Traumatic insemination refers to mating in which males pierce the female’s integument with his penis for insemination. Strepsiptera are often listed as an example for this mode of copulation. However, while traumatic insemination in Mengenillidae with free-living females is undisputed, its occurrence in Stylopidia with permanent endoparasitic females – 97% of the known species of Strepsiptera – has remained unclear. Rather, observations from a single study on Xenos vesparum (Xenidae) that questioned traumatic insemination in this species became generalized for Stylopidia. Here we show that integration of data from various imaging methods provides convincing evidence for traumatic insemination being phylogenetically widespread in Strepsiptera. Specifically, we provide the first evidence of injury wounds from traumatic insemination in species of Mengenillidae, Corioxenidae, Elenchidae, Halictophagidae and Xenidae. Using three-dimensional models of copulating pairs of Stylops ovinae (Stylopidae) and X. vesparum, we visualize the physical piercing of the female’s integument by the male’s penis. Finally, we show in species of Mengenillidae, Xenidae and Stylopidae that traumatic mating is associated with the injection of sperm in the female’s haemocoel. Our results significantly alter the understanding of the reproductive biology of Strepsiptera and imply that traumatic insemination has been the ancestral mode of copulation and retained in most, if not all, extant families.

The morphology of mature larvae of Sisyra nigra was studied and documented with a broad spectrum of techniques. Special emphasis is on the cephalic anatomy and on the digestive tract. Cephalic structures are highly modified, with numerous autapomorphic conditions, including a globular head capsule, an extended area with large cornea lenses, a massive tentorium, a strongly developed prepharyngeal pumping apparatus with a horizontal arrangement of dilators, a sharp bend between the prepharynx and pharynx, and an unusual filter apparatus at the entrance of the large crop. The thoracic and abdominal muscle sets, and the legs are largely unmodified. Postcephalic apomorphies are conspicuous tergal setiferous tubercles, trifid setiferous pleural projections, single pretarsal claws, zigzag‐shaped abdominal tracheal gills, and a dense vestiture of setae on the terminal abdominal segments. Mandibulo‐maxillary stylets curved outwards are an unusual apomorphy also found in the semiaquatic larvae of Osmylidae. Semiaquatic or aquatic habits and secondarily multisegmented antennae are potential synapomorphies of these two groups and Nevrorthidae (Osmyloidea). A sistergroup relationship between Sisyridae and Nevrorthidae suggests that fully aquatic habits of larvae may be a synapomorphy of both families. A specialized terminal antennal seta is a potential groundplan apomorphy of Neuroptera, with secondary loss in Nevrorthidae and Ithonidae + Myrmeleontiformia, respectively. A trumpet‐shaped empodium is likely an apomorphy of Neuroptera excluding Coniopterygidae and Osmyloidea, and the secondary loss an apomorphy of Ithonidae on one hand, and Myrmeleontiformia excl. Psychopsidae on the other.

The external and internal morphology of the primary larva of Mantispa aphavexelte was studied and documented with a broad spectrum of techniques. The cephalic anatomy, which is very similar to conditions found in other neuropteran immatures, is mainly affected by the formation of specialized mandibulo-maxillary sucking tubes and associated internal features. The postcephalic anatomy largely follows a generalized holometabolan pattern, with well-developed muscles in the thorax and the abdominal segments, an elongate, uncondensed ganglionic chain, and well-developed Malpighian tubules. The phylogenetic analysis of 70 larval characters suggests a weakly supported clade comprising Dilaridae, Berothidae, Rhachiberothidae and Mantispidae. Mantispoidea comprising Rhachiberothidae, Berothidae and paraphyletic Mantispidae is suggested by overlapping scales on antennae and maxillae, thoracic “trichobothria”, and hypermetamorphosis with scarabaeiform 2nd instar larvae. The relationships of the mantispoid terminals are not resolved, and Mantispa (Mantispinae) and Plega (Symphrasinae) are not retrieved as sister taxa. Spider parasitism and feeding in spider egg sacs is characteristic for Mantispinae, whereas immatures of Berothidae and non-mantispine mantispid larvae prey on the offspring of social or non-social aculeatan Hymenoptera, on termites, or on immatures living in soil or under bark, for example scarab larvae. Specializations of the legs like a trumpet-shaped empodium and a fixed tarsal pseudoclaw, enable mantispine larvae to cope with functional challenges linked with their specialized form of parasitism, like entering a host or penetrating its egg sack. The largely immobilized and straight sucking stylets are suitable for piercing eggs of spiders, but not for grasping prey. The terminal eversible attachment structure probably provides anchorage of the abdominal apex and thus may facilitate the penetration of the egg sac or membranes of the body surface of a spider. It may also facilitate boarding a passing suitable host. Very small size of the 1st instars and hypermetamorphosis are likely linked with parasitism. Even though the primary larvae of M. aphavexelte belong to the smallest immatures in Neuroptera, the effects of miniaturization are very limited.

As the only direct records of the history of evolution, it is critical to determine the geological source of biota-bearing fossils. Through the application of synchrotron-radiation micro-computed tomography (SR-µ-CT), Fourier-transformed infrared-spectroscopy (FT-IR), visual evaluation of ultraviolet fluorescence (UV-VS), radiocarbon dating (¹⁴C quantification), and historical sleuthing, we were able to identify and sort 161 (83 Baltic amber, 71 Copal and 7 Kauri gum pieces) individually numbered and largely mislabeled pieces of East African Defaunation resin (~145 years old) and copal (~390 years old), as well as Baltic amber (~35 million years old) from the Phyletisches Museum collection. Based on this collection, we define two new species: ‡Amphientomum knorrei Weingardt, Bock & Boudinot, sp. nov. (Psocodea: Amphientomidae, copal) and †Baltistena nigrispinata Batelka, Tröger & Bock, sp. nov. (Coleoptera: Mordellidae, Baltic amber). For selected taxa, we provide systematic reviews of the fossil record, including: Amphientomidae, for which we provide a key to all species of Amphientomum, extant and extinct, and recognize the junior synonymy of Am. ectostriolatum Li, 2002 (an unjustified emendation) under Am. ectostriolate Li, 1999 (syn. nov.); the fossil ant genus †Yantaromyrmex and the clades Dorylinae, Plagiolepidini, Camponotus, Crematogaster, and Pheidole (Formicidae); the Nevrorthidae (Neuroptera); and Doliopygus (Coleoptera: Curculionidae: Platypodinae). We synonymize Palaeoseopsis Enderlein, 1925 with Amphientomum Pictet, 1854, syn. nov. and transfer one species from Amphientomum, forming Lithoseopsis indentatum (Turner, 1975), comb. nov. To prevent the uncritical usage of unidentifiable fossils attributed to Camponotus for macroevolutionary analysis, we transfer 29 species to the form genus †Camponotites Steinbach, 1967, which we consider to be most useful as incertae sedis in the Formicinae. We treat †Ctt. ullrichi (Bachmayer, 1960), comb. nov. as unidentifiable hence invalid stat. nov. We also transfer †Ca. mengei Mayr, 1868 and its junior synonym †Ca. igneus Mayr, 1868 to a new genus, †Eocamponotus Boudinot, gen. nov., which is incertae sedis in the Camponotini. Concluding our revision of Camponotus fossils, we transfer †Ca. palaeopterus (Zhang, 1989) to Liometopum (Dolichoderinae), resulting in †L. palaeopterumcomb. nov. and the junior synonymy of †Shanwangella Zhang, 1989, syn. nov. under Liometopum Mayr, 1861. Because the type specimens of the genera †Palaeosminthurus Pierce & Gibron, 1962, stat. rev. and †Pseudocamponotus Carpenter, 1930 are unidentifiable due to poor preservation, we consider these taxa unidentifiable hence invalid stat. nov. To avoid unsupported use of the available fossils names attributed to Crematogaster for divergence dating calibration points, we transfer three species to a new collective taxon that is incertae sedis in Myrmicinae, †Incertogaster Boudinot, gen. nov., forming †In. aurora (LaPolla & Greenwalt, 2015), †In. praecursor (Emery, 1891), comb. nov., and †In. primitiva (Radchenko & Dlussky, 2019), comb. nov. Finally, we transfer †Ph. cordata (Holl, 1829) back to Pheidole, and designate a neotype from our copal collection based on all available evidence. All new species plus the neotype of ‡Ph. cordata are depicted with 3D cybertypes from our µ-CT scan data. We introduce the convention of a double dagger symbol (‡) to indicate fossils in copal or Defaunation resin, as these may yet be extant. To further contextualize our results, we provide a discussion of amber history and classification, as well as the Kleinkuhren locality, to which multiple specimens were attributed. We conclude with conspecti on key biological problems and increasing potential of µ-CT for phylogenetic paleontology.

Traumatic insemination describes an unusual form of mating during which a male penetrates the body wall of its female partner to inject sperm. Females unable to prevent traumatic insemination have been predicted to develop either traits of tolerance or of resistance, both reducing the fitness costs associated with the male-inflicted injury. The evolution of tolerance traits has previously been suggested for the bed bug. Here we present data suggesting that tolerance traits also evolved in females of the twisted-wing parasite speciesStylops ovinaeandXenos vesparum. Using micro-indentation experiments and confocal laser scanning microscopy, we found that females of both investigated species possess a uniform resilin-rich integument that is notably thicker at penetration sites than at control sites. As the thickened cuticle does not seem to hamper penetration by males, we hypothesise that thickening of the cuticle resulted in reduced penetration damage and loss of haemolymph and in improved wound sealing. To evaluate the evolutionary relevance of theStylops-specific paragenital organ and penis shape variation in the context of inter- and intraspecific competition, we conducted attraction and interspecific mating experiments, as well as a geometric-morphometric analysis ofS. ovinaeandX. vesparumpenises. We found thatS. ovinaefemales indeed attract sympatrically distributed congeneric males. However, only conspecific males were able to mate. In contrast, we did not observe any heterospecific male attraction byXenosfemales. We therefore hypothesise that the paragenital organ in the genusStylopsrepresents a prezygotic mating barrier that prevents heterospecific matings.