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Phenomenological and behavioural studies have greatly advanced the study of natural selection. Field studies of selection well appraise the natural situation, but is this also true for laboratory studies, which are typically more mechanistic? We compared precopulatory sexual selection (mating differential based on pairing success) in field and laboratory of several closely related, ecologically similar black scavenger dung flies (Diptera: Sepsidae). Selection on fore femur (sexual trait) and wing size (nonsexual trait) and shape varied considerably among seven species and continental populations in agreement with variation in their mating system and sexual size dimorphism. Selection on trait size was mostly positive or nil, but never significantly negative, implying mating advantages of large males in most species. Strongest selection was found in species/populations with male-biased size dimorphism, associating evolutionary shifts from female- to male-biased dimorphism with intensified sexual selection for large male size by adding male –male competition to a mating system previously driven primarily by female choice. Although sexual selection on shape was closely aligned with allometric shape variation, selection on fore femur shape was more consistent than selection on wing shape, which was absent in most species. Sexual selection intensities, but not necessarily the underlying behavioural mechanisms, were overall similar in field and laboratory, suggesting that laboratory assessments well represent the natural situation. If this conclusion can be generalized, it would lend credence to the strategy of using controlled laboratory mating studies to better understand natural selection, behaviour and ecology, at least for smaller animals that can be held in captivity.

BackgroundOxford Nanopore Technologies (ONT) long-read transcriptomes offer many advantages including long reads (>10kbp), end-to-end transcripts, structural variants, isoform-level resolution of genes and expression. However, uptake of ONT transcriptomics is still low, largely due to high error rates (2 to 13%) and reliance on reference databases that are unavailable for many non-model species. Additionally, bioinformatics tools and pipelines for de novo ONT transcriptomics are still in early stages of development. ResultsHere, we use de novo ONT GridION transcriptomics to discover novel genes from the male accessory glands (AG) of a widespread, non-model dung fly, Sepsis punctum. Insect AGs are of particular interest for this as they are hotspots for rapid evolution of novel reproductive genes, and they synthesize seminal fluid proteins that lack homology to any other known proteins. We implement a completely de novo ONT GridION transcriptome pipeline, incorporating quality-filtering and rigorous error-correction procedures, to characterize this novel gene set and to quantify their expression. Specifically, we compare these ONT genes and their expression against de novo lllumina HiSeq transcriptome data. We find 40 high-quality and high-confidence ONT genes that cross-verify against Illumina genes; twenty-six of which are novel and specific to S. punctum. Read count based expression quantification in ONT samples is highly congruent with Illumina’s Transcript per Million (TPM), both in overall pattern and within functional categories. Novel genes account for an average of 81% of total gene expression underscoring their functional importance in S. punctum AGs. Eighty percentage of these genes are secretory in nature, responsible for 74% total gene expression. Notably, median sequence similarities of ONT nucleotide and protein sequences match within-Illumina sequence similarities indicating that our de novo ONT transcriptome pipeline successfully mitigated sequencing errors. ConclusionsThis is the first study to adapt ONT transcriptomics for completely de novo characterization of novel genes in animals. Our study demonstrates that ONT long-reads, constituting a quarter of the number of bases sequenced at less than a third the cost of Illumina reads, can be a resource-friendly and cost-effective solution for end-to-end sequencing of unknown genes even in the absence of a reference database.

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.

The molecular basis for the evolution of novel phenotypes is a central question in evolutionary biology. In recent years, dung beetles have emerged as models for novel trait evolution as they possess distinct precopulatory traits such as sexually dimorphic horns on their head and thorax. Here, we use functional and evolutionary genomics to investigate the origins and the evolution of postcopulatory reproductive traits in male dung beetles. Male ejaculates that underlie postcopulatory sexual selection are excellent candidates to study novel trait evolution as they are complex, fast evolving, and often highly divergent in insects. We assemble de novo transcriptomes of male accessory glands and testes of a widespread dung beetle, Catharsius molossus, and we perform an evolutionary analysis of closely and distantly related insect genomes. Our results show there is rapid innovation at the genomic level even among closely related dung beetles. Genomic expansion and contraction drive the divergence of male reproductive traits and their functions. The birth of scores of completely novel reproductive genes is reinforced by the recruitment of these genes for high expression in male reproductive tissues, especially in the accessory glands. We find that male accessory glands of C. molossus are specialized for secretory function and express female, egg, and embryo-related genes as well as serine protease inhibitors, whilst the testes are specialized for spermatogenesis and sperm function. Finally, we touch upon putative functions of these evolutionary novelties using structure-function analysis as these proteins bear no homology to any other known proteins.

Variation in biotic and abiotic factors among populations affects individual behaviors by transforming the social landscape and shaping mating systems. Consequently, describing behaviors in natural populations requires consideration of the biological and physical factors that different individuals face. Here, we examined variation in socio-sexual and locomotor behaviors in a small, livebearing, freshwater fish, the pygmy halfbeak Dermogenys collettei, across natural populations in Singapore. The pygmy halfbeak is a surface feeding fish that spends most of the time near the water surface, making it ideal for non-invasive behavioral observations. We compared behaviors between sexes among 26 shoals while simultaneously accounting for environmental variation. We demonstrated that sexual interactions and locomotor behaviors differed among shoals with varying levels of canopy cover and water flow. Specifically, in areas with greater canopy cover, sexual interactions decreased, whereas time spent in a stationary position increased. Sexual interactions were more numerous in still water, where fish spent less time swimming. Variation in the expression of socio-sexual and locomotor behaviors were not associated with differences in the amount of aquatic vegetation, water depth or halfbeak shoal size. Agonistic interactions were robust to environmental effects, showing little variation among environments. However, there were strong sex effects, with males performing more agonistic behaviors and spending less time in a stationary position compared to females, regardless of the environment. Moreover, sexual interactions, measured as actively performed by males and passively received by females, were on average more frequent in males than in females. Our findings help us explore the proximal causes of intraspecific behavioral variation and suggest that fundamental information on socio-sexual behaviors from wild populations can lead to a better understanding of how sexual selection operates when the strength of natural selection varies across environments.

Background Wolbachia are intracellular bacterial endosymbionts found in most insect lineages. In mosquitoes, the influence of these endosymbionts on host reproduction and arboviral transmission has spurred numerous studies aimed at using Wolbachia infection as a vector control technique. However, there are several knowledge gaps in the literature and little is known about natural Wolbachia infection across species, their transmission modes, or associations between various Wolbachia lineages and their hosts. This study aims to address these gaps by exploring mosquito-Wolbachia associations and their evolutionary implications. Methods We conducted tissue-specific polymerase chain reaction screening for Wolbachia infection in the leg, gut and reproductive tissues of wild mosquitoes from Singapore using the Wolbachia surface protein gene (wsp) molecular marker. Mosquito-Wolbachia associations were explored using three methods—tanglegram, distance-based, and event-based methods—and by inferred instances of vertical transmission and host shifts. Results Adult mosquitoes (271 specimens) representing 14 genera and 40 species were screened for Wolbachia. Overall, 21 species (51.2%) were found positive for Wolbachia, including five in the genus Aedes and five in the genus Culex. To our knowledge, Wolbachia infections have not been previously reported in seven of these 21 species: Aedes nr. fumidus, Aedes annandalei, Uranotaenia obscura, Uranotaenia trilineata, Verrallina butleri, Verrallina sp. and Zeugnomyia gracilis. Wolbachia were predominantly detected in the reproductive tissues, which is an indication of vertical transmission. However, Wolbachia infection rates varied widely within a mosquito host species. There was no clear signal of cophylogeny between the mosquito hosts and the 12 putative Wolbachia strains observed in this study. Host shift events were also observed. Conclusions Our results suggest that the mosquito-Wolbachia relationship is complex and that combinations of transmission modes and multiple evolutionary events likely explain the observed distribution of Wolbachia diversity across mosquito hosts. These findings have implications for a better understanding of the diversity and ecology of Wolbachia and for their utility as biocontrol agents. Graphical Abstract

Sexual size dimorphism (SSD) arises when the net effects of natural and sexual selection on body size differ between the sexes. Quantitative SSD variation between taxa is common, but directional intraspecific SSD reversals are rare. We combined micro- and macroevolutionary approaches to study geographic SSD variation in closely related black scavenger flies. Common garden experiments revealed stark intra- and interspecific variation: Sepsis biflexuosa is monomorphic across the Holarctic, while S. cynipsea (only in Europe) consistently exhibits female-biased SSD. Interestingly, S. neocynipsea displays contrasting SSD in Europe (females larger) and North America (males larger), a pattern opposite to the geographic reversal in SSD of S. punctum documented in a previous study. In accordance with the differential equilibrium model for the evolution of SSD, the intensity of sexual selection on male size varied between continents (weaker in Europe), whereas fecundity selection on female body size did not. Subsequent comparative analyses of 49 taxa documented at least six independent origins of male-biased SSD in Sepsidae, which is likely caused by sexual selection on male size and mediated by bimaturism. Therefore, reversals in SSD and the associated changes in larval development might be much more common and rapid and less constrained than currently assumed.

Post-copulatory sexual selection (PSS), fuelled by female promiscuity, is credited with the rapid evolution of sperm quality traits across diverse taxa1. Yet, our understanding of the adaptive significance of sperm ornaments and the cryptic female preferences driving their evolution is extremely limited1,2. Here we review the evolutionary allometry of exaggerated sexual traits (for example, antlers, horns, tail feathers, mandibles and dewlaps), show that the giant sperm of some Drosophila species are possibly the most extreme ornaments3,4 in all of nature and demonstrate how their existence challenges theories explaining the intensity of sexual selection, mating-system evolution and the fundamental nature of sex differences5–9. We also combine quantitative genetic analyses of interacting sex-specific traits in D. melanogaster with comparative analyses of the condition dependence of male and female reproductive potential across species with varying ornament size to reveal complex dynamics that may underlie sperm-length evolution. Our results suggest that producing few gigantic sperm evolved by (1) Fisherian runaway selection mediated by genetic correlations between sperm length, the female preference for long sperm and female mating frequency, and (2) longer sperm increasing the indirect benefits to females. Our results also suggest that the developmental integration of sperm quality and quantity renders post-copulatory sexual selection on ejaculates unlikely to treat male–male competition and female choice as discrete processes.

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.

Reproductive traits often evolve rapidly, and some suggest that behavioural traits, in particular, can diverge faster than morphology, resulting in sexual isolation between populations/species. An earlier study of a Neotropical dung fly, Archisepsis diversiformis (Diptera: Sepsidae), reported anecdotally that two central American populations that were approximately 500 km apart (Costa Rica and Panama) differed in male courtship behaviour despite being morphologically similar. Here, I present results of an in-depth study designed (1) to test whether these two populations show qualitative and/or quantitative differences in mating behaviour and morphology, (2) to test whether individuals from either population show some degree of reproductive isolation and (3) to characterize population variation in a particularly fast evolving mitochondrial gene fragment, cytochrome oxidase c subunit I (COI), to estimate genetic differences between the two populations. Despite similarities in overall courtship, I identified behaviours that were clearly population specific, and report that both populations showed strong premating isolation in one-on-one crosses. However, after extended exposure in mass-container group crosses, individuals did produce adult F1 offspring, suggesting that isolation is incomplete. Surprisingly, morphometric analyses indicated that these two populations differed significantly in sexually monomorphic adult wing shape but differed only moderately in sexually dimorphic male forefemur shape, and not at all in male genital clasper shape. Finally, both populations were genetically similar, forming a single, monophyletic cluster with low uncorrected pairwise distances (COI threshold

Vertically transmitted reproductive parasites are both extraordinarily widespread and diverse in their effects on their invertebrate hosts. In addition to causing skewed population sex ratios via male-killing or feminization, such bacteria can further cause cytoplasmic incompatibility or parthenogenesis. Previous surveys show that the microbes Wolbachia and Spiroplasma are common in some dipteran families, e.g. Drosophilidae or Scathophagidae, and are known to be heritable symbionts and affect reproduction in the Diptera. However, little is known of Rickettsia infections and detailed surveys targeting other Dipteran families are lacking. Here 329 samples of 247 species of Diptera belonging to the Dolichopodidae, Empididae, and Hybotidae (superfamily Empidoidea) are surveyed for the presence of the endosymbionts Wolbachia, Spiroplasma, and Rickettsia. The superfamily Empidoidea contains numerous species, which have been the targets of intense research concerning reproductive traits involved in sexual selection. 151 of the species (i.e. ca. 61%) screened here, including species from key genera such as Dolichopus, Poecilobothrus or Empis, harboured one or more symbionts. Reproductive parasites are thus also common in the Empidoidae, yet effects on hosts remain unclear. Potential endosymbiont-host interactions in this group would hence be worthy of further investigation.

Theory predicts that males have a limited amount of resources to invest in reproduction, suggesting a trade-off between traits that enhance mate acquisition and those that enhance fertilization success. Here, we investigate the relationship between pre- and post-copulatory investment by comparing the mating behaviour and reproductive morphology of four European and five North American populations of the dung fly Sepsis punctum (Diptera) that display a reversal of sexual size dimorphism (SSD). We show that the geographic reversal in SSD between the continents (male biased in Europe, female biased in North America) is accompanied by differential investment in pre- vs. post-copulatory traits. We find higher remating rates in European populations, where larger males acquire more matings and consequently have evolved relatively larger testes and steeper hyper-allometry with body size. American populations, in sharp contrast, display much reduced, if any, effect of body size on those traits. Instead, North American males demonstrate an increased investment in mate acquisition prior to copulation, with more mounting attempts and a distinctive abdominal courtship display that is completely absent in Europe. When controlling for body size, relative female spermathecal size is similar on both continents, so we find no direct evidence for the co-evolution of male and female internal reproductive morphology. By comparing allopatric populations of the same species that apparently have evolved different mating systems and consequently SSD, we thus indirectly demonstrate differential investment in pre- vs. post-copulatory mechanisms increasing reproductive success.

The males of almost all sepsid species have strongly modified forelegs that are used to clamp the female's wingbase during mounting. Here, we describe a new species in the genus Perochaeta whose males have unmodified forelegs. We use DNA sequence data for ten genes to reconstruct the position of Perochaeta on the phylogenetic tree for Sepsidae, and reveal that the lack of foreleg armature in Perochaeta dikowi sp.n. is secondary. Through the study of the mating behaviour of the new species, we demonstrate that the loss of armature is correlated with a new mounting technique during which the males of P. dikowi do not use the foreleg to clamp the female's wingbase. Instead, the male approaches the female from behind and bends his abdomen forwards in order to establish genital contact. Our study shows how data from morphology, phylogenetics, and behavioural biology can complement each other to yield a deeper understanding of how changes in morphology and behaviour are correlated.

The complex, species-specific foreleg armature in males of the genus Themira (Diptera: Sepsidae) provides an ideal system for testing competing hypotheses for the evolution of sexually dimorphic character divergence. In sepsid flies, the male holds onto the female by clasping her wing base with his modified forelegs. In the present study, we document the male leg and the female wing morphology using scanning electron microscopy and confocal microscopy. We use a phylogenetic tree for Themira to reconstruct male foreleg and female wing evolution and demonstrate that the male legs have evolved elaborate structures with little or no corresponding changes in wing morphology. This lack of interspecific variation in female wings is not in agreement with the hypothesis of a morphological ‘evolutionary arms race’ between males and females. However, there is also no evidence for sex-specific wing differences in sensory organs on the wing base that may explain how females could assess males according to Eberhard's ‘cryptic female choice’ hypothesis. Finally, our study reveals the function of several novel morphological clasping structures and documents that the male foreleg characters in Themira are highly homoplastic. Male forelegs in two clades evolve considerably faster than in other species or clades. These two clades include Themira superba and Themira leachi, species that have some of the most dramatically modified forelegs known in Diptera. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 93, 227–238.

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.

Maternally transmitted reproductive parasites such as Wolbachia and Cardinium can drastically reshape reproduction in their hosts. Beyond skewing sex ratios towards females, these microbes can also cause cytoplasmic incompatibility. Wolbachia probably infects two thirds of insects, but far less is known about the occurrence or action of other bacteria with potentially similar effects. In contrast with the two more widespread reproductive parasites, Wolbachia and Spiroplasma, far less is known of infections with Cardinium (Bacteroidetes) and possible consequences in the Diptera. Here, in an extensive survey, 244 dipteran species from 67 genera belonging to the Dolichopodidae, Empididae, and Hybotidae were assessed for the presence of the microbe Cardinium. Although 130 of the species screened tested positive (ca. 53%), the presence of Cardinium could only be confirmed in 10 species (ca. 4%) based on analysis of sequences. Numerous additional sequences were found to be assignable to known or unknown Bacteroidetes. Considering the known issues concerning specificity of Cardinium primers and the phylogenetic uncertainties surrounding this microbe, the actual prevalence of this symbiont is worthy of further scrutiny. Potential directions for future research on Cardinium-host interactions in Diptera and in general are discussed.

A standardized bioassay previously developed with ivermectin for the yellow dung fly (Scathophagidae) and the face fly (Muscidae) was applied to test the response of 11 dung fly species to the presumably less toxic parasiticide moxidectin. The results were compared to existing data for the same species tested with ivermectin, albeit two new species (Scathophaga suilla and Musca domestica) were tested here with both the substances. Estimated lethal effect concentrations LC50 at which 50% of the flies died ranged more than tenfold from 0.012 mg moxidectin/kg fresh dung for Sepsis neocynipsea (Sepsidae) to 0.140 mg moxidectin/kg fresh dung for the house fly Musca domestica (Muscidae). In most of the species, we additionally revealed sub-lethal effects at lower moxidectin concentrations in terms of retarded growth and development and reduced body size. Mortality thresholds were about ten times higher for moxidectin than for ivermectin, hence moxidectin is indeed less toxic than ivermectin in absolute terms. Crucially, we obtained strong correlations among the 11 tested fly species in both lethal and sub-lethal responses to the two substances, such that species relatively sensitive to ivermectin were also relatively sensitive to moxidectin. Such correlations are expected if the two substances are structurally related and function in the same manner by disturbing ion channel transport. Methodologically speaking, all species used proved suitable for toxicological testing of parasiticides.

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.

Background The species-specificity of male genitalia has been well documented in many insect groups and sexual selection has been proposed as the evolutionary force driving the often rapid, morphological divergence. The internal female genitalia, in sharp contrast, remain poorly studied. Here, we present the first comparative study of the internal reproductive system of Sepsidae. We test the species-specificity of the female genitalia by comparing recently diverged sister taxa. We also compare the rate of change in female morphological characters with the rate of fast-evolving, molecular and behavioral characters. Results We describe the ectodermal parts of the female reproductive tract for 41 species representing 21 of the 37 described genera and define 19 morphological characters with discontinuous variation found in eight structures that are part of the reproductive tract. Using a well-resolved molecular phylogeny based on 10 genes, we reconstruct the evolution of these characters across the family [120 steps; Consistency Index (CI): 0.41]. Two structures, in particular, evolve faster than the rest. The first is the ventral receptacle, which is a secondary sperm storage organ. It accounts for more than half of all the evolutionary changes observed (7 characters; 61 steps; CI: 0.46). It is morphologically diverse across genera, can be bi-lobed or multi-chambered (up to 80 chambers), and is strongly sclerotized in one clade. The second structure is the dorsal sclerite, which is present in all sepsids except Orygma luctuosum and Ortalischema albitarse. It is associated with the opening of the spermathecal ducts and is often distinct even among sister species (4 characters; 16 steps; CI: 0.56). Conclusions We find the internal female genitalia are diverse in Sepsidae and diagnostic for all species. In particular, fast-evolving structures like the ventral receptacle and dorsal sclerite are likely involved in post-copulatory sexual selection. In comparison to behavioral and molecular data, the female structures are evolving 2/3 as fast as the non-constant third positions of the COI barcoding gene. They display less convergent evolution in characters (CI = 0.54) than the third positions or sepsid mating behavior (CICOI = 0.36; CIBEHAV = 0.45).

Our understanding of how fast mating behaviour evolves in insects is rather poor due to a lack of comparative studies among insect groups for which phylogenetic relationships are known. Here, we present a detailed study of the mating behaviour of 27 species of Sepsidae (Diptera) for which a well-resolved and supported phylogeny is available. We demonstrate that mating behaviour is extremely diverse in sepsids with each species having its own mating profile. We define 32 behavioural characters and document them with video clips. Based on sister species comparisons, we provide several examples where mating behaviour evolves faster than all sexually dimorphic morphological traits. Mapping the behaviours onto the molecular tree reveals much homoplasy, comparable to that observed for third positions of mitochondrial protein-encoding genes. A partitioned Bremer support (PBS) analysis reveals conflict between the molecular and behavioural data, but behavioural characters have higher PBS values per parsimony-informative character than DNA sequence characters.

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 thirteen tests performed in seven laboratories in Germany, the UK, Switzerland and Canada. Test results had an acceptable range of heterogeneity. The calculated concentration at which 50% egg-adult mortality was observed (Effect Concentration (EC50) averaged 20.8 +/- 19.1 (Standard Deviation (SD)) microg ivermectin / kg fresh dung (range: 6.33 - 67.5). Mortality was not observed below an average calculated No Observable Effect Concentration (NOEC) of 8.1 +/- 7.7 microg / kg. However, prolonged developmental time, and in a subset of tests reduced body size, was observed above an average calculated NOEC of 0.8 +/- 0.8 microg / kg. An oviposition choice test revealed further that yellow dung fly females do not discriminate among dung of different ivermectin concentrations. We conclude that the yellow dung fly is suitably sensitive, and the methods sufficiently repeatable, to support use of this standardized bioassay by the international community in the registration of new veterinary pharmaceuticals.

Due to their interesting biology, conspicuous sexual dimorphism and the ability to conduct experiments on species that breed under laboratory condition, sepsid flies (Diptera : Sepsidae) are becoming increasingly important model organisms in evolutionary biology. Accurate species boundaries and well supported phylogenetic hypotheses are thus of interest to many biologists. Here we resolve the conflict surrounding the taxonomic status of the European Sepsis nigripes Meigen, 1826, which is shown to be a valid species using morphological and molecular data applied to multiple species concepts. The species is also placed onto a phylogenetic tree for the genus Sepsis that includes most European and North American species. In addition, we assess the genetic variability between two populations of the Holarctic Sepsis luteipes Melander & Spuler, 1917 from Europe and North America and find conflicting evidence between morphology and DNA sequences. Different species concepts here yield different inferences, and if two species were to be accepted based on molecular data, Sepsis helvetica Munari, 1985 from Europe would have to be resurrected from synonymy. We provide high-resolution images for all species in order to aid in accurate identification. Both species are also added to Sepsidnet, the digital reference collection for Sepsidae (http://sepsidnet-rmbr.nus.edu.sg). Lastly, we discuss a field site in the Swiss Alps where 12 species of Sepsis occur sympatrically on the same pasture.