Effects of variation in resource acquisition during different stages of the life cycle on life‐history traits and trade‐offs in a burying beetle

Individual variation in resource acquisition should have consequences for life‐history traits and trade‐offs between them because such variation determines how many resources can be allocated to different life‐history functions, such as growth, survival and reproduction. Since resource acquisition can vary across an individual's life cycle, the consequences for life‐history traits and trade‐offs may depend on when during the life cycle resources are limited. We tested for differential and/or interactive effects of variation in resource acquisition in the burying beetle Nicrophorus vespilloides. We designed an experiment in which individuals acquired high or low amounts of resources across three stages of the life cycle: larval development, prior to breeding and the onset of breeding in a fully crossed design. Resource acquisition during larval development and prior to breeding affected egg size and offspring survival, respectively. Meanwhile, resource acquisition at the onset of breeding affected size and number of both eggs and offspring. In addition, there were interactive effects between resource acquisition at different stages on egg size and offspring survival. However, only when females acquired few resources at the onset of breeding was there evidence for a trade‐off between offspring size and number. Our results demonstrate that individual variation in resource acquisition during different stages of the life cycle has important consequences for life‐history traits but limited effects on trade‐offs. This suggests that in species that acquire a fixed‐sized resource at the onset of breeding, the size of this resource has larger effects on life‐history trade‐offs than resources acquired at earlier stages.     

Phenotypic constraints and community structure: Linking trade‐offs within and among species

Trade‐offs are central to many topics in biology, from the evolution of life histories to ecological mechanisms of species coexistence. Trade‐offs observed among species may reflect pervasive constraints on phenotypes that are achievable given biophysical and resource limitations. If so, then among‐species trade‐offs should be consistent with trade‐offs within species. Alternatively, trait variation among co‐occurring species may reflect historical contingencies during community assembly rather than within‐species constraints. Here, we test whether a key trade‐off between relative growth rate (RGR) and water‐use efficiency (WUE) among Sonoran Desert winter annual plants is apparent within four species representing different strategies in the system. We grew progeny of maternal families from multiple populations in a greenhouse common garden. One species, Pectocarya recurvata, displayed the expected RGR–WUE trade‐off among families within populations. For other species, although RGR and WUE often varied clinally among populations, among‐family variation within populations was lacking, implicating a role for past selection on these traits. Our results suggest that a combination of limited genetic variation in single traits and negative trait correlations could pose constraints on the evolution of a high‐RGR and high‐WUE phenotype within species, providing a microevolutionary explanation for phenotypes that influence community‐level patterns of abundance and coexistence.     

Constraints on the evolution of function‐valued traits: a study of growth in Tribolium castaneum

Growth trajectories often impact individual fitness. They are continuous by nature and so are amenable to analysis using a function‐valued (FV) trait framework to reveal their underlying genetic architecture. Previous studies have found high levels of standing additive genetic (co)variance for growth trajectories despite the expectation that growth should be responding to frequent strong directional selection. In this study, the FV framework is used to estimate the additive genetic covariance function for growth trajectories in larval Tribolium castaneum to address questions about standing additive genetic (co)variance and possible evolutionary constraints on growth and to predict responses to four plausible selection regimes. Results show that additive genetic (co)variance is high at the early ages, but decreases towards later ages in the larval period. A selection gradient function of the same size and in the same direction of the first eigenfunction of the G‐function should give the maximal response. However, evolutionary constraints may be acting to keep this maximal response from being realized, through either conflicting effects on survivability and fecundity of larger body size, few evolutionary directions having sufficient additive variance for a response, genetic trade‐offs with other traits or physiological regulatory mechanisms. More light may be shed on these constraints through the development of more sophisticated statistical approaches and implementation of additional empirical studies to explicitly test for specific types of constraints.     

Predators modify the temperature dependence of life‐history trade‐offs

Although life histories are shaped by temperature and predation, their joint influence on the interdependence of life‐history traits is poorly understood. Shifts in one life‐history trait often necessitate shifts in another—structured in some cases by trade‐offs—leading to differing life‐history strategies among environments. The offspring size–number trade‐off connects three traits whereby a constant reproductive allocation (R) constrains how the number (O) and size (S) of offspring change. Increasing temperature and size‐independent predation decrease size at and time to reproduction which can lower R through reduced time for resource accrual or size‐constrained fecundity. We investigated how O, S, and R in a clonal population of Daphnia magna change across their first three clutches with temperature and size‐independent predation risk. Early in ontogeny, increased temperature moved O and S along a trade‐off curve (constant R) toward fewer larger offspring. Later in ontogeny, increased temperature reduced R in the no‐predator treatment through disproportionate decreases in O relative to S. In the predation treatment, R likewise decreased at warmer temperatures but to a lesser degree and more readily traded off S for O whereby the third clutch showed a constant allocation strategy of O versus S with decreasing R. Ontogenetic shifts in S and O rotated in a counterclockwise fashion as temperature increased and more drastically under risk of predation. These results show that predation risk can alter the temperature dependence of traits and their interactions through trade‐offs.     

Host‐race formation: promoted by phenology,constrained by heritability

Host‐race formation is promoted by genetic trade‐offs in the ability of herbivores to use alternate hosts, including trade‐offs due to differential timing of host‐plant availability. We examined the role of phenology in limiting host‐plant use in the goldenrod gall fly (Eurosta solidaginis) by determining: (1) whether phenology limits alternate host use, leading to a trade‐off that could cause divergent selection on Eurosta emergence time and (2) whether Eurosta has the genetic capacity to respond to such selection in the face of existing environmental variation. Experiments demonstrated that oviposition and gall induction on the alternate host, Solidago canadensis, were the highest on young plants, whereas the highest levels of gall induction on the normal host, Solidago gigantea, occurred on intermediate‐age plants. These findings indicate a phenological trade‐off for host‐plant use that sets up the possibility of divergent selection on emergence time. Heritability, estimated by parent–offspring regression, indicated that host‐race formation is impeded by the amount of genetic variation, relative to environmental, for emergence time.     

Short‐ and long‐term costs of reproduction in a migratory songbird

Costs of reproduction represent a common life‐history trade‐off. Critical to understanding these costs in migratory species is the ability to track individuals across successive stages of the annual cycle. We assessed the effects of total number of offspring fledged and date of breeding completion on pre‐migratory body condition, the schedule of moult and annual survival in a migratory songbird, the Savannah Sparrow Passerculus sandwichensis. Between 2008 and 2010, moult was delayed for individuals that finished breeding later in the breeding period and resulted in reduced lean tissue mass during the pre‐migratory period, suggesting an indirect trade‐off between the timing of breeding completion and condition just prior to migration. Lean tissue mass decreased as the number of offspring fledged increased in 2009, a particularly cool and wet year, illustrating a direct trade‐off between reproductive effort and condition just prior to migration in years when weather is poor. However, using a 17‐year dataset from the same population, we found that parents that fledged young late in the breeding period had the highest survival and that number of offspring fledged did not affect survival, suggesting that individuals do not experience long‐term trade‐offs between reproduction and survival. Taken together, our results suggest that adult Savannah Sparrows pay short‐term costs of reproduction, but that longer‐term costs are mitigated by individual quality, perhaps through individual variation in resource acquisition.     

Trade‐off between fecundity and survival generates stabilizing selection on gall size

Complex interactions within multitrophic communities are fundamental to the evolution of individual species that reside within them. One common outcome of species interactions are fitness trade‐offs, where traits adaptive in some circumstances are maladaptive in others. Here, we identify a fitness trade‐off between fecundity and survival in the cynipid wasp Callirhytis quercusbatatoides that induces multichambered galls on the stem of its host plant Quercus virginiana. We first quantified this trade‐off in natural populations by documenting two relationships: a positive association between the trait gall size and fecundity, as larger galls contain more offspring, and a negative association between gall size and survival, as larger galls are attacked by birds at a higher rate. Next, we performed a field‐based experimental evolution study where birds were excluded from the entire canopy of 11 large host trees for five years. As a result of the five‐year release from avian predators, we observed a significant shift to larger galls per tree. Overall, our study demonstrates how two opposing forces of selection can generate stabilizing selection on a critical phenotypic trait in wild populations, and how traits can evolve rapidly in the predicted direction when conditions change.     

Functional trade‐offs increase species diversity in experimental plant communities

Functional trade‐offs have long been recognised as important mechanisms of species coexistence, but direct experimental evidence for such mechanisms is extremely rare. Here, we test the effect of one classical trade‐off – a negative correlation between seed size and seed number – by establishing microcosm plant communities with positive, negative and no correlation between seed size and seed number and analysing the effect of the seed size/number correlation on species richness. Consistent with theory, a negative correlation between seed size and seed number led to a higher number of species in the communities and a corresponding wider range of seed size (a measure of functional richness) by promoting coexistence of large‐ and small‐seeded species. Our study provides the first direct evidence that a seed size/number trade‐off may contribute to species coexistence, and at a wider context, demonstrates the potential role of functional trade‐offs in maintaining species diversity.     

STRONG SELECTION GENOME‐WIDE ENHANCES FITNESS TRADE‐OFFS ACROSS ENVIRONMENTS AND EPISODES OF SELECTION

Fitness trade‐offs across episodes of selection and environments influence life‐history evolution and adaptive population divergence. Documenting these trade‐offs remains challenging as selection can vary in magnitude and direction through time and space. Here, we evaluate fitness trade‐offs at the levels of the whole organism and the quantitative trait locus (QTL) in a multiyear field study of Boechera stricta (Brassicaceae), a genetically tractable mustard native to the Rocky Mountains. Reciprocal local adaptation was pronounced for viability, but not for reproductive components of fitness. Instead, local genomes had a fecundity advantage only in the high latitude garden. By estimating realized selection coefficients from individual‐level data on viability and reproductive success and permuting the data to infer significance, we examined the genetic basis of fitness trade‐offs. This analytical approach (Conditional Neutrality‐Antagonistic Pleiotropy, CNAP) identified genetic trade‐offs at a flowering phenology QTL (costs of adaptation) and revealed genetic trade‐offs across fitness components (costs of reproduction). These patterns would not have emerged from traditional ANOVA‐based QTL mapping. Our analytical framework can be applied to other systems to investigate fitness trade‐offs. This task is becoming increasingly important as climate change may alter fitness landscapes, potentially disrupting fitness trade‐offs that took many generations to evolve.     

The predictability and magnitude of life‐history divergence to ecological agents of selection: a meta‐analysis in livebearing fishes

Environments causing variation in age‐specific mortality – ecological agents of selection – mediate the evolution of reproductive life‐history traits. However, the relative magnitude of life‐history divergence across selective agents, whether divergence in response to specific selective agents is consistent across taxa and whether it occurs as predicted by theory, remains largely unexplored. We evaluated divergence in offspring size, offspring number, and the trade‐off between these traits using a meta‐analysis in livebearing fishes (Poeciliidae). Life‐history divergence was consistent and predictable to some (predation, hydrogen sulphide) but not all (density, food limitation, salinity) selective agents. In contrast, magnitudes of divergence among selective agents were similar. Finally, there was a negative, asymmetric relationship between offspring‐number and offspring‐size divergence, suggesting greater costs of increasing offspring size than number. Ultimately, these results provide strong evidence for predictable and consistent patterns of reproductive life‐history divergence and highlight the importance of comparing phenotypic divergence across species and ecological selective agents.     

HETEROGENEITY IN INDIVIDUAL QUALITY AND REPRODUCTIVE TRADE‐OFFS WITHIN SPECIES

Interspecifically, a reasonable body of evidence supports a trade‐off between offspring size and number. However, at the intraspecific level, a whole manner of phenotypic correlations between offspring size and number are observed. These correlations may be predicted when heterogeneity in resource availability, or quality, is considered. Making the assumption that maternal size is a proxy for resource availability, we meta‐analytically quantified four phenotypic reproductive correlations within numerous species: (1) maternal size and offspring size, (2) maternal size and offspring number, (3) offspring number and offspring size, and (4) offspring number and offspring size after controlling for maternal size. Within species, maternal size showed a positive correlation with both offspring size and number. Despite this consistency, no correlation between offspring size and number was found. After controlling for maternal size, however, offspring size and number showed a significant negative correlation. A phylogenetic component of our analysis accounted for little heterogeneity in the data, suggesting that our findings show remarkable consistency across taxa. Overall, our results support an observable phenotypic trade‐off between offspring size and number. However, this analysis also highlights the importance of considering quality when examining trade‐offs, a task that is not always straightforward as quality is context dependant.     

The trade‐off between clutch size and egg mass in tree swallows Tachycineta bicolor is modulated by female body mass

Egg production is a costly component of reproduction for female birds in terms of energy expenditure and maternal investment. Because resources are typically limited, clutch size and egg mass are expected to be constrained, and this putative trade‐off between offspring number and size is at the core of life history theory. Nevertheless, empirical evidence for this trade‐off is equivocal at best, as individual heterogeneity in resource acquisition and allocation may hamper the detection of the negative correlation between egg number and mass within populations. Here, we investigated how female body mass and landscape composition influences clutch size, egg mass, and the relationship between these two traits. To do so, we fitted linear mixed models using data from tree swallows Tachycineta bicolor breeding in a network of 400 nestboxes located along a gradient of agricultural intensity between 2004 and 2011. Our dataset comprised 1463 broods for clutch size analyses and 4371 eggs (from 847 broods laid between 2005–2008) for egg mass analyses. Our results showed that agricultural intensity negatively impacted clutch size, but not egg mass nor the relationship between these two traits. Female mass, on the other hand, modulated the trade‐off between clutch size and egg mass. For heavier females, both traits increased jointly, without evidence of a trade‐off. However, for lighter females, there was a clear negative relationship between clutch size and egg mass. This work shows that accounting for individual heterogeneity in body mass allows the detection of a clutch size/egg mass trade‐off that would have remained undetected otherwise. Identifying habitat and individual effects on resource allocation towards reproductive traits may help bridging the gap between predictions from theory and empirical evidence on life history trade‐offs.     

Genotype‐by‐environment interactions underlie the expression of pre‐ and post‐copulatory sexually selected traits in guppies

The role that genotype‐by‐environment interactions (GEIs) play in sexual selection has only recently attracted the attention of evolutionary biologists. Yet GEIs can have profound evolutionary implications by compromising the honesty of sexual signals, maintaining high levels of genetic variance underlying their expression and altering the patterns of genetic covariance among fitness traits. In this study, we test for GEIs in a highly sexually dimorphic freshwater fish, the guppy Poecilia reticulata. We conducted an experimental quantitative genetic study in which male offspring arising from a paternal half‐sibling breeding design were assigned to differing nutritional ‘environments’ (either high or low feed levels). We then determined whether the manipulation of diet quantity influenced levels of additive genetic variance and covariance for several highly variable and condition‐dependent pre‐ and post‐copulatory sexual traits. In accordance with previous work, we found that dietary limitation had strong phenotypic effects on numerous pre‐ and post‐copulatory sexual traits. We also report evidence for significant GEI for several of these traits, which in some cases (area of iridescence and sperm velocity) reflected a change in the rank order of genotypes across different nutritional environments (i.e. ecological crossover). Furthermore, we show that genetic correlations vary significantly between nutritional environments. Notably, a highly significant negative genetic correlation between iridescent coloration and sperm viability in the high food treatment broke down under dietary restriction. Taken together, these findings are likely to have important evolutionary implications for guppies; ecological crossover may influence sexual signal reliability in unstable (nutritional) environments and contribute towards the extreme levels of polymorphism in sexual traits typically reported for this species. Furthermore, the presence of environment‐specific genetic covariance suggests that trade‐offs measured in one environment may not be indicative of genetic constraints in others.     

Maternal investment increases with altitude in a frog on the Tibetan Plateau

Reproducing females can allocate energy between the production of eggs or offspring of different size or number, both of which can strongly influence fitness. The physical capacity to store developing offspring imposes constraints on maximum clutch volume, but individual females and populations can trade off whether more or fewer eggs or offspring are produced, and their relative sizes. Harsh environments are likely to select for larger egg or offspring size, and many vertebrate populations compensate for this reproductive investment through an increase in female body size. We report a different trade‐off in a frog endemic to the Tibetan Plateau, Rana kukunoris. Females living at higher altitudes (n = 11 populations, 2000–3500 m) produce larger eggs, but without a concomitant increase in female body size or clutch size. The reduced diel and seasonal activity at high altitudes may impose constraints on the maximum body size of adult frogs, by limiting the opportunity for energy accumulation. Simultaneously, producing larger eggs likely helps to increase the rate of embryonic development, causing tadpoles to hatch earlier. The gelatinous matrix surrounding eggs, more of which is produced by large females, may help buffer developing embryos from temperature fluctuations or offer protection from ultraviolet radiation. High‐altitude frogs on the Tibetan Plateau employ a reproductive strategy that favours large egg size independent of body size, which is unusual in amphibians. The harsh and unpredictable environmental conditions at high altitudes can thus impose strong and opposing selection pressures on adult and embryonic life stages, both of which can simultaneously influence fitness.     

The target of selection matters: An established resistance—development‐time negative genetic trade‐off is not found when selecting on development time

Trade‐offs are fundamental to evolutionary outcomes and play a central role in eco‐evolutionary theory. They are often examined by experimentally selecting on one life‐history trait and looking for negative correlations in other traits. For example, populations of the moth Plodia interpunctella selected to resist viral infection show a life‐history cost with longer development times. However, we rarely examine whether the detection of such negative genetic correlations depends on the trait on which we select. Here, we examine a well‐characterized negative genotypic trade‐off between development time and resistance to viral infection in the moth Plodia interpunctella and test whether selection on a phenotype known to be a cost of resistance (longer development time) leads to the predicted correlated increase in resistance. If there is tight pleiotropic relationship between genes that determine development time and resistance underpinning this trade‐off, we might expect increased resistance when we select on longer development time. However, we show that selecting for longer development time in this system selects for reduced resistance when compared to selection for shorter development time. This shows how phenotypes typically characterized by a trade‐off can deviate from that trade‐off relationship, and suggests little genetic linkage between the genes governing viral resistance and those that determine response to selection on the key life‐history trait. Our results are important for both selection strategies in applied biological systems and for evolutionary modelling of host–parasite interactions.     

Colonisation of toxic environments drives predictable life‐history evolution in livebearing fishes (Poeciliidae)

New World livebearing fishes (family Poeciliidae) have repeatedly colonised toxic, hydrogen sulphide‐rich waters across their natural distribution. Physiological considerations and life‐history theory predict that these adverse conditions should favour the evolution of larger offspring. Here, we examined nine poeciliid species that independently colonised toxic environments, and show that these fishes have indeed repeatedly evolved much larger offspring size at birth in sulphidic waters, thus uncovering a widespread pattern of predictable evolution. However, a second pattern, only indirectly predicted by theory, proved additionally common: a reduction in the number of offspring carried per clutch (i.e. lower fecundity). Our analyses reveal that this secondary pattern represents a mere consequence of a classic life‐history trade‐off combined with strong selection on offspring size alone. With such strong natural selection in extreme environments, extremophile organisms may commonly exhibit multivariate phenotypic shifts even though not all diverging traits necessarily represent adaptations to the extreme conditions.     

Temperature‐mediated trade‐offs and changes in life‐history integration in two slipper limpets (Gastropoda: Calyptraeidae) with planktotrophic development

Intraspecific variation in egg size and hatching size, and the genetic and environmental trade‐offs that contribute to variation, are the basis of the evolution of life histories. The present study examined both univariate and multivariate temperature‐mediated plasticity of life‐history traits, as well as temperature‐mediated trade‐offs in egg size and clutch size, in two planktotrophic species of marine slipper limpets, Crepidula. Previous work with two species of Crepidula with large eggs and lecithotrophic development has shown a significant effect of temperature on egg size and hatching size. To further examine the effect of temperature on egg size in Crepidula, the effects of temperature on egg size and hatching size, as well as the possible trade‐offs with other the life‐history features, were examined for two planktotrophic species: Crepidula incurva and Crepidula cf. marginalis. Field‐collected juveniles were raised at 23 or 28 °C and egg size, hatching size, capsules/brood, eggs/capsule, time to hatch, interbrood interval, and final body weight were recorded. Consistent with results for the lecithotrophic Crepidula, egg size and hatching size decreased with temperature in the planktotrophic species. The affects of maternal identity and individual brood account for more than half of the intraspecific variation in egg size and hatching size. Temperature also showed a significant effect on reproductive rate, with time to hatch and interbrood interval both decreasing with increasing temperature. However, temperature had contrasting effects on the number of offspring. Crepidula cf. marginalis has significantly more eggs/capsule and therefore more eggs per brood at 28 °C compared to 23 °C, although capsules/brood did not vary with temperature. Crepidula incurva, on the other hand, produced significantly more capsules/brood and more eggs per brood at the lower temperature, whereas the number of eggs/capsule did not vary with temperature. The phenotypic variance–covariance matrix of life‐history variables showed a greater response to temperature in C. incurva than in C. cf. marginalis, and temperature induced trade‐offs between offspring size and number differ between the species. These differences suggest that temperature changes as a result of seasonal upwelling along the coast of Panama will effect the reproduction and evolution of life histories of these two co‐occurring species differently. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, ?? , ??–??.     

Higher investment in flight morphology does not trade off with fecundity estimates in a poleward range‐expanding damselfly

1. Evolutionary increases in dispersal‐related traits are frequently documented during range expansions. Investment in flight‐related traits is energetically costly and a trade‐off with fecundity may be expected during range expansion. 2. However, in contrast to wing‐dimorphic species, this trade‐off is not general in wing‐monomorphic species. In the absence of a dispersal‐‐fecundity trade‐off, an increased investment in clutch size at the expansion front is expected possibly at a cost of reduced offspring size. 3. The study evaluated investment in female flight morphology and fecundity‐related traits (clutch size, hatchling size) and potential trade‐offs among these traits in replicated populations of the poleward range‐expanding damselfly Coenagrion scitulum. 4. Females at the expansion front had a higher relative thorax length, indicating an increased investment in flight; this can be explained by spatial sorting of dispersal ability or in situ natural selection at the expansion front. Edge females produced larger hatchlings, however, this pattern was totally driven by the population‐specific thermal larval regimes and could not be attributed to the range expansion per se. By contrast, clutch sizes did not differ between core and edge populations. There was no signal of a dispersal–fecundity trade‐off either for a trade‐off between clutch size and hatchling size. 5. These results indicate that evolution of a higher dispersal ability at the expansion front of C. scitulum does not trade off with investment in fecundity, hence a dispersal–fecundity trade‐off is unlikely to slow down range expansion of this species.     

Connecting thermal performance curve variation to the genotype: a multivariate QTL approach

Thermal performance curves (TPCs) are continuous reaction norms that describe the relationship between organismal performance and temperature and are useful for understanding trade‐offs involved in thermal adaptation. Although thermal trade‐offs such as those between generalists and specialists or between hot‐ and cold‐adapted phenotypes are known to be genetically variable and evolve during thermal adaptation, little is known of the genetic basis to TPCs – specifically, the loci involved and the directionality of their effects across different temperatures. To address this, we took a multivariate approach, mapping quantitative trait loci (QTL) for locomotor activity TPCs in the fly, Drosophila serrata, using a panel of 76 recombinant inbred lines. The distribution of additive genetic (co)variance in the mapping population was remarkably similar to the distribution of mutational (co)variance for these traits. We detected 11 TPC QTL in females and 4 in males. Multivariate QTL effects were closely aligned with the major axes genetic (co)variation between temperatures; most QTL effects corresponded to variation for either overall increases or decreases in activity with a smaller number indicating possible trade‐offs between activity at high and low temperatures. QTL representing changes in curve shape such as the ‘generalist–specialist’ trade‐off, thought key to thermal adaptation, were poorly represented in the data. We discuss these results in the light of genetic constraints on thermal adaptation.     

Genomic evidence that resource‐based trade‐offs limit host‐range expansion in a seed beetle

Trade‐offs have often been invoked to explain the evolution of ecological specialization. Phytophagous insects have been especially well studied, but there has been little evidence that resource‐based trade‐offs contribute to the evolution of host specialization in this group. Here, we combine experimental evolution and partial genome resequencing of replicate seed beetle selection lines to test the trade‐off hypothesis and measure the repeatability of evolution. Bayesian estimates of selection coefficients suggest that rapid adaptation to a poor host (lentil) was mediated by standing genetic variation at multiple genetic loci and involved many of the same variants in replicate lines. Sublines that were then switched back to the ancestral host (mung bean) showed a more gradual and variable (less repeatable) loss of adaptation to lentil. We were able to obtain estimates of variance effective population sizes from genome‐wide differences in allele frequencies within and between lines. These estimates were relatively large, which suggests that the contribution of genetic drift to the loss of adaptation following reversion was small. Instead, we find that some alleles that were favored on lentil were selected against during reversion on mung bean, consistent with the genetic trade‐off hypothesis.