Geographic Variation in Genomic Signals of Admixture Between Two Closely Related European Sepsid Fly Species.

The extent of interspecific gene flow and its consequences for the initiation, maintenance, and breakdown of species barriers in natural systems remain poorly understood. Interspecific gene flow by hybridization may weaken adaptive divergence, but can be overcome by selection against hybrids, which may ultimately promote reinforcement. An informative step towards understanding the role of gene flow during speciation is to describe patterns of past gene flow among extant species. We investigate signals of admixture between allopatric and sympatric populations of the two closely related European dung fly species Sepsis cynipsea and S. neocynipsea (Diptera: Sepsidae). Based on microsatellite genotypes, we first inferred a baseline demographic history using Approximate Bayesian Computation. We then used genomic data from pooled DNA of natural and laboratory populations to test for past interspecific gene flow based on allelic configurations discordant with the inferred population tree (ABBA-BABA test with D-statistic). Comparing the detected signals of gene flow with the contemporary geographic relationship among interspecific pairs of populations (sympatric vs. allopatric), we made two contrasting observations. At one site in the French Cevennes, we detected an excess of past interspecific gene flow, while at two sites in Switzerland we observed lower signals of past microsatellite genotypes gene flow among populations in sympatry compared to allopatric populations. These results suggest that the species boundaries between these two species depend on the past and/or present eco-geographic context in Europe, which indicates that there is no uniform link between contemporary geographic proximity and past interspecific gene flow in natural populations. Supplementary Information: The online version contains supplementary material available at 10.1007/s11692-023-09612-5.

Temperature-dependent melanism and phenoloxidase activity in the dimorphic sepsid fly Sepsis thoracica.

Climate is changing towards both higher average temperatures and more frequent and severe heat waves. Whereas numerous studies have investigated temperature effects on animal life histories, assessments of their immune function are limited. In the size- and colour-dimorphic black scavenger (or dung) fly Sepsis thoracica (Diptera: Sepsidae), we experimentally studied how developmental temperature and larval density influence phenoloxidase (PO) activity, a key enzyme in insect pigmentation, thermoregulation, and immunity. Flies from five latitudinal European populations were raised at three developmental temperatures (18, 24, 30 °C). PO activity increased with developmental temperature differently in the sexes and the two male morphs (black and orange), altering the sigmoid relationship between melanism, i.e. colouration and fly size. PO activity further positively correlated with larval rearing density, potentially because of higher risks of pathogen infection or greater developmental stress following stronger resource competition. Populations varied somewhat in PO activity, body size and colouration, however with no clear latitudinal pattern. Overall our results indicate that morph- and sex-specific PO activity, and thus likely immune function, in S. thoracica depends on temperature and larval density, modifying the underlying putative trade-off between immunity and body size. The strong dampening of the immune system of all morphs at cool temperatures suggests low-temperature stress in this warm-adapted species common in southern Europe. Our results also support the population density dependent prophylaxis hypothesis, which predicts higher investment in immunity when facing limited resource availability and increased pathogen infection probability.

Effective Ligand Design: Zinc Complexes with Guanidine Hydroquinoline Ligands for Fast Lactide Polymerization and Chemical Recycling.

Invited for this month's cover is the group of Sonja Herres-Pawlis at RWTH Aachen University. The image shows how the zinc guanidine complexes help a circular economy of bioplastics by mediating the ring-opening polymerization of lactide and the depolymerization of polylactide (PLA). The Research Article itself is available at 10.1002/cssc.202201075.

Effective Ligand Design: Zinc Complexes with Guanidine Hydroquinoline Ligands for Fast Lactide Polymerization and Chemical Recycling.

In this study, the synthesis of two new guanidine hydroquinoline ligands served as basis for six new zinc guanidine complexes. Two of these complexes showed very high activity in the lactide polymerization under industrial conditions. The lactide polymerization was demonstrated in solution and melt conditions observing high activity and molar masses up to 90 000 g mol-1 . Density functional theory studies elucidated the high activity of the complexes associated with the influence of the ligand backbone and the use of triflate counterions. On the way towards a circular economy, polymerization and depolymerization go hand in hand. So far, guanidine complexes have only shown their good activity in the ring opening polymerization of esters, and guanidine complexes with pure N donors have not been tested in recycling processes. Herein, the excellent ability of zinc guanidine complexes to catalyze both polymerization and depolymerization was demonstrated. The two most promising zinc complexes efficiently mediated the methanolysis of polylactide into methyl lactate under mild reaction conditions.

Growth rate mediates hidden developmental plasticity of female yellow dung fly reproductive morphology in response to environmental stressors.

Understanding how environmental variation influences even cryptic traits is important to clarify the roles of selection and developmental constraints in past evolutionary divergence and to predict future adaptation under environmental change. Female yellow dung flies (Scathophaga stercoraria) typically have three sperm storage compartments (3S), but occasionally four (4S). More spermathecae are thought to be a female adaptation facilitating sperm sorting after mating, but the phenotype is very rare in nature. We manipulated the flies' developmental environment by food restriction, pesticides, and hot temperatures to investigate the nature and extent of developmental plasticity of this trait, and whether spermatheca expression correlates with measures of performance and developmental stability, as would be expected if 4S expression is a developmental aberration. The spermathecal polymorphism of yellow dung fly females is heritable, but also highly developmentally plastic, varying strongly with rearing conditions. 4S expression is tightly linked to growth rate, and weakly positively correlated with fluctuating asymmetry of wings and legs, suggesting that the production of a fourth spermatheca could be a nonadaptive developmental aberration. However, spermathecal plasticity is opposite in the closely related and ecologically similar Scathophaga suilla, demonstrating that overexpression of spermathecae under developmental stress is not universal. At the same time, we found overall mortality costs as well as benefits of 4S pheno- and genotypes (also affecting male siblings), suggesting that a life history trade-off may potentially moderate 4S expression. We conclude that the release of cryptic genetic variation in spermatheca number in the face of strong environmental variation may expose hidden traits (here reproductive morphology) to natural selection (here under climate warming or food augmentation). Once exposed, hidden traits can potentially undergo rapid genetic assimilation, even in cases when trait changes are first triggered by random errors that destabilize developmental processes.

Heritable responses to combined effects of heat stress and ivermectin in the yellow dung fly.

In current times of global change, several sources of stress such as contaminants and high temperatures may act synergistically. The extent to which organisms persist in stressful conditions will depend on the fitness consequences of multiple simultaneously acting stressors and the genetic basis of compensatory genetic responses. Ivermectin is an antiparasitic drug used in livestock that is excreted in dung of treated cattle, causing severe negative consequences on non-target fauna. We evaluated the effect of a combination of heat stress and exposure to ivermectin in the yellow dung fly, Scathophaga stercoraria (Diptera: Scathophagidae). In a first experiment we investigated the effects of high rearing temperature on susceptibility to ivermectin, and in a second experiment we assayed flies from a latitudinal gradient to assess potential effects of local thermal adaptation on ivermectin sensitivity. The combination of heat and ivermectin synergistically reduced offspring survival, revealing severe effects of the two stressors when combined. However, latitudinal populations did not systematically vary in how ivermectin affected offspring survival, body size, development time, cold and heat tolerance. We also found very low narrow-sense heritability of ivermectin sensitivity, suggesting evolutionary constraints for responses to the combination of these stressors beyond immediate maternal or plastic effects. If the revealed patterns hold also for other invertebrates, the combination of increasing climate warming and ivermectin stress may thus have severe consequences for biodiversity. More generally, our study underlines the need for quantitative genetic analyses in understanding wildlife responses to interacting stressors that act synergistically and threat biodiversity.

Comprehensive thermal performance curves for yellow dung fly life history traits and the temperature-size-rule.

Ambient temperature strongly determines the behaviour, physiology, and life history of all organisms. The technical assessment of organismal thermal niches in form of now so-called thermal performance curves (TPC) thus has a long tradition in biological research. Nevertheless, several traits do not display the idealized, intuitive dome-shaped TPC, and in practice assessments often do not cover the entire realistic or natural temperature range of an organism. We here illustrate this by presenting comprehensive sex-specific TPCs for the major (juvenile) life history traits of yellow dung flies (Scathophaga stercoraria; Diptera: Scathophagidae). This concerns estimation of prominent biogeographic rules, such as the temperature-size-rule (TSR), the common phenomenon in ectothermic organisms that body size decreases as temperature increases. S. stercoraria shows an untypical asymptotic TPC of continuous body size increase with decreasing temperature without a peak (optimum), thus following the TSR throughout their entire thermal range (unlike several other insects presented here). Egg-to-adult mortality (our best fitness estimator) also shows no intermediate maximum. Both may relate to this fly entering pupal winter diapause below 12 °C. While development time presents a negative exponential relationship with temperature, development rate and growth rate typify the classic TPC form for this fly. The hitherto largely unexplored close relative S. suilla with an even more arctic distribution showed very similar responses, demonstrating large overlap among two ecologically similar, coexisting dung fly species, thus implying limited utility of even complete TPCs for predicting species distribution and coexistence.

Intraspecific mating system evolution and its effect on complex male secondary sexual traits: Does male-male competition increase selection on size or shape?

Sexual selection is generally held responsible for the exceptional diversity in secondary sexual traits in animals. Mating system evolution is therefore expected to profoundly affect the covariation between secondary sexual traits and mating success. Whereas there is such evidence at the interspecific level, data within species remain scarce. We here investigate sexual selection acting on the exaggerated male fore femur and the male wing in the common and widespread dung flies Sepsis punctum and S. neocynipsea (Diptera: Sepsidae). Both species exhibit intraspecific differences in mating systems and variation in sexual size dimorphism (SSD) across continents that correlates with the extent of male-male competition. We predicted that populations subject to increased male-male competition will experience stronger directional selection on the sexually dimorphic male foreleg. Our results suggest that fore femur size, width and shape were indeed positively associated with mating success in populations with male-biased SSD in both species, which was not evident in conspecific populations with female-biased SSD. However, this was also the case for wing size and shape, a trait often assumed to be primarily under natural selection. After correcting for selection on overall body size by accounting for allometric scaling, we found little evidence for independent selection on any of these size or shape traits in legs or wings, irrespective of the mating system. Sexual dimorphism and (foreleg) trait exaggeration is therefore unlikely to be driven by direct precopulatory sexual selection, but more so by selection on overall size or possibly selection on allometric scaling.

Comparative reproductive dormancy differentiation in European black scavenger flies (Diptera: Sepsidae).

Seasonality is a key environmental factor that regularly promotes life history adaptation. Insects invading cold-temperate climates need to overwinter in a dormant state. We compared the role of temperature and photoperiod in dormancy induction in the laboratory, as well as winter survival and reproduction in the field and the laboratory, of 5 widespread European dung fly species (Diptera: Sepsidae) to investigate their extent of ecological differentiation and thermal adaptation. Unexpectedly, cold temperature is the primary environmental factor inducing winter dormancy, with short photoperiod playing an additional role mainly in species common at high altitudes and latitudes (Sepsis cynipsea, neocynipsea, fulgens), but not in those species also thriving in southern Europe (thoracica, punctum). All species hibernate as adults rather than juveniles. S. thoracica had very low adult winter survivorship under both (benign) laboratory and (harsh) field conditions, suggesting flexible quiescence rather than genetically fixed winter diapause, restricting their distribution towards the pole. All other species appear well suited for surviving cold, Nordic winters. Females born early in the season reproduce before winter while late-born females reproduce after winter, fulgens transitioning earliest before winter and thoracica and punctum latest; a bet-hedging strategy of reproduction during both seasons occurs rarely but is possible physiologically. Fertility patterns indicate that females can store sperm over winter. Winter dormancy induction mechanisms of European sepsids are congruent with their geographic distribution, co-defining their thermal niches. Flexible adult winter quiescence appears the easiest route for insects spreading towards the poles to evolve the necessary overwinter survival.

Does thermal plasticity align with local adaptation? An interspecific comparison of wing morphology in sepsid flies.

Although genetic and plastic responses are sometimes considered as unrelated processes, their phenotypic effects may often align because genetic adaptation is expected to mirror phenotypic plasticity if adaptive, but run counter to it when maladaptive. Because the magnitude and direction of this alignment has further consequences for both the tempo and mode of adaptation, they are relevant for predicting an organisms' reaction to environmental change. To better understand the interplay between phenotypic plasticity and genetic change in mediating adaptive phenotypic variation to climate variability, we here quantified genetic latitudinal variation and thermal plasticity in wing loading and wing shape in two closely related and widespread sepsid flies. Common garden rearing of 16 geographical populations reared across multiple temperatures revealed that wing loading decreases with latitude in both species. This pattern could be driven by selection for increased dispersal capacity in the cold. However, although allometry, sexual dimorphism, thermal plasticity and latitudinal differentiation in wing shape all show similar patterns in the two species, the relationship between the plastic and genetic responses differed between them. Although latitudinal differentiation (south to north) mirrored thermal plasticity (hot to cold) in Sepsis punctum, there was no relationship in Sepsis fulgens. While this suggests that thermal plasticity may have helped to mediate local adaptation in S. punctum, it also demonstrates that genetic wing shape differentiation and its relation to thermal plasticity may be complex and idiosyncratic, even among ecologically similar and closely related species. Hence, genetic responses can, but do not necessarily, align with phenotypic plasticity induced by changing environmental selection pressures.

Comparative effects of the parasiticide ivermectin on survival and reproduction of adult sepsid flies.

Ivermectin is a veterinary pharmaceutical widely applied against parasites of livestock. Being effective against pests, it is also known to have lethal and sublethal effects on non-target organisms. While considerable research demonstrates the impact of ivermectin residues in livestock dung on the development and survival of dung feeding insect larvae, surprisingly little is known about its fitness effects on adults. We tested the impact of ivermectin on the survival of adult sepsid dung fly species (Diptera: Sepsidae) in the laboratory, using an ecologically relevant and realistic range of 69-1978 µg ivermectin/kg wet dung, and compared the sensitivities of larvae and adults in a phylogenetic framework. For one representative, relatively insensitive species, Sepsis punctum, we further investigated effects of ivermectin on female fecundity and male fertility. Moreover, we tested whether females can differentiate between ivermectin-spiked and non-contaminated dung in the wild. Adult sepsid flies exposed to ivermectin suffered increased mortality, whereby closely related species varied strongly in their sensitivity. Adult susceptibility to the drug correlated with larval susceptibility, showing a phylogenetic signal and demonstrating systemic variation in ivermectin sensitivity. Exposure of S. punctum females to even low concentrations of ivermectin lowered the number of eggs laid, while treatment of males reduced egg-to-adult offspring survival, presumably via impairment of sperm quality or quantity. The fitness impact was amplified when both parents were exposed. Lastly, sepsid flies did not discriminate against ivermectin-spiked dung in the field. Treatment of livestock with avermectins may thus have even more far-reaching sublethal ecological consequences than currently assumed via effects on adult dung-feeding insects.

Geographic clines in wing morphology relate to colonization history in New World but not Old World populations of yellow dung flies.

Geographic clines offer insights about putative targets and agents of natural selection as well as tempo and mode of adaptation. However, demographic processes can lead to clines that are indistinguishable from adaptive divergence. Using the widespread yellow dung fly Scathophaga stercoraria (Diptera: Scathophagidae), we examine quantitative genetic differentiation (QST ) of wing shape across North America, Europe, and Japan, and compare this differentiation with that of ten microsatellites (FST ). Morphometric analyses of 28 populations reared at three temperatures revealed significant thermal plasticity, sexual dimorphism, and geographic differentiation in wing shape. In North America morphological differentiation followed the decline in microsatellite variability along the presumed route of recent colonization from the southeast to the northwest. Across Europe, where S. stercoraria presumably existed for much longer time and where no molecular pattern of isolation by distance was evident, clinal variation was less pronounced despite significant morphological differentiation (QST >FST ). Shape vector comparisons further indicate that thermal plasticity (hot-to-cold) does not mirror patterns of latitudinal divergence (south-to-north), as might have been expected under a scenario with temperature as the major agent of selection. Our findings illustrate the importance of detailed phylogeographic information when interpreting geographic clines of dispersal traits in an adaptive evolutionary framework.

Critical weight mediates sex-specific body size plasticity and sexual dimorphism in the yellow dung fly Scathophaga stercoraria (Diptera: Scathophagidae).

Ultimate factors driving insect body size are rather well understood, while-apart from a few model species-the underlying physiological and developmental mechanisms received less attention. We investigate the physiological basis of adaptive size variation in the yellow dung fly Scathophaga stercoraria, which shows pronounced male-biased sexual size dimorphism and strong body size plasticity. We estimate variation of a major physiological threshold, the critical weight, which is the mass at which a larva initiates pupariation. Critical weight was associated with sexual size dimorphism and sex-specific plasticity, and is thus a likely target of selection on adult size. Detailed larval growth trajectories derived from individuals raised at two food and temperature treatments further reveal that sex-specific size plasticity is mediated by faster initial growth of males that later becomes reduced by higher male weight loss during the wandering stage. We further demonstrate that integral growth rates, which are typically calculated as simple ratios of egg-to-adult development time and adult weight, do not necessarily well reflect variation in instantaneous growth rates. We illustrate the importance of detailed assessments of ontogenetic growth trajectories for the understanding of adaptive size variation and discuss the mechanistic basis of size determination in shaping sex-specific phenotypic plasticity.

Stage- and sex-specific heat tolerance in the yellow dung fly Scathophaga stercoraria.

Thermal tolerance varies at all hierarchical levels of biological organization: among species, populations, individuals, and even within individuals. Age- or developmental stage- and sex-specific thermal effects have received relatively little attention in the literature, despite being crucial for understanding thermal adaptation in nature and responses to global warming. We document stage- and sex- specific heat tolerance in the yellow dung fly Scathophaga stercoraria (Diptera: Scathophagidae), a species common throughout the northern hemisphere that generally favours cool climates. Exposure of eggs to temperatures up to 32°C did not affect larval hatching rate, but subsequent egg-to-adult survival at a benign temperature was reduced. Permanent transfer from benign (18°C) to hot temperatures (up to 31°C) at different larval and pupal stages strongly decreased egg-to-adult survival, though survival continuously improved the later the transfer occurred. Temporary transfer for only two days increased mortality more weakly, survival being lowest when temperature stress was imposed early during the larval or pupal stages. Adult flies provided with sugar and water tolerated 31°C longer than previously thought (5 days in males to 9 days in females). Eggs were thus less susceptible to thermal stress than larvae, pupae or adults, in agreement with the hypothesis that more mobile stages require less physiological protection against heat because they can behaviourally thermoregulate. The probability of mating, of laying a clutch, and hatching success were generally independently reduced by exposure of females or males to warm temperatures (24°C) during the juvenile or adult stages, with some interactions evident. High temperature stress thus affects survival differentially depending on when it occurs during the juvenile or the pre-reproductive adult life stage, and affects reproductive success via the mating behaviour of both sexes, female physiology in terms of oviposition, and fertility via sperm and/or egg quality. Our results illustrate that temperature stress, even when moderate and temporary, during early development can have profound lethal and non-lethal fitness-consequences later in life.

Ivermectin sensitivity is an ancient trait affecting all ecdysozoa but shows phylogenetic clustering among sepsid flies.

Avermectins are potent and popular veterinary pharmaceuticals used globally to fight parasites of livestock and humans. By disturbing ion channel transport through the membrane, avermectins are effective against endo- and ectoparasitic round and horsehair worms (Nematoida), insects, or ticks (Arthropoda), but not against Plathelminthes, including flatworms (Trematoda) and tapeworms (Cestoda), or segmented worms (Annelida). Unfortunately, excreted avermectins have strong nontarget effects on beneficial arthropods such as the insect community decomposing livestock dung, ultimately impeding this important ecosystem function to the extent that regulators mandate standardized eco-toxicological tests of dung organisms worldwide. We show that the ancient phylogenetic pattern and qualitative mechanism of avermectin sensitivity is conserved and compatible with most recent phylogenomic hypotheses grouping the Nematoida with the Arthropoda as Ecdysozoa (molting animals). At the species level, we demonstrate phylogenetic clustering in ivermectin sensitivities of 23 species of sepsid dung flies (Diptera: Sepsidae). This clustered 500-fold quantitative variation in sensitivity may indicate recent lineage-specific responses to selection, but more likely reflects pre-existing genetic variation with pleiotropic effects on eco-toxicological responses to pollutants. Regardless, our results question the common practice in eco-toxicology of choosing single test species to infer detrimental effects on entire species communities, which should ideally assess a representative taxonomic sample.

Standardized laboratory tests with 21 species of temperate and tropical sepsid flies confirm their suitability as bioassays of pharmaceutical residues (ivermectin) in cattle dung.

Veterinary pharmaceuticals excreted in the dung of treated livestock can have strong non-target effects on the dung organism community. We report results of ecotoxicological tests with ivermectin for 21 species of temperate (Europe, North America) and tropical (Asia, Central America) black scavenger flies (Diptera: Sepsidae), using standardized methods developed previously for the yellow dung fly and the face fly. Our study documents great variation in ivermectin sensitivity of more than two orders of magnitude among species and even populations within species: estimated lethal effect concentrations LC(50) (at which 50% of the flies died) ranged from 0.05 to 18.55 µg/kg dung fresh weight (equivalent to 0.33-132.22 µg/kg dung dry weight). We also show that controlled laboratory tests can--within reasonable limits-be extended to the field or to laboratory settings without climate control, as obtained LC(50) were roughly similar. In addition to lethal effects, our study revealed relevant sub-lethal effects at lower ivermectin concentrations in terms of prolonged development, smaller body size and reduced juvenile growth rate. Finally, oviposition choice experiments showed that females generally do not discriminate against dung containing ivermectin residues. We conclude that sepsid flies are well suited test organisms for pharmaceutical residues in the dung of livestock due to their ease and speed of rearing and handling, particularly in the tropics, where high-tech laboratory equipment is often not available.

Sexual selection accounts for the geographic reversal of sexual size dimorphism in the dung fly, sepsis punctum (Diptera: Sepsidae).

Sexual size dimorphism (SSD) varies widely across and within species. The differential equilibrium model of SSD explains dimorphism as the evolutionary outcome of consistent differences in natural and sexual selection between the sexes. Here, we comprehensively examine a unique cross-continental reversal in SSD in the dung fly, Sepsis punctum. Using common garden laboratory experiments, we establish that SSD is male-biased in Europe and female-biased in North America. When estimating sexual (pairing success) and fecundity selection (clutch size of female partner) on males under three operational sex ratios (OSRs), we find that the intensity of sexual selection is significantly stronger in European versus North American populations, increasing with male body size and OSR in the former only. Fecundity selection on female body size also increases strongly with egg number and weakly with egg volume, however, equally on both continents. Finally, viability selection on body size in terms of intrinsic (physiological) adult life span in the laboratory is overall nil and does not vary significantly across all seven populations. Although it is impossible to prove causality, our results confirm the differential equilibrium model of SSD in that differences in sexual selection intensity account for the reversal in SSD in European versus North American populations, presumably mediating the ongoing speciation process in S. punctum.

Lethal and sublethal toxic effects of a test chemical (ivermectin) on the yellow dung fly (Scathophaga stercoraria) based on a standardized international ring test.

A standardized bioassay using the yellow dung fly, Scathophaga stercoraria L. (Diptera: Scathophagidae), was developed to test the lethal and sublethal toxicity of parasiticide residues in livestock dung. The repeatability of the bioassay was assessed for the parasiticide ivermectin in 13 tests performed by seven laboratories in Germany, the United Kingdom, Switzerland, and Canada. Test results had an acceptable range of heterogeneity. The calculated median effective concentration for 50% (EC50) egg-to-adult mortality was 20.9 +/- 19.1 microg ivermectin/kg dung fresh weight (FW) (mean +/- standard deviation; range, 6.33-67.5 microg/kg). Mortality was not observed below a calculated no-observable-effect concentration (NOEC) of 8.1 +/- 7.7 microg/kg FW. However, prolonged development time (and, in a subset of tests, reduced body size) was observed above a calculated NOEC of 0.8 +/- 0.8 microg/kg FW. An oviposition site choice test revealed that yellow dung fly females do not discriminate among dung of different ivermectin concentrations. Thus, the yellow dung fly is suitably sensitive, and the methods are sufficiently repeatable, to support use of this standardized bioassay by the international community in the registration of new veterinary pharmaceuticals.

Development of polymorphic microsatellite markers for the dung fly (Sepsis cynipsea).

The polyandrous fly Sepsis cynipsea has been used extensively in studies of sexual selection and local adaptation. We isolated and characterized 11 novel microsatellite markers for S. cynipsea from a genomic library and screened 32 flies for polymorphism. All microsatellite markers show high allelic diversity with an average of 9.64 alleles per locus. Two microsatellites were found likely to be X-linked. These novel markers will significantly advance studies of sexual selection and evolutionary genetics of S. cynipsea and related species, especially given the low numbers of markers currently available in this family.