines. Our study suggests that a conceptual model involving only two variables, (1) a minimum genome size of the population, and (2) a vector containing information on additional elements that may increase genome size in this population (size, number, and meiotic segregation behaviour), can effectively address most scenarios of short-term evolutionary change of genome size in a population.

latworms can very rapidly attach to and detach from many substrates. In the presented work, we analysed the adhesive system of the marine proseriate flatworm Minona ileanae. We used light-, scanning- and transmission electron microscopy to analyse the morphology of the adhesive organs, which are located at the ventral side of the tail-plate. We performed transcriptome sequencing and differential RNA-seq for the identification of tail-specific transcripts. Using in situ hybridization expression screening, we identified nine transcripts that were expressed in the cells of the adhesive organs. Knock-down of five of these transcripts by RNA interference led to a reduction of the animal's attachment capacity. Adhesive proteins in footprints were confirmed using mass spectrometry and antibody staining. Additionally, lectin labelling of footprints revealed the presence of several sugar moieties. Furthermore, we determined a genome size of about 560 Mb for M. ileanae. We demonstrated the potential of Oxford Nanopore sequencing of genomic DNA as a cost-effective tool for identifying the number of repeats within an adhesive protein and for combining transcripts that were fragments of larger genes. A better understanding of the molecules involved in flatworm bioadhesion can pave the way towards developing innovative glues with reversible adhesive properties. This article is part of the theme issue ‘Transdisciplinary approaches to the study of adhesion and adhesives in biological systems’., Philosophical Transactions of the Royal Society B: Biological Sciences, 374 (1784), ISSN:0962-8436, ISSN:1471-2970, ISSN:0080-4622

Background The causes and consequences of genome size variation across Eukaryotes, which spans five orders of magnitude, have been hotly debated since before the advent of genome sequencing. Previous studies have mostly examined variation among larger taxonomic units (e.g., orders, or genera), while comparisons among closely related species are rare. Rotifers of the Brachionus plicatilis species complex exhibit a seven-fold variation in genome size and thus represent a unique opportunity to study such changes on a relatively short evolutionary timescale. Here, we sequenced and analysed the genomes of four species of this complex with nuclear DNA contents spanning 110–422 Mbp. To establish the likely mechanisms of genome size change, we analysed both sequencing read libraries and assemblies for signatures of polyploidy and repetitive element content. We also compared these genomes to that of B. calyciflorus, the closest relative with a sequenced genome (293 Mbp nuclear DNA content). Results Despite the very large differences in genome size, we saw no evidence of ploidy level changes across the B. plicatilis complex. However, repetitive element content explained a large portion of genome size variation (at least 54%). The species with the largest genome, B. asplanchnoidis, has a strikingly high 44% repetitive element content, while the smaller B. plicatilis genomes contain between 14 and 25% repetitive elements. According to our analyses, the B. calyciflorus genome contains 39% repetitive elements, which is substantially higher than previously reported (21%), and suggests that high repetitive element load could be widespread in monogonont rotifers. Conclusions Even though the genome sizes of these species are at the low end of the metazoan spectrum, their genomes contain substantial amounts of repetitive elements. Polyploidy does not appear to play a role in genome size variations in these species, and these variations can be mostly explained by changes in repetitive element content. This contradicts the naïve expectation that small genomes are streamlined, or less complex, and that large variations in nuclear DNA content between closely related species are due to polyploidy. Electronic supplementary material The online version of this article (10.1186/s12864-019-5859-y) contains supplementary material, which is available to authorized users.

Three well-defined groups, consisting of 15 species, have recently been ascribed to organisms historically identified as the Brachionus plicatilis species complex. One of these groups, the large clade, is composed of two named species (Brachionus plicatilis s.s. and Brachionus manjavacas) and two species identifiers (B. ‘Nevada’ and B. ‘Austria’). B. ‘Austria’ has been confirmed to be B. asplanchnoidis. As no type specimen exists for this species, and the original taxonomic description is lacking in detail, we give a detailed account of this species using material from Obere Halbjochlacke in Austria where B. ‘Austria’ was first identified genetically. Our analysis of B. asplanchnoidis populations was of global scope, an approach that revealed a great degree of morphological variability. However, combining aspects of both the dorsal and ventral surfaces clearly discriminated B. asplanchnoidis from the rest of the large-type members. This approach may prove useful in taxonomic studies of other cryptic species with relatively few morphological features. We also observed a geographic pattern of genetic divergence within B. asplanchnoidis. Average uncorrected COI divergences for a 554-bp fragment of the COI gene ranged from 3.9% within species to 17.5% between species of the large clade and indicate deep divisions within the cryptic species complex.

Genome size in the rotifer Brachionus asplanchnoidis, which belongs to the B. plicatilis species complex, is greatly enlarged and extremely variable (205–407 Mbp). Such variation raises the question whether large genome size differences among individuals might cause reproductive barriers, which could trigger speciation within this group by restricting gene flow across populations. To test this hypothesis, we used B. asplanchnoidis clones from three geographic populations and conducted assays to quantify reproductive isolation among clones differing in genome size, and we examined the population structure of all three populations using amplified fragment length polymorphisms (AFLPs). AFLPs indicated that these populations were genetically separated, but we also found hints of natural gene flow. Clones from different populations with genome size differences of up to 1.7-fold could interbred successfully in the laboratory and give rise to viable, fertile ‘hybrid’ offspring. Genome sizes of these ‘hybrids’ were intermediate between those of their parents, and fitness in terms of male production, population growth, and egg development time was not negatively affected. Thus, we found no evidence for reproductive isolation or nascent speciation within B. asplanchnoidis. Instead, our results suggest that gene flow within this species can occur despite a remarkably large range of genome sizes.

Understanding patterns and processes in biological diversity is a critical task given current and rapid environmental change. Such knowledge is even more essential when the taxa under consideration are important ecological and evolutionary models. One of these cases is the monogonont rotifer cryptic species complex Brachionus plicatilis, which is by far the most extensively studied group of rotifers, is widely used in aquaculture, and is known to host a large amount of unresolved diversity. Here we collate a dataset of previously available and newly generated sequences of COI and ITS1 for 1273 isolates of the B. plicatilis complex and apply three approaches in DNA taxonomy (i.e. ABGD, PTP, and GMYC) to identify and provide support for the existence of 15 species within the complex. We used these results to explore phylogenetic signal in morphometric and ecological traits, and to understand correlation among the traits using phylogenetic comparative models. Our results support niche conservatism for some traits (e.g. body length) and phylogenetic plasticity for others (e.g. genome size).

Supplementary Figures 1-13; Supplementary Table 1 and 2; Materials and Methods: Details on Mass Spectrometry sample preparation and data analysis, Transcriptome assembly and differential gene expression analyses, Transmission Electron Microscopy (TEM) and Element Analysis, Scanning Electron Microscopy, Whole mount in situ hybridization (WISH) and RNA interference, Oxford Nanopore sequencing, Flow cytometric measurement of genome size

Over the last decades, monogonont rotifers have proven to be excellent experimental models for life history studies. Their short life cycle and the ease of culturing rotifers facilitate experimental studies over one or several generations. Clonal reproduction allows the same genotype to be replicated over an essentially unlimited number of factors and factor combinations. Altogether this has led to a myriad of studies on how different factors can affect life history variables in rotifers. The purpose of this review is to give an overview on the main life history traits of the rotifer life cycle and to summarize the basic sources of variation of these life history traits, i.e., phenotypic plasticity (external factors affecting life history variables of individual genotypes within one generation), genetic variation (genotypic differences among individuals), and other sources of variation (maternal effects, transgenerational effects, developmental stochasticity).

A long-standing question in evolutionary biology is how sexual reproduction has persisted in eukaryotic lineages. As cyclical parthenogens, monogonont rotifers are a powerful model for examining this question, yet the molecular nature of sexual reproduction in this lineage is currently understudied. To examine genes involved in meiosis, we generated partial genome assemblies for 2 distantly related monogonont species, Brachionus calyciflorus and B. manja vacas. Here we present an inventory of 89 meiotic genes, of which 80 homologs were identified and annotated from these assemblies. Using phylogenetic analysis, we show that several meiotic genes have undergone relatively recent duplication events that appear to be specific to the monogonont lineage. Further, we compare the expression of “meiosis-specific” genes involved in recombination and all annotated copies of the cell cycle regulatory gene CDC20 between obligate parthenogenetic (OP) and cyclical parthenogenetic (CP) strains of B. caly ciflorus. We show that “meiosis-specific” genes are expressed in both CP and OP strains, whereas the expression of one of the CDC20 genes is specific to cyclical parthenogenesis. The data presented here provide insights into mechanisms of cyclical parthenogenesis and establish expectations for studies of obligate asexual relatives of monogononts, the bdelloid rotifer lineage.

Current theory proposes that sex can increase genetic variation and produce high fitness genotypes if genetic associations between alleles at different loci are non-random. In case beneficial and deleterious alleles at different loci are in linkage disequilibrium, sex may i) recombine beneficial alleles of different loci, ii) liberate beneficial alleles from genetic backgrounds of low fitness, or iii) recombine deleterious mutations for more effective elimination. In our study, we found that the first mechanism dominated the initial phase of adaptive evolution in Brachionus calyciflorus rotifers during a natural selection experiment. We used populations that had been locally adapted to two environments previously, creating a linkage disequilibrium between beneficial and deleterious alleles at different loci in a combined environment. We observed the highest fitness increase when several beneficial alleles of different loci could be recombined, while the other mechanisms were ineffective. Our study thus provides evidence for the hypothesis that sex can speed up adaptation by recombination between beneficial alleles of different loci, in particular during early stages of adaptive evolution in our system. We also suggest that the benefits of sex might change over time and state of adaptive progress.

Facultative sex combines sexual and asexual reproduction in the same individual (or clone) and allows for a large diversity of life-history patterns regarding the timing, frequency and intensity of sexual episodes. In addition, other life-history traits such as a diapause stage may become linked to sex. Here, we develop a matrix modelling framework for addressing the cost of sex in facultative sexuals, in constant, periodic and stochastically fluctuating environments. The model is parametrized using life-history data from Brachionus calyciflorus , a facultative sexual rotifer in which sex and diapause are linked. Sexual propensity was an important driver of costs in constant environments, in which high costs (always > onefold, and sometimes > twofold) indicated that asexuals should outcompete facultative sexuals. By contrast, stochastic environments with high temporal autocorrelation favoured facultative sex over obligate asex, in particular, if the penalty to fecundity in ‘bad’ environments was large. In such environments, obligate asexuals were constrained by their life cycle length (i.e. time from birth to last reproductive adult age class), which determined an upper limit to the number of consecutive bad periods they could tolerate. Nevertheless, when facultative asexuals with different sexual propensities competed simultaneously against each other and asex, the lowest sex propensity was the most successful in stochastic environments with positive autocorrelation. Our results suggest that a highly specific mechanism (i.e. diapause linked to sex) can alone stabilize facultative sex in these animals, and protect it from invasion of both asexual and pure sexual strategies. This article is part of the themed issue ‘Weird sex: the underappreciated diversity of sexual reproduction’.

Summary 1. Population size is often regulated by density dependence, that is negative feedbacks between growth and population density. Several density-dependent mechanisms may operate simultaneously in a population. 2. In this study, I focus on two different mechanisms of density-dependent population regulation, resource exploitation (RE) and density-dependent sexual reproduction (DDS). 3. I analyse both mechanisms in clonal populations of the rotifer Brachionus calyciflorus, which differ in the investment in sex because of a polymorphism at a single Mendelian locus. Some clones were cyclical parthenogens (CP) and possessed both mechanisms of population regulation (RE + DDS), while other clones were obligate parthenogens (OP) and thus lacking the DDS mechanism. 4. Equilibrium population size was considerably lower in CP clones, compared with OP clones, regardless of the exact measurement variable for population size (numbers of individuals or total biovolume/biomass). Interestingly, the decrease in population size was most pronounced in CP clones that heavily invested in sexual reproduction. 5. This suggests that the DDS mechanism can significantly contribute to population regulation and that genotypes lacking this mechanism (because of a mutation in genes affecting this trait) reach substantially higher population sizes. Apparently, the DDS mechanism operates already at much lower population densities than the RE, causing CP populations to stop growing before they are limited by resources. 6. As these differences in population regulation were caused by genetic variation within a single species and as rapid selective sweeps by OP clones are common in B. calyciflorus, this study provides an example for an eco-evolutionary feedback on an important ecological variable – equilibrium population size.

Transitions to obligate asexuality have been documented in almost all metazoan taxa, yet the conditions favoring such transitions remained largely unexplored. We address this problem in the rotifer Brachionus calyciflorus. In this species, a polymorphism at a single locus, op, can result in transitions to obligate parthenogenesis. Homozygotes for the op allele reproduce strictly by asexual reproduction, whereas heterozygous clones (þ/op) and wild-type clones (þ/þ) are cyclical parthenogens that undergo sexual reproduction at high population densities. Here, we examine dosage effects of the op allele by analyzing various life-history characteristics and population traits in 10 clones for each of the 3 possible genotypes (op/ op, þ/op, and þ/þ). For most traits, we found that op/op clones differed significantly (P , 0.05) from the 2 cyclical parthenogenetic genotypes (þ/þ and þ/op). By contrast, the 2 cyclical parthenogenetic genotypes were almost indistinguishable, except that heterozygote individuals were slightly but significantly smaller in body size compared with wild-type individuals. Overall, this indicates that the op allele is selectively neutral in the heterozygous state. Thus, selective sweeps of this allele in natural populations would first require conditions favoring the generation of homozygotes. This may be given by inbreeding in very small populations or by double mutants in very large populations.

This study provides the first analysis of genome size diversity in Monogonont rotifers. Measurements were made using flow cytometry, with Drosophila melanogaster and chicken erythrocytes as internal standards. Nuclear DNA content (“2C”— assuming diploid genomes) in eight different species of four different genera ranged almost fourfold, from 0.12 to 0.46 pg. A comparison with previously published values for Bdelloid rotifers suggested that the genomes of Monogononts are significantly smaller than those of Bdelloids. When compared to other Metazoans, Monogonont rotifers seem to have relatively small genomes. For instance, the C-values of the two species with the smallest genomes, Brachionus dimidiatus and Synchaeta pectinata, were only 0.06 and 0.085 pg, respectively. Various explanations for genome size diversity within Monogononta are discussed.

Zooplankton organisms with short generation times, such as rotifers, are ideal models to study general ecological and evolutionary questions on the population level, because meaningful experiments can often be completed within a couple of weeks. Yet biological analysis of such populations is often extremely time consuming, owing to abundance estimation by counting, measuring body size, or determining the investment into sexual versus asexual reproduction. An automated system for sampling and analyzing experimental rotifer populations is described. It relies on image analysis of digital photographs taken from subsamples of the culture. The system works completely autonomously for up to several weeks and can sample up to 12 cultures at time intervals down to a few hours. It allows quantitative analysis of female population density at a precision equivalent to that of conventional methods (i.e., manual counts of samples fixed in Lugol solution), and it can also recognize males, which allows detecting temporal variation of sexual reproduction in such cultures. Another parameter that can be automatically measured with the image analysis system is female body size. This feature may be useful for studies of population productivity and/or in competition experiments with clones of different body size. In this article, I describe the basic setup of the system and tests on the efficiency of data collection, and show some example data sets on the population dynamics of different strains of the rotifer Brachionus calyciflorus.

Transitions to asexuality have occurred in many animals and plants, yet the biological mechanisms causing such transitions have often remained unclear. Cyclical parthenogens, such as cladocerans, rotifers or aphids often give rise to obligate asexual lineages. In many rotifers, chemical signals that accumulate during population crowding trigger the induction of sexual stages. In this study, I tested two hypotheses on the origin of obligate parthenogenesis in the rotifer Brachionus calyciflorus: (i) that obligate parthenogens have lost the responsiveness to the sexual signal; and (ii) that obligate parthenogens have lost the ability to produce the sexual signal. Pairwise cross-induction assays among three obligate parthenogenetic strains and two cyclically parthenogenetic (sexual) strains were used to test these hypotheses. I found that obligate parthenogens can induce sexual reproduction in sexual strains, but not vice versa. This demonstrates that obligate parthenogens do still produce the sexual signal, but have lost responsiveness to that signal.

The high prevalence of sexual reproduction is considered a paradox mainly for two reasons. First, asexuals should enjoy various growth benefits because they seemingly rid themselves of the many inefficiencies of sexual reproduction—the so-called costs of sex. Second, there seems to be no lack of asexual origins because losses of sexual reproduction have been described in almost every larger eukaryotic taxon. Current attempts to resolve this paradox concentrate on a few hypotheses that provide universal benefits that would compensate for these costs and give sexual reproduction a net advantage. However, are new asexual lineages really those powerful invaders that could quickly displace their sexual ancestors? Research on the costs of sex indicates that sex is often stabilized by highly lineage-specific mechanisms. Two main categories can be distinguished. First are beneficial traits that evolved within a particular species and became tightly associated with sex (e.g., a mating system that involves sexual selection, or a sexual diapausing stage that allows survival through harsh periods). If such traits are absent in asexuals, simple growth efficiency considerations will not capture the fitness benefits gained by skipping sexual reproduction. Second, lineage-specific factors might prevent asexuals from reaching their full potential (e.g., dependence on fertilization in sperm-dependent parthenogens). Such observations suggest that the costs of sex are highly variable and often lower than theoretical considerations suggest. This has implications for the magnitude of universal benefits required to resolve the paradox of sex.

SUMMARY 1. The effect of temperature on the outcome of resource competition between two planktonic rotifers (Synchaeta pectinata and Brachionus calyciflorus) was investigated in laboratory experiments. In addition to the competition experiments, several physiological variables and their temperature-dependence were characterised, including ingestion rate and starvation tolerance. 2. Because of a lower threshold food level (TFL) for population growth for the food algae Cryptomonas erosa, Synchaeta was predicted to be the superior competitor at low temperatures (12 � C). In contrast, Brachionus had a lower TFL at 20 � C and was predicted to be competitively superior at this temperature. 3. In both rotifer species, ingestion rates increased with temperature, but the increase was much more pronounced in Brachionus. Ingestion rates of Brachionus at temperatures from 8 to 24 � C were always higher than in Synchaeta (up to 4.6-fold). 4. Starvation resistance reduced with temperature in both rotifer species. At all temperatures investigated (12, 16 and 20 � C) Brachionus could survive starvation for longer than Synchaeta. This difference was strongest at 12 � C (5.8 days versus 2.5 days). 5. In the first competition experiment, food was supplied at 48 h-intervals. Brachionus displaced Synchaeta at both experimental temperatures (12 and 20 � C). Competitive exclusion of Synchaeta at the lower temperature was probably because of large fluctuations in algal densities that resulted from the long intervals between feeding, a condition that favoured Brachionus because of its higher starvation resistance. 6. In the second competition experiment, one third of the food suspension was renewed every 8 h, resulting in a much better approximation to a continuous resource supply. At 12 � C Synchaeta and Brachionus coexisted for more than 1 month and the densities of both rotifer species were significantly lower in the presence of their competitor. In contrast to expectations, Brachionus was able to persist even when Cryptomonas concentrations fell below its TFL. This was probably because Brachionus was using detritus and associated bacteria as additional food sources, which were present in the cultures during the later phase of the experiment. 7. Autocorrelation analysis of the temporal changes in egg ratios revealed significant periodic cycles in Synchaeta during the second competition experiment. A possible explanation for this is the fecundity schedule of Synchaeta, in which reproduction is highly concentrated in a few age classes. According to demographic theory, such a life cycle feature can cause slower convergence to a stable age distribution.

produced only 0.3% mictic offspring. Conditioned water isolated from exponentially growing mass cultures induced significantly higher levels of mixis in the offspring of individually cultured females, when compared with untreated control medium (15.7 versus 1.4% mixis). In S. tremula, the propensity of females to respond to the mixis chemical decreased strongly with age. The highest proportion of mictic offspring (up to 63%) was produced by females of the youngest adult age class (� 24-48 h old). Females older than 3 days were virtually unresponsive to the mixis stimulus.

The defining feature of the life cycle in monogonont rotifers such as Brachionus plicatilis (Muller) is alternation of asexual and sexual reproduction (mixis). Why sex is maintained in such life cycles is an important unsolved evolutionary question and one especially amenable to experimental analysis. Mixis is induced by a chemical signal produced by the rotifers which accumulates to threshold levels at high population densities. The chemical features of this signal were characterized using size exclusion, enzymatic degradation, protease protection assays, selective binding to anion ion exchange and C3 reversed phase HPLC columns, and the sequence of 17 N-terminal amino acids. These studies were carried out over two years beginning in 2003 using B. plicatilis Russian strain. When rotifer-conditioned medium was treated with proteinase K, its mixis-inducing ability was reduced by 70%. Proteinase K was added to medium auto-conditioned by 1 female ml−1 where typically 17% of daughters became mictic and mixis was reduced to 1%. A cocktail of protease inhibitors added to conditioned medium significantly reduced degradation of the mixis signal by natural proteases. Conditioned medium subjected to ultrafiltration retained mixis-inducing activity in the >10 kDa fraction, but the

Induction of mixis (sexual reproduction) in rotifers of the genus Brachionus is believed to be triggered by a chemical that is released into the water and accumulates at high population densities. However, direct and conclusive evidence for this hypothesis is thus far lacking. In this study, two mass cultures of the rotifer Brachionus plicatilis were monitored as they grew from low to high population densities. Conditioned water was prepared daily from these cultures, and females were exposed in a bioassay, which consisted of juvenile Brachionus, cultured individually in large volumes that would normally suppress mixis. Conditioned water induced mixis in the bioassay at rates comparable to those found in the mass cultures. Both in bioassay and mass cultures, mixis was essentially absent in the beginning of the experiment, when population densities were very low. The first mictic females appeared at densities of 0.1 females ml-1, and their proportion increased rapidly as the populations grew to ≫1 female ml-1. The maximum rates of mixis in the bioassay were highly significant and reached 51% of those observed in the mass cultures. These results strongly support the hypothesis that mixis in Brachionus plicatilis is induced by a density-dependent chemical cue.

Summary 1 Larger body size at low temperatures is a commonly observed phenomenon in ectothermic organisms. The mechanisms that lead to this pattern and its possible adaptive significance were studied in laboratory experiments using the parthenogenetically reproducing rotifer Synchaeta pectinata. 2 At low temperatures of 4 °C mean body volume was 46% larger than in individuals cultured at 12 °C. Egg volume was 35% larger in low vs high temperatures. 3 Larger body size at low temperatures was caused by two mechanisms. First, when exposed to low temperatures, mothers laid larger eggs and the hatchlings of these eggs developed into larger adults (irrespective of temperature). Second, individuals cultured at low temperatures grew to a larger body size during their juvenile phase. The former mechanism had a greater influence on adult size than the latter. 4 The production of larger eggs at low temperatures seemed to be due to a higher reproductive investment into individual offspring as it occurred independently of differences in maternal size. 5 Life table experiments showed that offspring from small eggs (produced at high temperatures) had a significantly higher population growth rate than offspring from large eggs, when cultured at high temperatures. This was mainly due to an increase in fertility during the first days of adult life.

SUMMARY 1. The effect of the addition of emulsions with different fatty acid composition to a semi-natural zooplankton community was studied in enclosures. 2. The reactions of different taxa in the zooplankton community to the addition of the emulsions were different. The copepods showed almost no reaction, nor did the selective cladocerans (Bosmina) or rotifers (Synchaeta or Polyarthra). The non-selective filterfeeding cladocerans Daphnia and Ceriodaphnia, and the rotifer Keratella, showed responses to the addition of the emulsions. 3. Keratella showed the highest density in the enclosures with high amounts of highly unsaturated fatty acids added, whereas both Daphnia and Ceriodaphnia reached the highest numbers in the enclosures where we added emulsions of saturated fatty acids only. 4. Our results suggest that different taxa may be limited by different factors, even though they use similar food sources. Hence, we conclude that it is very difficult to generalize on the limiting factors in aquatic systems.

The planktonic rotifer Ascomorpha ovalis feeds on large dinoflagellates (e.9. Ceratium sp., Peridinium sp.) and is able to extract their cell contents by means of its virgate mastax. This paper presents the results of experiments on the feeding behaviour of laboratory-cultured Ascomorpha with Ceratium furcoides as food algae. Ascomorpha are three times larger than their prey Ceratium (by volume), but with regard to total length, their prey was even 20Y" larger. Ascomorplla showed a hyperbolic functional response curve with a plateau of the feeding rate at 8 Ceratium cells animal-r dayf when concentrations of Ceratium were >100 cells ml-r. The mean handling time (time for capturing and extracting one Ceratium cell) was 3 min. The shape of the functional response was better described by a curvilinear model than by a rectilinear model. However, handling times cannot be responsible for this, since they were too short to set limits on ingestion rates. At low food concen- trations, encounter rates with prey seemed to limit the feeding rates of Ascomorpha, whereas at medium to high food concentrations, satiation effects (lower attack rates) seemed to set limits on the feeding rates. Ascomorpha showed a significant decrease in the exploitation of single Ceratium cells at high prey concentrations. This decrease could be explained by a satur4tion effect in which the partly filled guts of Ascomorpha did not permit the total extraction of the contents of a Ceratium cell.

Background Although Daphnia is increasingly recognized as a model for ecological genomics and biomedical research, there is, as of yet, no high-resolution genetic map for the genus. Such a map would provide an important tool for mapping phenotypes and assembling the genome. Here we estimate the genome size of Daphnia magna and describe the construction of an SNP array based linkage map. We then test the suitability of the map for life history and behavioural trait mapping. The two parent genotypes used to produce the map derived from D. magna populations with and without fish predation, respectively and are therefore expected to show divergent behaviour and life-histories. Results Using flow cytometry we estimated the genome size of D. magna to be about 238 mb. We developed an SNP array tailored to type SNPs in a D. magna F2 panel and used it to construct a D. magna linkage map, which included 1,324 informative markers. The map produced ten linkage groups ranging from 108.9 to 203.6 cM, with an average distance between markers of 1.13 cM and a total map length of 1,483.6 cM (Kosambi corrected). The physical length per cM is estimated to be 160 kb. Mapping infertility genes, life history traits and behavioural traits on this map revealed several significant QTL peaks and showed a complex pattern of underlying genetics, with different traits showing strongly different genetic architectures. Conclusions The new linkage map of D. magna constructed here allowed us to characterize genetic differences among parent genotypes from populations with ecological differences. The QTL effect plots are partially consistent with our expectation of local adaptation under contrasting predation regimes. Furthermore, the new genetic map will be an important tool for the Daphnia research community and will contribute to the physical map of the D. magna genome project and the further mapping of phenotypic traits. The clones used to produce the linkage map are maintained in a stock collection and can be used for mapping QTLs of traits that show variance among the F2 clones., BMC Genomics, 15, ISSN:1471-2164

The ubiquity of sexual reproduction is an evolutionary puzzle because asexuality should have major reproductive advantages. Theoretically, transitions to asexuality should confer substantial benefits in population growth and lead to rapid displacement of all sexual ancestors. So far, there have been few rigorous tests of one of the most basic assumptions of the paradox of sex: that asexuals are competitively superior to sexuals immediately after their origin. Here I examine the fitness consequences of very recent transitions to obligate parthenogenesis in the cyclical parthenogenetic rotifer Brachionus calyciflorus. This experimental system differs from previous animal models, since obligate parthenogens were derived from the same maternal genotype as cyclical parthenogens. Obligate parthenogens had similar fitness compared with cyclical parthenogens in terms of the intrinsic rate of increase (calculated from life tables). However, population growth of cyclical parthenogens was predicted to be much lower: sexual female offspring do not contribute to immediate population growth in Brachionus, since they produce either males or diapausing eggs. Hence, if cyclical parthenogens constantly produce a high proportion of sexual offspring, there is a cost of sex, and obligate parthenogens can invade. This prediction was confirmed in laboratory competition experiments.

Background Studies on genome size variation in animals are rarely done at lower taxonomic levels, e.g., slightly above/below the species level. Yet, such variation might provide important clues on the tempo and mode of genome size evolution. In this study we used the flow-cytometry method to study the evolution of genome size in the rotifer Brachionus plicatilis, a cryptic species complex consisting of at least 14 closely related species. Results We found an unexpectedly high variation in this species complex, with genome sizes ranging approximately seven-fold (haploid '1C' genome sizes: 0.056-0.416 pg). Most of this variation (67%) could be ascribed to the major clades of the species complex, i.e. clades that are well separated according to most species definitions. However, we also found substantial variation (32%) at lower taxonomic levels - within and among genealogical species - and, interestingly, among species pairs that are not completely reproductively isolated. In one genealogical species, called B. 'Austria', we found greatly enlarged genome sizes that could roughly be approximated as multiples of the genomes of its closest relatives, which suggests that whole-genome duplications have occurred early during separation of this lineage. Overall, genome size was significantly correlated to egg size and body size, even though the latter became non-significant after controlling for phylogenetic non-independence. Conclusions Our study suggests that substantial genome size variation can build up early during speciation, potentially even among isolated populations. An alternative, but not mutually exclusive interpretation might be that reproductive isolation tends to build up unusually slow in this species complex.

Zooplankton organisms with short generation times, such as rotifers, are ideal models to study general ecological and evolutionary questions on the population level, because meaningful experiments can often be completed within a couple of weeks. Yet biological analysis of such populations is often extremely time consuming, owing to abundance estimation by counting, measuring body size, or determining the investment into sexual versus asexual reproduction. An automated system for sampling and analyzing experimental rotifer populations is described. It relies on image analysis of digital photographs taken from subsamples of the culture. The system works completely autonomously for up to several weeks and can sample up to 12 cultures at time intervals down to a few hours. It allows quantitative analysis of female population density at a precision equivalent to that of conventional methods (i.e., manual counts of samples fixed in Lugol solution), and it can also recognize males, which allows detecting temporal variation of sexual reproduction in such cultures. Another parameter that can be automatically measured with the image analysis system is female body size. This feature may be useful for studies of population productivity and/or in competition experiments with clones of different body size. In this article, I describe the basic setup of the system and tests on the efficiency of data collection, and show some example data sets on the population dynamics of different strains of the rotifer Brachionus calyciflorus.

Background: Asexuality has major theoretical advantages over sexual reproduction, yet newly formed asexual lineages rarely endure. The success, or failure, of such lineages is affected by their mechanism of origin, because it determines their initial genetic makeup and variability. Most previously described mechanisms imply that asexual lineages are randomly frozen subsamples of a sexual population. Methodology/Principal Findings: We found that transitions to obligate parthenogenesis (OP) in the rotifer Brachionus calyciflorus, a small freshwater invertebrate which normally reproduces by cyclical parthenogenesis, were controlled by a simple Mendelian inheritance. Pedigree analysis suggested that obligate parthenogens were homozygous for a recessive allele, which caused inability to respond to the chemical signals that normally induce sexual reproduction in this species. Alternative mechanisms, such as ploidy changes, could be ruled out on the basis of flow cytometric measurements and genetic marker analysis. Interestingly, obligate parthenogens were also dwarfs (approximately 50% smaller than cyclical parthenogens), indicating pleiotropy or linkage with genes that strongly affect body size. We found no adverse effects of OP on survival or fecundity. Conclusions/Significance: This mechanism of inheritance implies that genes causing OP may evolve within sexual populations and remain undetected in the heterozygous state long before they get frequent enough to actually cause a transition to asexual reproduction. In this process, genetic variation at other loci might become linked to OP genes, leading to non-random associations between asexuality and other phenotypic traits.

When compared to most other multicellular animals, rotifers are all relatively small, short-lived and fast-reproducing organisms. However among and within different rotifer species there is a large variation in life history patterns. This review accounts for such variation in rotifers, with a strong focus on monogonont rotifers. As the life cycle of monogonont rotifers involves both asexual and sexual reproduction, life history patterns can be examined on the level of the genetic individual, which includes all asexual females, sexual females and males that originated from one resting egg. This concept has been applied successfully in many areas, for example in predicting optimal levels of mictic reproduction or sex allocation theory. The benefits and implications of the view of the genetic individual are discussed in detail. Rotifer life histories can also be viewed on the level of physiological individuals. A large part of this review deals with the life histories of individual amictic females and addresses life history traits like body size, egg size and resource allocation patterns. It asks which trade-offs exist among those traits, how these traits change under the influence of environmental factors like food availability or temperature, and whether these changes can be interpreted as adaptive.

The numerical response of populations to different food concentrations in an important parameter to be determined for a mechanistic approach to interspecific competition. Theory predicts that the species with the lowest food level (TFL) should always be the superior competitor if only one food source is offered. However, TFLs are not species specific constants but may change along environmental gradients such as food size or temperature.

All monogonont rotifers have a diapause stage (=resting egg, RE), which allows them to endure unfavourable periods and to disperse across non-aquatic boundaries. However, recent research has shown that REs may often develop spontaneously within a few days, which seems to partly offset these adaptive explanations. In this study, I determined the minimum duration of RE development in a Brachionus calyciflorus Pallas 1766 strain in relation to other life-history variables. RE development took at least 72 h, but 50 % of those REs that hatched within the first week did so in a much synchronized manner, within 72–82 h. By contrast, amictic egg development took 11 h, while REs that had been previously stored in the cold/dark for weeks, hatched within 21 h if exposed to warm/light conditions. I discuss the fitness consequences of such fast RE development and whether it can ameliorate the costs of sex in this system. I also propose a conceptual model of RE development, which assumes two obligatory phases of pre- and post-diapause development and a facultative phase of dormancy. The latter phase may be missing in spontaneously developing REs.

Background Sexual reproduction is a widely studied biological process because it is critically important to the genetics, evolution, and ecology of eukaryotes. Despite decades of study on this topic, no comprehensive explanation has been accepted that explains the evolutionary forces underlying its prevalence and persistence in nature. Monogonont rotifers offer a useful system for experimental studies relating to the evolution of sexual reproduction due to their rapid reproductive rate and close relationship to the putatively ancient asexual bdelloid rotifers. However, little is known about the molecular underpinnings of sex in any rotifer species. Results We generated mRNA-seq libraries for obligate parthenogenetic (OP) and cyclical parthenogenetic (CP) strains of the monogonont rotifer, Brachionus calyciflorus, to identify genes specific to both modes of reproduction. Our differential expression analysis identified receptors with putative roles in signaling pathways responsible for the transition from asexual to sexual reproduction. Differential expression of a specific copy of the duplicated cell cycle regulatory gene CDC20 and specific copies of histone H2A suggest that such duplications may underlie the phenotypic plasticity required for reproductive mode switch in monogononts. We further identified differential expression of genes involved in the formation of resting eggs, a process linked exclusively to sex in this species. Finally, we identified transcripts from the bdelloid rotifer Adineta ricciae that have significant sequence similarity to genes with higher expression in CP strains of B. calyciflorus. Conclusions Our analysis of global gene expression differences between facultatively sexual and exclusively asexual populations of B. calyciflorus provides insights into the molecular nature of sexual reproduction in rotifers. Furthermore, our results offer insight into the evolution of obligate asexuality in bdelloid rotifers and provide indicators important for the use of monogononts as a model system for investigating the evolution of sexual reproduction.