Unlocking the "Black box": internal female genitalia in Sepsidae (Diptera) evolve fast and are species-specific

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)

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 di- morphism 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 labo- ratory 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

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

Size rather than complexity of sexual ornaments prolongs male metamorphosis and explains sexual size dimorphism in sepsid flies

Male sexual ornaments often evolve rapidly and are thought to be costly, thus contributing to sexual size dimorphism. However, little is known about their developmental costs, and even less about costs associated with structural complexity. Here, we quantified the size and complexity of three morphologically elaborate sexually dimorphic male ornaments that starkly differ across sepsid fly species (Diptera: Sepsidae): (i) male forelegs range from being unmodified, like in most females, to being adorned with spines and large cuticular protrusions; (ii) the fourth abdominal sternites are either unmodified or are converted into complex de novo appendages; and (iii) male genital claspers range from small and simple to large and complex (e.g. bifurcated). We tracked the development of 18 sepsid species from egg to adult to determine larval feeding and pupal metamorphosis times of both sexes. We then statistically explored whether pupal and adult body size, ornament size and/or ornament complexity are correlated with sex-specific development times. Larval growth and foraging periods of male and female larvae did not differ, but the time spent in the pupal stage was ca 5% longer for sepsid males despite emerging 9% smaller than females on average. Surprisingly, we found no evidence that sexual trait complexity prolongs pupal development beyond some effects of trait size. Evolving more complex traits thus does not incur developmental costs at least in this system

Metabarcoding mosquitoes: MinION sequencing of bulk samples gives accurate species profiles for vector surveillance (Culicidae)

Mosquitoes (Family: Culicidae) are dominant vectors of pathogens, and their surveillance has been incorporated into major disease control programs worldwide. However, routine, species-level identification of mosquitoes is often a bottleneck for management, and Next Generation Sequencing (NGS) platforms and DNA metabarcoding can revolutionize this process. MinION nanopore technologies promise on-site sequencing and rapid sample processing rates ideal for time-sensitive biosurveillance. Here, we benchmark the results of DNA metabarcoding on the MinION against the Illumina MiSeq platform, which is known for its higher sequencing accuracy. We used metazoan COI mini-barcode primers to carry out DNA metabarcoding of mosquito bulk samples caught during a real vector survey, then compared the mosquito species profiles recovered on each sequencing platform. We also tested the influence of using different trap lures, storage methods, and pooling different specimen body parts on the number of species recovered. We report that mosquito species-level identifications were highly congruent between MinION and Illumina (93% overlap). We also find that CO2 gas cylinders outperformed biogenic CO2 sources significantly, by two-fold. Notably, we demonstrated the feasibility of detecting zoonotic reservoirs and pathogen signals from mosquito bulk samples. We present the first use of DNA metabarcoding on the MinION for vector surveillance and discuss future applications

Morphology, molecules and mating behavior: an integrative study of population divergence and speciation in widespread sepsid flies (Sepsidae: Diptera)

Europäische und nordamerikanische Populationen der weit verbreiteten Schwingfliege Sepsis punctum, gehören zur gleichen Art, unterscheiden sich aber im Paarungsverhalten und Geschlechtsdimorphismus, eine in dieser Ausprägung bislang einzigartige Situation. Experimentelle Laboruntersuchungen zeigten, dass der Paarungsvorteil grosser Männchen in Europa sehr stark ist, während Weibchen in Nordamerika einen Balztanz bevorzugen und die Grösse weniger wichtig ist. Mittels molekularer Methoden konnte starke populationsgenetische Differenzierung dokumentiert werden. Genetische Isolation und systematische Unterschiede im sexuellen Selektionsdruck auf Verhalten und Morphologie erklären die kontinentalen Unterschiede zwischen den Populationen und weisen auf Artbildung hin. General summary This work explores processes of selection and speciation acting on diverging populations in widespread sepsid flies. The main focus was on Sepsis punctum where European and North American populations differed greatly in mating behavior and sexual size dimorphism, that is unique to this species. Laboratory experiments show that large male body size is a strong mating advantage in Europe but in North America females prefer a courtship behavior and male size is less important. Molecular methods also suggest that there is strong genetic differences between populations. This integrative research, including detailed behavioral, morphological and molecular data, shows that genetic isolation and systematic differences in selection on reproductive traits explain the continental differences in this species

Behavioural barriers to reproduction may evolve faster than sexual morphology among populations of a dung fly (Sepsidae)

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 <1 %), suggesting that marked behavioural differences between both populations have evolved quickly but without complete isolation

A comparative study of mating behaviour and sexual dimorphisms in sepsidae (Dlptera)

Master'sMASTER OF SCIENC

Rapid genomic evolution drives the diversification of male reproductive genes in dung beetles

10.1093/gbe/evab172Genome Biology and Evolutio

Sexual selection on male size drives the evolution of male-biased sexual size dimorphism via the prolongation of male development

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