Maternal age effects on offspring lifespan and reproduction vary within a species.

Across diverse taxa, offspring from older mothers have decreased lifespan and fitness. Little is known about the extent to which maternal age effects vary among genotypes for a given species, however, except for studies of a few arthropod species. To investigate the presence and degree of intraspecific variability in maternal age effects, we compared lifespan, reproductive schedule, and lifetime reproductive output of offspring produced by young, middle-aged, and old mothers in four strains of rotifers in the Brachionus plicatilis species complex. We found significant variability among strains in the magnitude and direction of maternal age effects on offspring life history traits. In one strain, offspring of young mothers lived 20% longer than offspring of old mothers, whereas there were no significant effects of maternal age on lifespan for other strains. Depending on strain, advanced maternal age had positive effects, negative effects, or no effect on lifetime reproductive output. Across strains, older mothers produced offspring that had higher maximum daily reproduction early in life. The effects of maternal age on offspring vital rates could not be explained by changes in trade-offs between lifespan and reproduction. This study documents intraspecific variability in maternal age effects in an additional clade. Investigating intraspecific variability is critical for understanding the ubiquity of maternal age effects and their role in the evolution of life history and aging.

A free and user-friendly software protocol for the quantification of microfauna swimming behavior.

Characterizing swimming behavior can provide a holistic assessment of the health, physiology and ecology of microfaunal species when done in conjunction with measuring other biological parameters. However, tracking and quantifying microfauna swimming behavior using existing automated tools is often difficult due to the animals' small size or transparency, or because of the high cost, expertise, or labor needed for the analysis. To address these issues, we created a cost-effective, user-friendly protocol for behavior analysis that employs the free software packages HitFilm and ToxTrac along with the R package 'trajr' and used the method to quantify the behavior of rotifers. This protocol can be used for other microfaunal species for which investigators may face similar issues in obtaining measurements of swimming behavior.

Highly efficient CRISPR-mediated gene editing in a rotifer.

Rotifers have been studied in the laboratory and field for over 100 years in investigations of microevolution, ecological dynamics, and ecotoxicology. In recent years, rotifers have emerged as a model system for modern studies of the molecular mechanisms of genome evolution, development, DNA repair, aging, life history strategy, and desiccation tolerance. However, a lack of gene editing tools and transgenic strains has limited the ability to link genotype to phenotype and dissect molecular mechanisms. To facilitate genetic manipulation and the creation of reporter lines in rotifers, we developed a protocol for highly efficient, transgenerational, CRISPR-mediated gene editing in the monogonont rotifer Brachionus manjavacas by microinjection of Cas9 protein and synthetic single-guide RNA into the vitellaria of young amictic (asexual) females. To demonstrate the efficacy of the method, we created knockout mutants of the developmental gene vasa and the DNA mismatch repair gene mlh3. More than half of mothers survived injection and produced offspring. Genotyping these offspring and successive generations revealed that most carried at least 1 CRISPR-induced mutation, with many apparently mutated at both alleles. In addition, we achieved precise CRISPR-mediated knock-in of a stop codon cassette in the mlh3 locus, with half of injected mothers producing F2 offspring with an insertion of the cassette. Thus, this protocol produces knockout and knock-in CRISPR/Cas9 editing with high efficiency, to further advance rotifers as a model system for biological discovery.

SAG-RAD: A Method for Single-Cell Population Genomics of Unicellular Eukaryotes.

Sequencing of reduced representation libraries enables genotyping of many individuals for population genomic studies. However, high amounts of DNA are required, and the method cannot be applied directly on single cells, preventing its use on most microbes. We developed and implemented the analysis of single amplified genomes followed by restriction-site-associated DNA sequencing to bypass labor-intensive culturing and to avoid culturing bias in population genomic studies of unicellular eukaryotes. This method thus opens the way for addressing important questions about the genetic diversity, gene flow, adaptation, dispersal, and biogeography of hitherto unexplored species.

Maternal age effects on offspring lifespan and reproduction vary within a species.

Across diverse taxa, offspring from older mothers have decreased lifespan and fitness. Little is known about whether such maternal age effects vary among genotypes for a given species, however. We compared maternal age effects among four strains of rotifers in the Brachionus plicatilis species complex. For each strain, we measured lifespan, reproductive schedule, and lifetime reproductive output of offspring produced by young, middle-aged, and old mothers. We found unexpected variability among strains in the magnitude and direction of maternal age effects on offspring life history traits. In one strain, offspring of young mothers lived 20% longer than offspring of old mothers, whereas there were no significant effects of maternal age on lifespan for the other strains. Across strains, advanced maternal age had positive effects, negative effects, or no effect on lifetime reproductive output. For all but one strain, older mothers produced offspring that had higher maximum daily reproduction early in life. Maternal age effects appear to be genetically determined traits, not features of life history strategy or due to accumulation of age-related damage in the germline. Investigating intraspecific variability is critical for understanding the ubiquity of maternal age effects and their role in the evolution of life history and aging.

The Contributions of Maternal Age Heterogeneity to Variance in Lifetime Reproductive Output.

AbstractVariance among individuals in fitness components reflects both genuine heterogeneity between individuals and stochasticity in events experienced along the life cycle. Maternal age represents a form of heterogeneity that affects both the mean and the variance of lifetime reproductive output (LRO). Here, we quantify the relative contribution of maternal age heterogeneity to the variance in LRO using individual-level laboratory data on the rotifer Brachionus manjavacas to parameterize a multistate age × maternal age matrix model. In B. manjavacas, advanced maternal age has large negative effects on offspring survival and fertility. We used multistate Markov chains with rewards to quantify the contributions to variance in LRO of heterogeneity and of the stochasticity inherent in the outcomes of probabilistic transitions and reproductive events. Under laboratory conditions, maternal age heterogeneity contributes 26% of the variance in LRO. The contribution changes when mortality and fertility are reduced to mimic more ecologically relevant environments. Over the parameter space where populations are near stationarity, maternal age heterogeneity contributes an average of 3% of the variance. Thus, the contributions of maternal age heterogeneity and individual stochasticity can be expected to depend strongly on environmental conditions; over most of the parameter space, the variance in LRO is dominated by stochasticity.

Sex-specific aging in animals: Perspective and future directions.

Sex differences in aging occur in many animal species, and they include sex differences in lifespan, in the onset and progression of age-associated decline, and in physiological and molecular markers of aging. Sex differences in aging vary greatly across the animal kingdom. For example, there are species with longer-lived females, species where males live longer, and species lacking sex differences in lifespan. The underlying causes of sex differences in aging remain mostly unknown. Currently, we do not understand the molecular drivers of sex differences in aging, or whether they are related to the accepted hallmarks or pillars of aging or linked to other well-characterized processes. In particular, understanding the role of sex-determination mechanisms and sex differences in aging is relatively understudied. Here, we take a comparative, interdisciplinary approach to explore various hypotheses about how sex differences in aging arise. We discuss genomic, morphological, and environmental differences between the sexes and how these relate to sex differences in aging. Finally, we present some suggestions for future research in this area and provide recommendations for promising experimental designs.

Brachionus rotifers as a model for investigating dietary and metabolic regulators of aging.

Because every species has unique attributes relevant to understanding specific aspects of aging, using a diversity of study systems and a comparative biology approach for aging research has the potential to lead to novel discoveries applicable to human health. Monogonont rotifers, a standard model for studies of aquatic ecology, evolutionary biology, and ecotoxicology, have also been used to study lifespan and healthspan for nearly a century. However, because much of this work has been published in the ecology and evolutionary biology literature, it may not be known to the biomedical research community. In this review, we provide an overview of Brachionus rotifers as a model to investigate nutritional and metabolic regulators of aging, with a focus on recent studies of dietary and metabolic pathway manipulation. Rotifers are microscopic, aquatic invertebrates with many advantages as a system for studying aging, including a two-week lifespan, easy laboratory culture, direct development without a larval stage, sexual and asexual reproduction, easy delivery of pharmaceuticals in liquid culture, and transparency allowing imaging of cellular morphology and processes. Rotifers have greater gene homology with humans than do established invertebrate models for aging, and thus rotifers may be used to investigate novel genetic mechanisms relevant to human lifespan and healthspan. The research on caloric restriction; dietary, pharmaceutical, and genetic interventions; and transcriptomics of aging using rotifers provide insights into the metabolic regulators of lifespan and health and suggest future directions for aging research. Capitalizing on the unique biology of Brachionus rotifers, referencing the vast existing literature about the influence of diet and drugs on rotifer lifespan and health, continuing the development of genetic tools for rotifers, and growing the rotifer research community will lead to new discoveries a better understanding of the biology of aging.

A demographic and evolutionary analysis of maternal effect senescence.

Maternal effect senescence-a decline in offspring survival or fertility with maternal age-has been demonstrated in many taxa, including humans. Despite decades of phenotypic studies, questions remain about how maternal effect senescence impacts evolutionary fitness. To understand the influence of maternal effect senescence on population dynamics, fitness, and selection, we developed matrix population models in which individuals are jointly classified by age and maternal age. We fit these models to data from individual-based culture experiments on the aquatic invertebrate, Brachionus manjavacas (Rotifera). By comparing models with and without maternal effects, we found that maternal effect senescence significantly reduces fitness for B. manjavacas and that this decrease arises primarily through reduced fertility, particularly at maternal ages corresponding to peak reproductive output. We also used the models to estimate selection gradients, which measure the strength of selection, in both high growth rate (laboratory) and two simulated low growth rate environments. In all environments, selection gradients on survival and fertility decrease with increasing age. They also decrease with increasing maternal age for late maternal ages, implying that maternal effect senescence can evolve through the same process as in Hamilton's theory of the evolution of age-related senescence. The models we developed are widely applicable to evaluate the fitness consequences of maternal effect senescence across species with diverse aging and fertility schedule phenotypes.

Transglutaminase Activity Determines Nuclear Localization of Serotonin Immunoreactivity in the Early Embryos of Invertebrates and Vertebrates.

Serotonin (5-HT) is a key player in many physiological processes in both the adult organism and developing embryo. One of the mechanisms for 5-HT-mediated effects is covalent binding of 5-HT to the target proteins catalyzed by transglutaminases (serotonylation). Despite the implication in a variety of physiological processes, the involvement of serotonylation in embryonic development remains unclear. Here we tested the hypothesis that 5-HT serves as a substrate for transglutaminase-mediated transamidation of the nuclear proteins in the early embryos of both vertebrates and invertebrates. For this, we demonstrated that the level of serotonin immunoreactivity (5-HT-ir) in cell nuclei increases upon the elevation of 5-HT concentration in embryos of sea urchins, mollusks, and teleost fish. Consistently, pharmacological inhibition of transglutaminase activity resulted in the reduction of both brightness and nuclear localization of anti-5-HT staining. We identified specific and bright 5-HT-ir within nuclei attributed to a subset of different cell types: ectodermal and endodermal, macro- and micromeres, and blastoderm. Western blot and dot blot confirmed the presence of 5-HT-ir epitopes in the normal embryos of all the species examined. The experimental elevation of 5-HT level led to the enhancement of 5-HT-ir-related signal on blots in a species-specific manner. The obtained results demonstrate that 5-HT is involved in transglutaminase-dependent monoaminylation of nuclear proteins and suggest nuclear serotonylation as a possible regulatory mechanism during early embryonic development. The results reveal that this pathway is conserved in the development of both vertebrates and invertebrates.

Maternal age alters offspring lifespan, fitness, and lifespan extension under caloric restriction.

Maternal age has a negative effect on offspring lifespan in a range of taxa and is hypothesized to influence the evolution of aging. However, the mechanisms of maternal age effects are unknown, and it remains unclear if maternal age alters offspring response to therapeutic interventions to aging. Here, we evaluate maternal age effects on offspring lifespan, reproduction, and the response to caloric restriction, and investigate maternal investment as a source of maternal age effects using the rotifer, Brachionus manjavacas, an aquatic invertebrate. We found that offspring lifespan and fecundity decline with increasing maternal age. Caloric restriction increases lifespan in all offspring, but the magnitude of lifespan extension is greater in the offspring from older mothers. The trade-off between reproduction and lifespan extension under low food conditions expected by life history theory is observed in young-mother offspring, but not in old-mother offspring. Age-related changes in maternal resource allocation to reproduction do not drive changes in offspring fitness or plasticity under caloric restriction in B. manjavacas. Our results suggest that the declines in reproduction in old-mother offspring negate the evolutionary fitness benefits of lifespan extension under caloric restriction.

Congeneric variability in lifespan extension and onset of senescence suggest active regulation of aging in response to low temperature.

Lifespan extension under low temperature is well conserved across both endothermic and exothermic taxa, but the mechanism underlying this change in aging is poorly understood. Low temperature is thought to decrease metabolic rate, thus slowing the accumulation of cellular damage from reactive oxygen species, although recent evidence suggests involvement of specific cold-sensing biochemical pathways. We tested the effect of low temperature on aging in 11 strains of Brachionus rotifers, with the hypothesis that if the mechanism of lifespan extension is purely thermodynamic, all strains should have a similar increase in lifespan. We found differences in change in median lifespan ranging from a 6% decrease to a 100% increase, as well as differences in maximum and relative lifespan extension and in mortality rate. Low temperature delays reproductive senescence in most strains, suggesting an extension of healthspan, even in strains with little to no change in lifespan. The combination of low temperature and caloric restriction in one strain resulted in an additive lifespan increase, indicating these interventions may work via non- or partially-overlapping pathways. The known low temperature sensor TRPA1 is present in the rotifer genome, but chemical TRPA1 agonists did not affect lifespan, suggesting that this gene may be involved in low temperature sensation but not in chemoreception in rotifers. The congeneric variability in response to low temperature suggests that the mechanism of low temperature lifespan extension is an active genetic process rather than a passive thermodynamic one and is dependent upon genotype.

Genome-wide transcriptomics of aging in the rotifer Brachionus manjavacas, an emerging model system.

BACKGROUND: Understanding gene expression changes over lifespan in diverse animal species will lead to insights to conserved processes in the biology of aging and allow development of interventions to improve health. Rotifers are small aquatic invertebrates that have been used in aging studies for nearly 100 years and are now re-emerging as a modern model system. To provide a baseline to evaluate genetic responses to interventions that change health throughout lifespan and a framework for new hypotheses about the molecular genetic mechanisms of aging, we examined the transcriptome of an asexual female lineage of the rotifer Brachionus manjavacas at five life stages: eggs, neonates, and early-, late-, and post-reproductive adults. RESULTS: There are widespread shifts in gene expression over the lifespan of B. manjavacas; the largest change occurs between neonates and early reproductive adults and is characterized by down-regulation of developmental genes and up-regulation of genes involved in reproduction. The expression profile of post-reproductive adults was distinct from that of other life stages. While few genes were significantly differentially expressed in the late- to post-reproductive transition, gene set enrichment analysis revealed multiple down-regulated pathways in metabolism, maintenance and repair, and proteostasis, united by genes involved in mitochondrial function and oxidative phosphorylation. CONCLUSIONS: This study provides the first examination of changes in gene expression over lifespan in rotifers. We detected differential expression of many genes with human orthologs that are absent in Drosophila and C. elegans, highlighting the potential of the rotifer model in aging studies. Our findings suggest that small but coordinated changes in expression of many genes in pathways that integrate diverse functions drive the aging process. The observation of simultaneous declines in expression of genes in multiple pathways may have consequences for health and longevity not detected by single- or multi-gene knockdown in otherwise healthy animals. Investigation of subtle but genome-wide change in these pathways during aging is an important area for future study.

Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions.

Rotifers are microscopic cosmopolitan zooplankton used as models in ecotoxicological and aging studies due to their several advantages such as short lifespan, ease of culture, and parthenogenesis that enables clonal culture. However, caution is required when measuring their survival time as it is affected by maternal age and maternal feeding conditions. Here we provide a protocol for powerful and reproducible measurement of the survival time in Brachionus rotifers following a careful synchronization of culture conditions over several generations. Empirically, poor synchronization results in early mortality and a gradual decrease in survival rate, thus resulting in weak statistical power. Indeed, under such conditions, calorie restriction (CR) failed to significantly extend the lifespan of B. plicatilis although CR-induced longevity has been demonstrated with well-synchronized rotifer samples in past and present studies. This protocol is probably useful for other invertebrate models, including the fruitfly Drosophila melanogaster and the nematode Caenorhabditis elegans, because maternal age effects have also been reported in these species.

Rotifers as experimental tools for investigating aging.

Comparative biogerontology has much to contribute to the study of aging. A broad range of aging rates has evolved to meet environmental challenges, and understanding these adaptations can produce valuable insights into aging. The supra Phylum Lophotrochozoa is particularly understudied and has several groups that have intriguing patterns of aging. Members of the lophotrochozoan phylum Rotifera are particularly useful for aging studies because cohort life tables can be conducted with them easily, and biochemical and genomic tools are available for examining aging mechanisms. This paper reviews a variety of caloric restriction regimens, small molecule inhibitors, and dietary supplements that extend rotifer lifespan, as well as important interactions between caloric restriction and genotype, antioxidant supplements, and TOR and JNK pathways, and the use of RNAi to identify key genes involved in modulating the aging response. Examples of how rapamycin and JNK inhibitor exposure keeps mortality rates low during the reproductive phase of the life cycle are presented, and the ease of conducting life table experiments to screen natural products from red algae for life extending effects is illustrated. Finally, experimental evolution to produce longer-lived rotifer individuals is demonstrated, and future directions to determine the genetic basis of aging are discussed.

Maternal caloric restriction partially rescues the deleterious effects of advanced maternal age on offspring.

While many studies have focused on the detrimental effects of advanced maternal age and harmful prenatal environments on progeny, little is known about the role of beneficial non-Mendelian maternal inheritance on aging. Here, we report the effects of maternal age and maternal caloric restriction (CR) on the life span and health span of offspring for a clonal culture of the monogonont rotifer Brachionus manjavacas. Mothers on regimens of chronic CR (CCR) or intermittent fasting (IF) had increased life span compared with mothers fed ad libitum (AL). With increasing maternal age, life span and fecundity of female offspring of AL-fed mothers decreased significantly and life span of male offspring was unchanged, whereas body size of both male and female offspring increased. Maternal CR partially rescued these effects, increasing the mean life span of AL-fed female offspring but not male offspring and increasing the fecundity of AL-fed female offspring compared with offspring of mothers of the same age. Both maternal CR regimens decreased male offspring body size, but only maternal IF decreased body size of female offspring, whereas maternal CCR caused a slight increase. Understanding the genetic and biochemical basis of these different maternal effects on aging may guide effective interventions to improve health span and life span.

Patterns of intraspecific variability in the response to caloric restriction.

Caloric restriction (CR) is cited as the most robust means of increasing lifespan across a range of taxa, yet there is a high degree of variability in the response to CR, both within and between species. To examine the intraspecific evolutionary conservation of lifespan extension by CR, we tested the effects of chronic caloric restriction (CCR) at multiple food levels and of intermittent fasting (IF) in twelve isolates from the Brachionus plicatilis species complex of monogonont rotifers. While CCR generally increased or did not change lifespan and total fecundity, IF caused increased, unchanged, or decreased lifespan, depending upon the isolate, and decreased total fecundity in all but one isolate. Lifespan under ad libitum (AL) feeding varied among isolates and predicted the lifespan response to CR: longer-lived isolates under AL were less likely to have a significant increase in lifespan under CCR and were more likely to have a significantly shortened lifespan under IF. Lifespan under AL conditions and the response to CR were not correlated with hydroperiodicity of native habitat or with time in culture. Lack of trade-off between lifespan and fecundity under CCR, and differences in lifespan and fecundity under CCR and IF, even when average food intake was similar, suggest that longevity changes are not always directly determined by energy intake and that CCR and IF regimens extend lifespan through diverse genetic mechanisms.

Life-span extension by caloric restriction is determined by type and level of food reduction and by reproductive mode in Brachionus manjavacas (Rotifera).

We measured life span and fecundity of three reproductive modes in a clone of the monogonont rotifer Brachionus manjavacas subjected to chronic caloric restriction (CCR) over a range of food concentrations or to intermittent fasting (IF). IF increased life span 50%-70% for all three modes, whereas CCR increased life span of asexual females derived from sexually or asexually produced eggs, but not that of sexual females. The main effect of CR on both asexual modes was to delay death at young ages, rather than to prevent death at middle ages or to greatly extend maximum life span; in contrast CR in sexual females greatly increased the life span of a few long-lived individuals. Lifetime fecundity did not decrease with CCR, suggesting a lack of resource allocation trade-off between somatic maintenance and reproduction. Multiple outcomes for a clonal lineage indicate that different responses are established through epigenetic programming, whereas differences in life-span allocations suggest that multiple genetic mechanisms mediate life-span extension.

The mate recognition protein gene mediates reproductive isolation and speciation in the Brachionus plicatilis cryptic species complex.

BACKGROUND: Chemically mediated prezygotic barriers to reproduction likely play an important role in speciation. In facultatively sexual monogonont rotifers from the Brachionus plicatilis cryptic species complex, mate recognition of females by males is mediated by the Mate Recognition Protein (MRP), a globular glycoprotein on the surface of females, encoded by the mmr-b gene family. In this study, we sequenced mmr-b copies from 27 isolates representing 11 phylotypes of the B. plicatilis species complex, examined the mode of evolution and selection of mmr-b, and determined the relationship between mmr-b genetic distance and mate recognition among isolates. RESULTS: Isolates of the B. plicatilis species complex have 1-4 copies of mmr-b, each composed of 2-9 nearly identical tandem repeats. The repeats within a gene copy are generally more similar than are gene copies among phylotypes, suggesting concerted evolution. Compared to housekeeping genes from the same isolates, mmr-b has accumulated only half as many synonymous differences but twice as many non-synonymous differences. Most of the amino acid differences between repeats appear to occur on the outer face of the protein, and these often result in changes in predicted patterns of phosphorylation. However, we found no evidence of positive selection driving these differences. Isolates with the most divergent copies were unable to mate with other isolates and rarely self-crossed. Overall the degree of mate recognition was significantly correlated with the genetic distance of mmr-b. CONCLUSIONS: Discrimination of compatible mates in the B. plicatilis species complex is determined by proteins encoded by closely related copies of a single gene, mmr-b. While concerted evolution of the tandem repeats in mmr-b may function to maintain identity, it can also lead to the rapid spread of a mutation through all copies in the genome and thus to reproductive isolation. The mmr-b gene is evolving rapidly, and novel alleles may be maintained and increase in frequency via asexual reproduction. Our analyses indicate that mate recognition, controlled by MMR-B, may drive reproductive isolation and allow saltational sympatric speciation within the B. plicatilis cryptic species complex, and that this process may be largely neutral.

Genetic determinants of mate recognition in Brachionus manjavacas (Rotifera).

BACKGROUND: Mate choice is of central importance to most animals, influencing population structure, speciation, and ultimately the survival of a species. Mating behavior of male brachionid rotifers is triggered by the product of a chemosensory gene, a glycoprotein on the body surface of females called the mate recognition pheromone. The mate recognition pheromone has been biochemically characterized, but little was known about the gene(s). We describe the isolation and characterization of the mate recognition pheromone gene through protein purification, N-terminal amino acid sequence determination, identification of the mate recognition pheromone gene from a cDNA library, sequencing, and RNAi knockdown to confirm the functional role of the mate recognition pheromone gene in rotifer mating. RESULTS: A 29 kD protein capable of eliciting rotifer male circling was isolated by high-performance liquid chromatography. Two transcript types containing the N-terminal sequence were identified in a cDNA library; further characterization by screening a genomic library and by polymerase chain reaction revealed two genes belonging to each type. Each gene begins with a signal peptide region followed by nearly perfect repeats of an 87 to 92 codon motif with no codons between repeats and the final motif prematurely terminated by the stop codon. The two Type A genes contain four and seven repeats and the two Type B genes contain three and five repeats, respectively. Only the Type B gene with three repeats encodes a peptide with a molecular weight of 29 kD. Each repeat of the Type B gene products contains three asparagines as potential sites for N-glycosylation; there are no asparagines in the Type A genes. RNAi with Type A double-stranded RNA did not result in less circling than in the phosphate-buffered saline control, but transfection with Type B double-stranded RNA significantly reduced male circling by 17%. The very low divergence between repeat units, even at synonymous positions, suggests that the repeats are kept nearly identical through a process of concerted evolution. Information-rich molecules like surface glycoproteins are well adapted for chemical communication and aquatic animals may have evolved signaling systems based on these compounds, whereas insects use cuticular hydrocarbons. CONCLUSION: Owing to its critical role in mating, the mate recognition pheromone gene will be a useful molecular marker for exploring the mechanisms and rates of selection and the evolution of reproductive isolation and speciation using rotifers as a model system. The phylogenetic variation in the mate recognition pheromone gene can now be studied in conjunction with the large amount of ecological and population genetic data being gathered for the Brachionus plicatilis species complex to understand better the evolutionary drivers of cryptic speciation.