Yoko Matsumura

Insect morphology in the age of phylogenomics: innovative techniques and its future role in systematics

A brief account of the history of insect morphology is given. Different techniques and analytical methods used in current projects on insect morphology and phylogeny and their optimized combined application are described. These include fixation, dissection, maceration, histology (microtome sectioning), scanning electron microscopy (SEM), transmission electron microscopy (TEM), serial block‐face scanning electron microscopy (SBFSEM), focused ion beam scanning electron microscopy (FIB/SEM), confocal laser scanning microscopy (CLSM), bleaching, micro‐computed tomography (μCT), computer‐based three‐dimensional reconstruction, focus stacking of digital images, geometric morphometrics and the storage of morphological metadata. The role of insect morphology in the “age of phylogenomics” is discussed.

100 years Zoraptera : a phantom in insect evolution and the history of its investigation

Zoraptera are a cryptic and enigmatic group of insects. The species diversity is lower than in almost all other groups of Hexapoda, but may be distinctly higher than presently known. Several new species were described from different regions recently. The systematic placement was discussed controversially since the group was discovered 100 years ago. Affinities with Isoptera and Psocoptera were discussed in earlier studies. A sister group relationship with Acercaria (Psocodea, Thysanoptera, Hemiptera) was proposed by W. Hennig, for the first time based on a strictly phylogenetic argumentation. More recent studies consistently suggest a placement among the “lower neopteran orders” (Polyneoptera). Close affinities to Dictyoptera were proposed and alternatively a sister group relationship with Embioptera or with Embioptera + Phasmatodea (Eukinolabia), respectively. The precise placement is still controversial and the intraordinal relationships are largely unclear. Recent transcriptome analyses tentatively suggest a clade Zoraptera + Dermaptera as sister group of all other polyneopteran orders. The oldest fossils are from Cretaceous amber. An extinct genus from this era may be the sister group of all the remaining zorapterans. The knowledge of the morphology, development and features related to the reproductive system greatly increased in recent years. The general body morphology is very uniform, whereas the genitalia differ strongly between species. This is likely due to different kinds of selection, i.e. sexual selection in the case of the genital organs. The mating pattern also differs profoundly within the order. A unique external sperm transfer occurs in Zorotypus impolitus. A species-level phylogeny and more investigations of the reproductive system should have high priority.

Giant spermatozoa and a huge spermatheca: a case of coevolution of male and female reproductive organs in the ground louse Zorotypus impolitus (Insecta, Zoraptera).

The male and female genital apparatus of the recently discovered ground louse Zorotypus impolitus were examined using light and electron microscopy. The rounded testes and a large seminal vesicle are connected with a complex of four accessory glands by a long tapering ejaculatory duct. Two accessory glands have the same whitish coloration, whereas the third one is pale blue, and the elongated and cylindrical fourth one translucent. The sperm are the largest known in Hexapoda, 3mm long and 3μm wide, with a volume of ca. 21,000μm(3); the ratio between the diameter of the axoneme and the width of the main body of the sperm ranges between 1:10 and 1:13. The exceptional width of the spermatozoa is due to an extreme enlargement of the mitochondrial derivatives and accessory bodies. A single sperm is contained in a small globular spermatophore (100μm). The highly unusual external transfer correlates with an atypical mating behavior. The male produces several to many spermatophores during the mating process. As in other zorapterans the ovaries are panoistic and the eggs bear two micropyles. An exceptionally large apical spermathecal receptacle is present; it is connected with the vagina by a long spermathecal duct, which varies structurally along its course. A correlation between the sperm size and the size of the spermatheca is likely. Ultrastructural features of different species support two strikingly different models of male and female reproductive apparatus in the small order Zoraptera. This is in stark contrast to the extreme uniformity of their external morphology. It is likely that sexual selection played a decisive role in the evolution of the reproductive system.

Evolution of a giant intromittent organ in Scydmaeninae (Coleoptera: Staphylinidae): functional morphology of the male postabdomen in Mastigini.

We compared the postabdominal architecture of Mastigini with extremely long (Stenomastigus) or short (Palaeostigus) aedeagus. A novel mode of copulation was discovered: males of Stenomastigus insert a paramere between the females abdomen and elytra, and the intromission is stabilized by several structures of both sexes. The intrinsic aedeagal mechanism is indicated as responsible for inflating the endophallus, and the long flagellum does not penetrate the ductus spermathecae during copulation. The structure of the flagellum suggests that it is primarily responsible for the sperm transfer. Asymmetrical postabdominal rotators of the aedeagus were only found in Stenomastigus; they presumably facilitate the withdrawal of the genitalia; their origin as bundles separated from larger muscles is postulated. We discuss a scenario in which the evolution of elongated genitalia was facilitated by the lack of structural constraints and existing preadaptations. Benefits of stabilizing the copulation and intromission are indicated as the driving force for the evolution of extremely long aedeagi, while the short aedeagi might have the advantage of freedom of movements facilitating the initiation of copulation by males. Disruptive selection is suggested as a working hypothesis to further investigate mechanisms that have played a role in the evolution of genital structures of Mastigini.

The intermediate sperm type and genitalia of Zorotypus shannoni Gurney: evidence supporting infraordinal lineages in Zoraptera (Insecta)

Abstract The sperm ultrastructure and the male and female genital apparatus of Zorotypus shannoni were examined and documented in detail, mainly using transmission electron microscopy micrographs. The findings suggest an evolutionary trend shared with Z. hubbardi and Z. impolitus. The three species are characterized by enlarged mitochondrial derivatives and related modifications. Giant sperm are probably a synapomorphy of Z. hubbardi and Z. impolitus, whereas an intermediate condition of this feature is found in Z. shannoni. The monophyletic origin of Z. caudelli, Z. magnicaudelli, Z. huxleyi and Z. weidneri is suggested by characteristically modified axonemes. The presence of extra-acrosomal material is also an unusual feature for Zoraptera, but this condition also occurs in the majority of polyneopteran groups. The long and convoluted female spermathecal duct with secretory and duct-forming cells is a constant feature in Zoraptera. The enlarged seminal receptacle suggests an evolutionary link between the male genital structures and the sperm size on one hand, and the size of the female spermatheca on the other. The small and otherwise uniform group Zoraptera exhibits a remarkable variation of sperm types and genital structures, suggesting the impact of different types of selection. It is likely that cryptic female choice plays a major role in shaping the genital apparatus.

Male penile propulsion into spiraled spermathecal ducts of female chrysomelid beetles: A numerical simulation approach.

Genital diversification in animals is an interesting evolutionary phenomenon. Sexual selection is the main driving force behind the diversification. However, evolutionary mechanisms that have established and maintained variations in genitalia shape parameters observed in related species are not well understood. Here, for the first time, we used numerical simulations to test the hypothesis that variations in female spermathecal duct shapes among related beetle species mechanically interfere with penile propulsion in varying ways. Our numerical simulations showed that high curvature of the spiraled spermathecal ducts of the female have effects with a threshold-based interaction on male penile insertion. The relative size of spirals observed in the beetle, Cassida rubiginosa, studied here is not small enough to interfere with penile propulsion. But the model revealed that propulsion is impeded by the presence of reverse turns in spermathecal ducts. This type of morphology leads to an increase in the velocity of the propulsion but also to an increase in the propulsion energy cost for males. Our results showed that quantitative differences in spermathecal duct shape can mediate qualitative differences in penile motion. This explains, in part, the mechanism behind origin and maintenance of genital divergence among closely related species in general.

Stiffness gradient of the beetle penis facilitates propulsion in the spiraled female spermathecal duct

It is well known that sexual selection is the main driving force of substantial diversity of genitalia found in animals. However, how it facilitates the diversity is still largely unknown, because genital morpho/physical features and motions/functional morphology of the structures in sexual intercourse are not linked for the vast majority of organisms. Here we showed the presence of material gradient and numerically studied an effect of stiffness gradient of the beetle penis during its propulsion through the female duct. We found that stiffness gradient on the penis essentially affects its propulsion. Microscopic investigation suggests the possibility that the tip of the hyper-elongated penis is softer than the rest of it, and our numerical model confirms that this type of distribution of stiffness gradient aids in faster propulsion than other types. This result indicates that previously ignored physical properties of genital materials are of crucial importance in evolutionary studies of genitalia.

Parallel evolution of novelties: extremely long intromittent organs in the leaf beetle subfamily Criocerinae.

Extreme elongation of a part of the intromittent organ, the flagellum, has occurred several times in Criocerinae (Chrysomelidae). These leaf beetles have acquired a specialized pocket to store the flagellum in the abdominal cavity, at the same time allowing a quick control of movements of this structure during copulation. We investigated the morphogenesis of the intromittent organs of species with and without a flagellum to discuss the evolutionary background of parallel evolution of novel structures. We found that the specialized pocket is formed by the invagination of an epidermal layer and a resultant rotation of the primary gonopore. Invagination itself is a well‐known phenomenon in morphogenetic processes, which leads us to hypothesize that the novelty is formed by co‐opting a previously acquired genetic system. A large open‐space is present within the intromittent organ during the entire morphogenesis in species without a flagellum, and the invagination in the species with a flagellum grows in the corresponding area. This means that there are no physical impediments for the growth of a large pocket. In addition the sites of muscular attachments in the species with a flagellum are also different from those without it. The differentiation of muscles is completed immediately before adult emergence, which means the muscles are adjustable during the entire morphogenesis in this group. Simple modifications probably based on a co‐option of previously acquired genetic systems, the potential space for adding a new element, and an adjustable factor in morphogenesis of the intromittent organ facilitate the parallel evolution of the extreme elongation.

Penetration mechanics of a beetle intromittent organ with bending stiffness gradient and a soft tip

An apical region of a hyper-long penis in a beetle has flexibility gradient aiding in penetration of a coiled female duct. Hyper-elongated structures and their penetration are widespread among insects, for example, intromittent organs, ovipositors, and piercing-sucking mouthparts. The penetration of thin structures with high aspect ratio without buckling and rupturing is mechanically very challenging. However, this problem is economically solved in nature, and the solutions might be helpful for, for example, in the development of harmless catheters. We focus on the penetration process of a hyper-elongated structure of a cassidine beetle intromittent organ, termed a flagellum. We applied a three-point bending test for the flagellum to measure its bending stiffness along the entire flagellum. We demonstrated the bending stiffness gradient, in which the basal half is relatively stiff and the apical half is softer, whose good performance during copulation had been previously numerically demonstrated. The stiffness gradient is the result of the flagellum shape, which is cylindrical and tapered toward the tip. Moreover, the curved tip comprises a harder outer curve and a softer inner curve. Considering the findings of preceding studies, the flagellum works in the following way: (i) the bending stiffness gradient supports the flagellum, easily fitting to a shape of a highly coiled spermathecal duct, (ii) the stiffness property of the very tip may make the tip tougher, and (iii) the curled tip and homogeneously cylindrical shape of the organ help the very tip to fit the shape of the spermathecal duct of the female. Our study shows that the apparently simple flagellum penetration is achieved with numerous elaborate mechanical adaptations.

A remarkable new species of Zoraptera, Zorotypus asymmetristernum sp. n., from Kenya (Insecta, Zoraptera, Zorotypidae)

A new species of order Zoraptera, Zorotypus asymmetristernum Mashimo, n. sp., is described from Kakamega, Kenya, with its major diagnostic features and characteristics of the egg described and illustrated. The new species represents the sixth zorapteran species from the Afrotropic ecozone. A brief discussion on vestigial eye spots of apteron individuals and a key to the species of the Afrotropic ecozone are provided.