The development of extremely large male genitalia under spatial limitation.

Extensive research in evolutionary biology has focused on the exaggeration of sexual traits; however, the developmental basis of exaggerated sexual traits has only been determined in a few cases. The evolution of exaggerated sexual traits may involve the relaxation of constraints or developmental processes mitigating constraints. Ground beetles in the subgenus Ohomopterus (genus Carabus) have species-specific genitalia that show coevolutionary divergence between the sexes. Here, we examined the morphogenesis of the remarkably enlarged male and female genitalia of Carabus uenoi by X-ray microcomputed tomography. The morphogenetic processes generating the male and female genitalia at the pupal stage were qualitatively similar to those in closely related species with standard genital sizes. Higher growth rates contributed to the exaggeration of both the male and female genital parts of C. uenoi, possibly related to a gene network commonly upregulated in both sexes. Additionally, the length of the copulatory piece (CP), the enlarged male genital part stored in the aedeagus (AD), reached close to that of the AD at the later developmental stages and thereafter decelerated to grow in parallel with the AD, suggesting a structural constraint on the CP by the outer AD. Then, unlike related species, the lengths of the CP and AD increased at eclosion, suggesting a mechanism leading to further elongation of the male genitalia. These observations suggest that a developmental process allows continuous growth of the male genitalia even under the spatial limitation. These results revealed the spatio-temporal dynamics of the development of exaggerated genital structures under structural constraints.

Microsurgical manipulation reveals pre-copulatory function of key genital sclerites.

The copulatory organs of male insects are generally complex, species-specific arrangements of hardened sclerotized plates bound together by flexible, less sclerotized cuticle. Their extensive morphological diversification is a recurrent pattern in the evolutionary radiation of animals, yet a clear consensus as to what selection pressures drive this divergence is still to emerge. In part, this stems from the fact that the function of individual sclerites that integrate to form the aedeagus are poorly understood. In insects, the male copulatory organ is often bounded by two lateral parameres tipped with setae. In a number of species, these have been observed to brush against the terminal abdominal sclerites of the female, suggesting a role in pre- and/or post-copulatory female choice. However, in the absence of experimental manipulation, their function remains elusive. Here, we used microsurgery to reduce paramere length and show that males with one or both paramere tips removed were less likely to achieve genital coupling than sham-operated male control groups. Where treatment males did achieve copulation, surgical removal of the paramere tip(s) had no detectable effect on copulation duration or on the outcome of sperm competition. Surgical manipulation of the end-plate, a genital sclerite that covers the ostium of the median lobe (the non-intromittent section of the aedeagus), resulted in near-complete failure of males to achieve copulation. Our experimental manipulations show that the parameres and end-plate function during pre-copulatory sexual interactions and thus most likely evolved in response to sexual selection occurring prior to insemination.

Bayesian species delimitation in Pleophylla chafers (Coleoptera) - the importance of prior choice and morphology.

BACKGROUND: Defining species units can be challenging, especially during the earliest stages of speciation, when phylogenetic inference and delimitation methods may be compromised by incomplete lineage sorting (ILS) or secondary gene flow. Integrative approaches to taxonomy, which combine molecular and morphological evidence, have the potential to be valuable in such cases. In this study we investigated the South African scarab beetle genus Pleophylla using data collected from 110 individuals of eight putative morphospecies. The dataset included four molecular markers (cox1, 16S, rrnL, ITS1) and morphometric data based on male genital morphology. We applied a suite of molecular and morphological approaches to species delimitation, and implemented a novel Bayesian approach in the software iBPP, which enables continuous morphological trait and molecular data to be combined. RESULTS: Traditional morphology-based species assignments were supported quantitatively by morphometric analyses of the male genitalia (eigenshape analysis, CVA, LDA). While the ITS1-based delineation was also broadly congruent with the morphospecies, the cox1 data resulted in over-splitting (GMYC modelling, haplotype networks, PTP, ABGD). In the most extreme case morphospecies shared identical haplotypes, which may be attributable to ILS based on statistical tests performed using the software JML. We found the strongest support for putative morphospecies based on phylogenetic evidence using the combined approach implemented in iBPP. However, support for putative species was sensitive to the use of alternative guide trees and alternative combinations of priors on the population size (θ) and rootage (τ 0 ) parameters, especially when the analysis was based on molecular or morphological data alone. CONCLUSIONS: We demonstrate that continuous morphological trait data can be extremely valuable in assessing competing hypotheses to species delimitation. In particular, we show that the inclusion of morphological data in an integrative Bayesian framework can improve the resolution of inferred species units. However, we also demonstrate that this approach is extremely sensitive to guide tree and prior parameter choice. These parameters should be chosen with caution - if possible - based on independent empirical evidence, or careful sensitivity analyses should be performed to assess the robustness of results. Young species provide exemplars for investigating the mechanisms of speciation and for assessing the performance of tools used to delimit species on the basis of molecular and/or morphological evidence.

Directional mitochondrial introgression and character displacement due to reproductive interference in two closely related Pterostichus ground beetle species.

Reproductive interference due to interspecific hybridization can lead to character displacement among related species with overlapping ranges. However, no studies have examined which reproductive traits are most important in reducing reproductive interference. We conducted molecular analyses of two nuclear genes (28S and Wingless) and a mitochondrial gene (COI) from two closely related ground beetle species, Pterostichus thunbergi and Pterostichus habui (Coleoptera: Carabidae), with overlapping distributions. In addition, we examined four reproductive traits (body size, organ morphologies of intromittent and non-intromittent male genital organs, and female reproductive period) in sympatric and allopatric habitats. We compared male genital morphology using geometric morphometric analysis. The species determined by morphology were classified into separate groups based on the phylogenetic tree constructed by the nuclear gene (Wingless). However, according to the mitochondrial genes examined, P. thunbergi was not monophyletic, whereas at the sympatric sites, these species formed a monophyletic clade. This incongruence suggests that interspecific hybridization and subsequent mitochondrial introgression from P. habui to P. thunbergi have occurred. Concerning genital morphology, both of the intromittent and nonintromittent organs of P. thunbergi differed more from P. habui at the sympatric sites than between allopatric sites, suggesting reproductive character displacement. Pterostichus thunbergi, which likely arrived in P. habui habitat in small numbers, would have experienced stronger selection pressures than P. habui.

Revision of fleas of the genus Plocopsylla belonging to the 'angusticeps-lewisi' complex in the Andean biogeographic region, with the description of a new species.

In Argentina, the Andean biogeographic region accommodates the most diverse population of fleas in the country. The Craneopsyllinae (Siphonaptera: Stephanocircidae) represent one of the most commonly found subfamilies in this region and show some endemism and high diversity. Plocopsylla is the most diverse genus of Craneopsyllinae; it includes 10 species mainly distributed in the Patagonian subregion, which parasitize sigmodontine rodents (Rodentia: Cricetidae). We describe and illustrate the morphology of the aedeagus in species of Plocopsylla that belong to the 'angusticeps-lewisi' complex. This character is of diagnostic value in differentiating among species. A new species of this complex, Plocopsylla (Plocopsylla) linardii sp. n., is described and identified by the shape and chaetotaxy of the distal arm of sternite IX, as well as by the shape of the median dorsal lobe of the aedeagus. New host associations for this complex and range extensions for most of its species are reported. Plocopsylla (P.) silewi is recorded for the first time in Argentina. The southern limits of the distributions of Plocopsylla (P.) lewisi and Plocopsylla (P.) wilesi are extended to Santa Cruz Province. The angusticeps-lewisi complex is found for the first time in San Juan Province. The information may be useful in epidemiological studies of flea-borne diseases.

The paralobe: A new diagnostic and synapomorphic character for the genera Paulipalpina Gnaspini & Peck, 1996 and Parapaulipalpina Gnaspini, 1996 (Leiodidae: Cholevinae: Ptomaphagini).

In insect taxa with homogeneous external morphology, genital structures often emerge as essential traits for interspecific differentiation. In the tribe Ptomaphagini (Coleoptera, Leiodidae, Cholevinae), precise identification often depends on analyzing the male genital morphology, even at the genus level. Here, we present a new character for diagnosing the genera Paulipalpina Gnaspini & Peck, 1996 and Parapaulipalpina Gnaspini, 1996. This feature, which we dub 'paralobe', is a projection arising from the internal surface of the right lobe of the aedeagal apex. Based on its absence in other beetles, including other Ptomaphagini, we recognize it as a putative synapomorphy for those genera. The recognition of this previously overlooked structure adds important information for understanding the sequence of changes that occurred in the male genitalia among the genera of Ptomaphagini.

Taxonomic review of the genus Ponyalis Fairmaire, 1899 (Coleoptera, Lycidae), with descriptions of six new species from China.

The lycid genus Ponyalis Fairmaire, 1899 is reviewed. Six new species are described from China, including P.longicornis sp. nov., P.truncata sp. nov., P.dabieshanensis sp. nov., P.hainanensis sp. nov., P.quadricollimima sp. nov., and P.zhejiangensis sp. nov. Nine previously known species, including P.alternata (Pic, 1927), P.fukiensis (Bocak, 1999), P.gracilis (Bocak, 1999), P.himalejica (Bourgeois, 1885), P.klapperichi (Bocak, 1999), P.laticornis Fairmaire, 1899, P.nigrohumeralis (Pic, 1939), P.quadricollis (Kiesenwetter, 1874), and P.variabilis Li, Bocak & Pang, 2015 are illustrated with images of the habitus and aedeagi to make the comparisons with the new species. In addition, a distribution map and an identification key to all 24 species of Ponyalis are provided.

Ultrastructure of aedeagus, spermatheca and ovipositor of Julodis ehrenbergii Laporte (Coleoptera: Buprestidae: Julodinae) by scanning electron microscope.

The paper presents unknown ultrastructure observed by scanning electron microscope (SEM) of aedeagus, spermatheca and ovipositor of Julodis ehrenbergii Laporte (Coleoptera: Buprestidae: Julodinae) from Turkey. The studied specimens were collected from Çankiri and Çorum provinces which are new provincial records for Turkey, in July and August 2021. The genus Julodis Eschscholtz includes 49 species in the Palearctic region, while it is represented by seven species in Turkey. One of them, J. ehrenbergii Laporte is represented by only the nominate subspecies in Turkey. As known, aedeagus, spermatheca and ovipositor are taxonomically important structures. Before the present study, however, there are no work on these structures of J. ehrenbergii Laporte. For this reason, ultrastructural and detailed investigations of aedeagus, spermatheca and ovipositor of J. ehrenbergii Laporte from Turkey were firstly studied with SEM and stereo microscope to obtain new diagnostic characters in the genus Julodis Eschscholtz. The female spermatheca is found insignificant and not diagnostic but aedeagus is found important for diagnosis. In addition, the ultrastructural characters of the ovipositor can also be useful for diagnosis. Photos in SEM as well as photos in the stereo microscope are also given in the text. HIGHLIGHTS: Ultrastructural and detailed investigations of aedeagus, spermatheca and ovipositor of Julodis ehrenbergii Laporte from Turkey were firstly studied with scanning electron microscope (SEM) and stereo microscope to obtain new diagnostic characters in the genus Julodis Eschscholtz. New diagnostic characters that can be distinguished by SEM have been revealed in aedeagus, spermatheca and ovipositor structures, which are used for morphological differentiation of species.

Evidence for Reconsidering the Taxonomic Status of Closely Related Oligonychus Species in punicae Complex (Acari: Prostigmata: Tetranychidae).

To elucidate the taxonomic problems in species delineation within the Oligonychus punicae complex (O. punicae, O. mangiferus, and O. vitis) (Acari: Prostigmata: Tetranychidae), we performed morphological and molecular investigations on mite samples, collected from different hosts/countries. Thirty-nine samples of punicae complex, collected from Egypt, Pakistan, and Saudi Arabia (SA), did not show any considerable morphological differences in females and males. All 39 samples of the punicae complex resembled the original description of O. punicae, while the claimed Mexican O. punicae was distinctively different based on male aedeagus. Molecularly, the low nucleotide diversity ranged from 0% to 2.1% (ITS2-rDNA) and 0% to 1% (COI-mtDNA), and was observed among various DNA sequences of the punicae complex from Egypt, India, Israel, Pakistan, and SA, confirming their identity as one species. The high genetic divergence ranged from 17.2% to 18.8% (ITS2) and 9.2% to 10.2% (COI), observed between the claimed Mexican O. punicae and all other sequences of the punicae complex, indicating that the Mexican sample do not belong to O. punicae. Basing our findings on both morphological and molecular data, we can conclude that O. mangiferus and O. vitis are synonymized with O. punicae. Additionally, this study reveals that the claimed Mexican O. punicae needs to be re-identified.

Population structure, adaptation and divergence of the meadow spittlebug, Philaenus spumarius (Hemiptera, Aphrophoridae), revealed by genomic and morphological data.

Understanding patterns of population differentiation and gene flow in insect vectors of plant diseases is crucial for the implementation of management programs of disease. We investigated morphological and genome-wide variation across the distribution range of the spittlebug Philaenus spumarius (Linnaeus, 1758) (Hemiptera, Auchenorrhyncha, Aphrophoridae), presently the most important vector of the plant pathogenic bacterium Xylella fastidiosa Wells et al., 1987 in Europe. We found genome-wide divergence between P. spumarius and a very closely related species, P. tesselatus Melichar, 1899, at RAD sequencing markers. The two species may be identified by the morphology of male genitalia but are not differentiated at mitochondrial COI, making DNA barcoding with this gene ineffective. This highlights the importance of using integrative approaches in taxonomy. We detected admixture between P. tesselatus from Morocco and P. spumarius from the Iberian Peninsula, suggesting gene-flow between them. Within P. spumarius, we found a pattern of isolation-by-distance in European populations, likely acting alongside other factors restricting gene flow. Varying levels of co-occurrence of different lineages, showing heterogeneous levels of admixture, suggest other isolation mechanisms. The transatlantic populations of North America and Azores were genetically closer to the British population analyzed here, suggesting an origin from North-Western Europe, as already detected with mitochondrial DNA. Nevertheless, these may have been produced through different colonization events. We detected SNPs with signatures of positive selection associated with environmental variables, especially related to extremes and range variation in temperature and precipitation. The population genomics approach provided new insights into the patterns of divergence, gene flow and adaptation in these spittlebugs and led to several hypotheses that require further local investigation.

Discovery of the genus Platycotylus Olliff, 1883 (Coleoptera, Tenebrionidae) in Japan: Description of a new and remarkable species.

The genus Platycotylus Olliff, 1883 (Coleoptera: Tenebrionidae) is recorded from Japan (Nakanoshima Island, Tokara Islands) for the first time, through the discovery of a new and remarkable species, Platycotylusmerkli sp. nov., which is described herein. The male of this new species can be distinguished from all known males of other congeneric species by its long and asymmetrical epistomal horn. Although this new species is most similar to Platycotylusparvicollis (Pic, 1923), for which a male has not been examined, it can be distinguished from this species by its simple sparse pronotal punctation, smaller eyes, and acutely produced temples.

A new species of the genus Rhaphidosoma Amyot et Serville, 1843 (Heteroptera, Reduviidae), with data on its chromosome complement.

A new species, Rhaphidosomapaganicum sp. nov. (Heteroptera: Reduviidae: Harpactorinae: Rhaphidosomatini), is described from the Dry Zone of Myanmar. It is the fifth species of Rhaphidosoma Amyot et Serville, 1843, known from the Oriental Region, and the first record of the genus for Myanmar and Indochina. The structure of the external and internal terminalia of the male and female is described and illustrated in detail. The completely inflated endosoma is described for the first time in reduviids. The complex structure of the ductus seminis is shown; it terminates with a voluminous seminal chamber which opens with a wide secondary gonopore and may be a place where spermatophores are formed. The new species is compared with all congeners from the Oriental Region and Western Asia. It is characterised by the absence of distinct tubercles on the abdominal tergites of the male, the presence only two long tubercles and small rounded ones on the abdominal tergites VII and VI, respectively, in the female, the presence of short fore wing vestiges which are completely hidden under longer fore wing vestiges, and other characters. In addition to the morphological description, an account is given of the male karyotype and the structure of testes of Rh.paganicum sp. nov. and another species of Harpactorinae, Polididusarmatissimus Stål, 1859 (tribe Harpactorini). It was found that Rh.paganicum sp. nov. has a karyotype comprising 12 pairs of autosomes and a multiple sex chromosome system (2n♂=24A+X1X2X3Y), whereas P.armatissimus has a karyotype comprising five pairs of autosomes and a simple sex chromosome system (2n♂=10A+XY). The males of these species were found to have seven and nine follicles per testis, respectively. FISH mapping of 18S ribosomal DNA (major rDNA) revealed hybridisation signals on two of the four sex chromosomes (Y and one of the Xs) in Rh.paganicum sp. nov. and on the largest pair of autosomes in P.armatissimus. The presence of the canonical "insect" (TTAGG) n telomeric repeat was detected in the chromosomes of both species. This is the first application of FISH in the tribe Raphidosomatini and in the genus Polididus Stål, 1858.

A new species and new records of Malagasy Megalopinus Eichelbaum (Coleoptera, Staphylinidae, Megalopsidiinae), with notes on the sperm pump in rove beetles.

A new Malagasy megalopsidiine rove beetle, Megalopinus puthzi sp. n., is described; the male morphological structures, including the aedeagus, are described for M. lemur Puthz for the first time, and a new record for M. heissi Puthz is given. The distribution and diversity of the sperm pump within Staphylinidae is discussed; the pump is for the first time reported to occur in Megalopsidiinae, Steninae, and Solieriinae; examples in Euaesthetinae and Scydmaeninae are also illustrated.

Sporades jaechi sp. nov. with comments on classification of the New Caledonian genus Sporades Fauvel (Coleoptera, Carabidae, Trechini, Trechodina).

Sporades jaechi sp. nov. from Poum, New Caledonia is newly described and shown to be a member of the monophyletic Sporades subgenus Perileptosporades Deuve, 2010. Sporades millei Giachino and S. schuhi Donabauer are newly recognized as members of the subgenus Perileptosporades, and a key to its species is provided. Although Perileptosporades can be defined monophyletically relative to the rest of the genus, genitalic variation among species assigned to the nominate subgenus Sporades Fauvel, 1882 leave monophyly of that taxon ambiguously supported. Several morphological characters of long- standing use have been proposed to define the mutual monophyly of Sporades s. l. and its putative adelphotaxon, Trechodes Blackburn, 1901. Increasing knowledge concerning the diversity of male genitalic characters among Sporades spp. lends support to a recently proposed molecular phylogenetic hypothesis positing that Sporades evolved from within Trechodes. The consequences of the alternate phylogenetic hypotheses on their attendant nomenclature are discussed. An additional locality record for S. sexpunctatus Fauvel expands the known distribution of this species to include most of Grande Terre, New Caledonia.

New findings on the functional morphology of the male genitalia of the blood-feeding bug Rhodnius prolixus in the light of a different observation on living tissue.

Among insect species, the male genitalia evolved many different anatomical features to ensure reproductive success. Males of primitive insects do not copulate but transfer sperm packaged in a spermatophore to the female, whereas males of the most advanced insects copulate and deliver free sperm to the storage organ, the spermatheca, in the female reproductive tract. Based on previous studies on several species of triatomine bugs (Rhodnius prolixus included), Triatominae are thought to use an intermediate form of sperm transfer in which the male genitalia have evolved to carry out both copulation and spermatophore formation. Previous observations on chemically fixed tissue of R. prolixus appear to show that the penis, the aedeagus, contains a much-folded internal cuticle which is a collapsed sac that everts from the aedeagus during copulation for the formation and delivery of the spermatophore directly to the female genital chamber. However, observations on living tissue of these structures in R. prolixus show that this internal cuticle functions as a tube through which male secretions pass through the aedeagus to the vagina. The organization of this cuticle in the context of the various anatomical structures of the aedeagus, its different morpho-functional meaning during sperm transfer in R. prolixus, the use of living versus chemically fixed tissue and a new understanding of previous observations are discussed.

Spinosyns Cause Aedeagus Eversion in Carpophilus spp. (Coleoptera: Nitidulidae).

During an assessment of various insecticides against sap beetles, Carpophilus sp., it was noted that at least some males everted their reproductive structures (referred to as aedeagus from here forward) when treated with a spinosyn insecticide, spinetoram. This response to spinosyns or other insecticides is not documented in the literature even though sap beetles have been included in numerous insecticide assays and spinosyn insecticides have been in widespread commercial use for >20 yr. Additionally, other insect species have not been documented to show a similar aedeagus eversion response to spinosyns or other insecticides. The objective of this study was to further examine eversion of the aedeagus when sap beetles were exposed to different rates of two spinosyn insecticides, spinetoram and spinosad. Additionally, we examined whether a similar response would occur in other insects when three species of Coccinellidae (Coleoptera) and one species of Curculionidae (Coleoptera) were exposed to spinetoram. Our results show that male sap beetles respond to both spinosyns by everting the aedeagus, females did not have an analogous response. No similar response was observed for the assayed species of Coccinellidae or Curculionidae.

Microperla qinlinga Chen reviewed with supplementary illustrations and the description of the nymph.

Microperla qinlinga Chen, 2019 was recently described, but details of the aedeagus were not included in the original description. In this paper, the male aedeagus and nymph of M. qinlinga are described for the first time. Additional information concerning the genus Microperla Chu, 1928 is discussed.

Additions to the genus Hemacroneuria Enderlein (Plecoptera: Perlidae) from China.

Two new species of the perlid genus Hemacroneuria Enderlein, 1909, H. elongata sp. nov. and H. ovalis sp. nov. are described from Sichuan and Zhejiang provinces of China, respectively. A poorly known Sichuan species, H. spatulata (Wu 1948) comb. nov. is transferred from Kiotina Klapálek, 1907 to Hemacroneuria, and an additional species known only from a female is reported by an informal designation. Aedeagal structures of Hemacroneuria males are described in detail for the first time and are useful for the separation of the genus from other genera of the Acroneuriinae. The taxonomic relationships of the new species are discussed.

An illustrated atlas for male genitalia of the New World Polistes Latreille, 1802 (Vespidae: Polistinae).

The male genitalia of 58 species of the New World Polistes are described: 29 of Polistes (Aphanilopterus), 12 of Polistes (Epicnemius), 13 of Polistes (Fuscopolistes), one of Polistes (Onerarius) and three of Polistes (Palisotius). Comments and remarks are also provided.

Notes on Ditomina, with description of two new genera and a new species (Coleoptera, Carabidae) from the Middle East and India.

Two new monotypical genera of the subtribe Ditomina are described: Parapenthus gen. n. for Ditomus solitarius Peyron, 1858 from the Middle East, which was treated previously either within Penthus Chaudoir, 1843 or within Penthophonus Reitter, 1900, and Indocarterus gen. n. for I. inexspectatus sp. n. from the west of the Indian state of Maharashtra (type locality: Wai env., 70 km S of Pune). The possible genesis of Indocarterus gen. n. is discussed because it is the single genus of the subtribe distributed in the Oriental (Indo-Malayan) region in isolation from the other Ditomina which all occur in the West Palaearctic. It is assumed that the ancestor of this genus was isolated in the Indian Peninsula from the Tethyan area probably during the early stages of the diversification of Ditomina. In addition, the enigmatic genus Proditomus Schauberger, 1934 and its single species, P. mirus Schauberger, 1934, known only from the female holotype, are re-described and illustrated. Setation of the parameres and medial lobe in Ditomina is described for the first time. Previously, setae on the aedeagus of Carabidae, mostly on the parameres, were observed only in many basal lineages and, as exception, within Harpalinae, in some Lebiini and Panagaeini. A key to the genera and subgenera of Ditomina is provided.