Till evolution do us part: The diversity of symbiotic associations across populations of Philaenus spittlebugs.

Symbiotic bacteria have played crucial roles in the evolution of sap-feeding insects and can strongly affect host function. However, their diversity and distribution within species are not well understood; we do not know to what extent environmental factors or associations with other species may affect microbial community profiles. We addressed this question in Philaenus spittlebugs by surveying both insect and bacterial marker gene amplicons across multiple host populations. Host mitochondrial sequence data confirmed morphology-based identification of six species and revealed two divergent clades of Philaenus spumarius. All of them hosted the primary symbiont Sulcia that was almost always accompanied by Sodalis. Interestingly, populations and individuals often differed in the presence of Sodalis sequence variants, suggestive of intra-genome 16S rRNA variant polymorphism combined with rapid genome evolution and/or recent additional infections or replacements of the co-primary symbiont. The prevalence of facultative endosymbionts, including Wolbachia, Rickettsia, and Spiroplasma, varied among populations. Notably, cytochrome I oxidase (COI) amplicon data also showed that nearly a quarter of P. spumarius were infected by parasitoid flies (Verralia aucta). One of the Wolbachia operational taxonomic units (OTUs) was exclusively present in Verralia-parasitized specimens, suggestive of parasitoids as their source and highlighting the utility of host gene amplicon sequencing in microbiome studies.

Expanding the Chromosomal Evolution Understanding of Lygaeioid True Bugs (Lygaeoidea, Pentatomomorpha, Heteroptera) by Classical and Molecular Cytogenetic Analysis.

The Lygaeoidea comprise about 4660 species in 790 genera and 16 families. Using standard chromosome staining and FISH with 18S rDNA and telomeric (TTAGG)n probes, we studied male karyotypes and meiosis in 10 species of Lygaeoidea belonging to eight genera of the families Blissidae, Cymidae, Heterogastridae, Lygaeidae, and Rhyparochromidae. Chromosome numbers were shown to range from 12 to 28, with 2n = 14 being predominant. All species have an XY system and all but one have a pair of m-chromosomes. The exception is Spilostethus saxatilis (Lygaeidae: Lygaeinae); in another species of Lygaeinae, Thunbergia floridulus, m-chromosomes were present, which represents the first finding for this subfamily. All species have an inverted sequence of sex chromosome divisions ("post-reduction"). The 18S rDNA loci were observed on one or both sex chromosomes in Kleidocerys resedae and Th. floridulus, respectively (Lygaeidae), while on an autosomal bivalent in all other species. The rDNA loci tended to be close to the end of the chromosome. Using (TTAGG)n-FISH, we were able to show for the first time that the Lygaeoidea lack the canonical "insect" telomere motif (TTAGG)n. We speculate that this ancestral motif is absent from the entire infraorder Pentatomomorpha being replaced by some other telomere repeat motif sequences.

Highly divergent karyotypes and barcoding of the East African genus Gonatoxia Karsch (Orthoptera: Phaneropterinae).

East Africa is a hotspot of biodiversity of many orthopteran taxa, including bushcrickets. Gonatoxia Karsch, 1889 species are fully alate Phaneropterinae, which are perfectly adapted to the foliage of forests. We examined five species using combined cytogenetic and molecular data to determine the inter- and intraspecific genetic diversity. The variation in the diploid number of chromosomes in males ranged from 2n = 28 + X0 and 26 + X0 to 2n = 6 + X0. Fluorescence in situ hybridization showed from one to many 18S rDNA loci as well as interstitial sequences, especially in G. helleri. 18S rDNA loci coincided with active NOR and C-banding patterns. The isolation of populations of the species explains differences in the number of chromosomes (G. maculata), chromosomal polymorphism and chromosomal heterozygosity (G. helleri). Our molecular phylogeny based on the COI locus supported the monophyly of the genus Gonatoxia and separateness of the five examined species in accordance with their morphological features and chromosome numbers as well as the species' distribution.

A revision of the shield-back katydid genus Neduba (Orthoptera: Tettigoniidae: Tettigoniinae: Nedubini).

The Nearctic shield-back katydid genus Neduba is revised. Species boundaries were demarcated by molecular phylogenetic analysis, morphology, quantitative analysis of calling songs, and karyotypes. Nine previously described species are redescribed: N. carinata, N. castanea, N. convexa, N. diabolica, N. extincta, N. macneilli, N. propsti, N. sierranus, and N. steindachneri, and twelve new species are described: N. ambagiosa sp. n., N. arborea sp. n., N. cascadia sp. n., N. duplocantans sp. n., N. inversa sp. n., N. longiplutea sp. n., N. lucubrata sp. n., N. oblongata sp. n., N. prorocantans sp. n., N. radicata sp. n., N. radocantans sp. n., and N. sequoia sp. n. We chose a lectotype for N. steindachneri and transferred N. picturata from a junior synonym of N. diabolica to a junior synonym of N. steindachneri. Diversification in this relict group reflects cycles of allopatric isolation and secondary contact amidst the tumultuous, evolving geography of western North America. The taxonomy and phylogenies presented in this revision lay the groundwork for studies of speciation, biogeography, hybrid zones, and behavioral evolution. Given that one Neduba species is already extinct from human environmental disturbance, we suggest conservation priorities for the genus.

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.

Rapid chromosomal evolution in the bush-cricket Gonatoxia helleri Hemp, 2016 (Orthoptera, Phaneropterinae).

Gonatoxia helleri Hemp, 2016 is one of the most widespread bush-crickets of the genus Gonatoxia Karsch, 1889 in East Africa. This species with seven large chromosomes (2n♂ = 7) differs from other representatives of the genus Gonatoxia drastically by its reduced chromosome number, the asymmetrical karyotype including karyomorphs rarely found in tettigoniids, as well as in irregularities in the course of meiosis. To better understand the origin of such an exceptional karyotype, chromosomes of 29 specimens from four populations/localities were studied using classical techniques, such as C-banding, silver impregnation, fluorochrome double staining and fluorescence in situ hybridization (FISH) technique with 18S rDNA and (TTAGG) n telomeric probes. FISH showed many 18S rDNA loci as well as interstitial telomeric sequences, where chromosome morphology varied in these components in terms of quantity and distribution. The 18S rDNA loci coincided with active NORs and C-banding patterns. We suggest that a combination of Robertsonian rearrangements and/or multiple common tandem fusions involving the same chromosomes contributed to the formation of this karyotype/karyomorphs. The results are the first step towards a better understanding of chromosomal reorganization and evolution within the genus Gonatoxia. Low chromosome number, together with the incidence of chromosomal polymorphism that is higher in G. helleri than previously reported in bush-crickets, implies that this species can be a valuable new model for cytogenetic and speciation studies. Our findings suggest that chromosomal translocations lead to diversification and speciation in this species and could be the driving force of adaptive radiation.

Variation in the number of testicular follicles and ovarioles among 18 lacewing species of the families Myrmeleontidae, Ascalaphidae, and Nemopteridae (Insecta, Neuroptera, Myrmeleontiformia).

The representatives of the lacewing families Myrmeleontidae, Ascalaphidae, and Nemopteridae (the suborder Myrmeleontiformia) were studied with reference to the number of testicular follicles in males and the number of ovarioles in females. We have found that the number of follicles is highly variable, at least in the first two families. In the comparatively more fully explored family Myrmeleontidae, the species studied have three to several hundred follicles per testis, the dominant values being six and five. In Ascalaphidae, two main patterns were revealed: testes with a low number of follicles (six and twelve per testis) and testes with multiple follicles (several dozens). Moreover, differences in the follicle number were often observed both between males of the same species and different testes of a male. In Nemopteridae, considered a sister group to the [Myrmeleontidae + Ascalaphidae] clade, the testes in males were found to consist of six or five follicles each. This implies that a low number of follicles, most likely six, is an ancestral trait in Myrmeleontiformia. All other numbers are thus the derived traits and are probably due to a simple oligomerization or a simple polymerization, the latter process having been very intensive in the evolution of the suborder. Conversely, females were found to have ten ovarioles per ovary in each of the three families studied.

Cytogenetic Characterization of Eight Odonata Species Originating from the Curonian Spit (the Baltic Sea, Russia) Using C-Banding and FISH with 18S rDNA and Telomeric (TTAGG)n Probes.

We studied the karyotypes of 8 dragonfly species originating from the Curonian Spit (the Baltic Sea, Russia) using C-banding and FISH with 18S rDNA and "insect" telomeric (TTAGG)n probes. Our results show that Leucorrhinia rubicunda, Libellula depressa, L. quadrimaculata, Orthetrum cancellatum, Sympetrum danae, and S. vulgatum from the family Libellulidae, as well as Cordulia aenea and Epitheca bimaculata from the family Corduliidae share 2n = 25 (24 + X) in males, with a minute pair of m-chromosomes being present in every karyotype except for that of C. aenea. Major rDNA clusters are located on one of the large pairs of autosomes in all the species. No hybridization signals were obtained by FISH with the (TTAGG)n probe in the examined species with the only exception of S. vulgatum. In this species, clear signals were detected at the ends of almost all chromosomes. This finding raises the possibility that in Odonata the canonical "insect" (TTAGG)n telomeric repeat is in fact present but in very low copy number and is consequently difficult to detect by in situ hybridization. We conclude that more work needs to be done to answer questions about the organization of telomeres in this very ancient and thus phylogenetically important insect order.

Karyotype stability in the family Issidae (Hemiptera, Auchenorrhyncha) revealed by chromosome techniques and FISH with telomeric (TTAGG)n and 18S rDNA probes.

We report several chromosomal traits in 11 species from 8 genera of the planthopper family Issidae, the tribes Issini, Parahiraciini and Hemisphaeriini. All species present a 2n = 27, X(0) chromosome complement known to be ancestral for the family. The karyotype is conserved in structure and consists of a pair of very large autosomes; the remaining chromosomes gradually decrease in size and the X chromosome is one of the smallest in the complement. For selected species, analyses based on C-, AgNOR- and CMA3-banding techniques were also carried out. By fluorescence in situ hybridization, the (TTAGG) n probe identified telomeres in all species, and the major rDNA loci were detected on the largest pair of autosomes. In most species, ribosomal loci were found in an interstitial position while in two species they were located in telomeric regions suggesting that chromosomal rearrangements involving the rDNA segments occurred in the evolution of the family Issidae. Furthermore, for 8 species the number of testicular follicles is provided for the first time.

Characterization of Heterochromatin in the Salivary Gland Chromosomes of Chironomus riparius Meigen and Chironomuspiger Strenzke (Diptera, Chironomidae) by Differential and Fluorochrome Staining.

The constitutive heterochromatin of two homosequential sibling species, Chironomus riparius and Chironomus piger, was studied. The salivary gland chromosomes of both species were analyzed using three staining methods: orcein and C band staining combined with DAPI and CMA3 fluorochrome staining. Both species have the chromosome set 2n = 8, with the same banding pattern and chromosome arm combinations: AB, CD, EF, G, but they differed in number and distribution of heterochromatic bands, AT-rich sequences (DAPI+) and GC-rich sequences (CMA3+). In the polytene chromosomes of C. piger, C-bands were found in centromeres only. They contain two types of repetitive DNA sequences: DAPI+ (very weak) and CMA3+ sequences. However, the polytene chromosomes of C. riparius have many interstitial heterochromatic bands in addition to the centromeric heterochromatin. Some of these bands contain both AT-rich and GC-rich sequences, while others are either AT-rich (DAPI+) or GC-rich (CMA3+). Therefore, these closely related species differ both in the content and localization of constitutive heterochromatin. The species-specific organization of the constitutive heterochromatin can be used as an additional cytogenetic marker for species differentiation.

Relations of Wolbachia Infection with Phylogeography of Philaenus spumarius (Hemiptera: Aphrophoridae) Populations Within and Beyond the Carpathian Contact Zone.

Wolbachia is the most widespread intracellular α-proteobacteria maternally inherited endosymbiont of insects and nematodes. These bacteria are associated with a number of different reproductive phenotypes of their hosts. Relatively few studies have dealt with distribution of infections across populations and with the influence of these bacteria on host genetic diversification and speciation. The aims of this study are to determine the distribution and rate of infection and to characterize the Wolbachia strains associated with Philaenus spumarius spittlebug (Hemiptera) by using multilocus sequencing typing (MLST) analysis and host phylogeography. The results showed that infection rate was significantly different between members of both main mitochondrial phylogenetic lineages of P. spumarius. We detected much higher infection rates of Wolbachia in P. spumarius populations from the north-east clade than the south-west clade. Moreover, the frequency of these infections varied within and outside the contact zone known from the Carpathians. Given the reproductive alterations which are often associated with this endosymbiont, Wolbachia probably maintain genetic differentiation of its hosts in its contact zone in the Carpathians. This is one of the first studies demonstrating the presence of Wolbachia across a large part of the range of insect species, including the contact zone. The spread of Wolbachia in P. spumarius populations can potentially cause speciation by compromising the potential reproductive barrier between infected and uninfected populations. We discuss possible implications of Wolbachia infection inducing cytoplasmic incompatibility in the population dynamics of this spittlebug but confirm that more studies are also required.

The secondary contact zone of phylogenetic lineages of the Philaenus spumarius (Hemiptera: Aphrophoridae): an example of incomplete allopatric speciation.

Previous studies on the phylogeography of the meadow spittlebug Philaenus spumarius (L.) (Hemiptera: Aphrophoridae) suggest the existence of a contact zone of its main phylogenetic lineages along mountain chains in Europe and western Asia. This study presents a detailed examination of the population genetics of P. spumarius within the Carpathian Mountains. The main objective was to determine whether the populations inhabiting that area consist of individuals belonging to different genetic units and whether the observed pattern could be an example of secondary contact zone which formed after incomplete allopatric speciation. Specimens from six transects across the Carpathian arc were examined. The mitochondrial phylogeography of the meadow spittlebug in the examined area clearly shows that individuals from both main clades meet and mix there. Representatives of all three main EF1-α clades were also found. The present distribution of the main clades with a zone of overlap along the mountain ranges may suggest that these phylogenetic lineages form a young hybrid zone. Moreover, a limited number of individuals were shown to possess heteroplasmic mitochondrial DNA, which gives additional support to intraspecific hybridization. P. spumarius could be used in future work as an excellent model species in investigating population genetics, intraspecific hybridization, and speciation in progress.

A comparative study of genome organization and inferences for the systematics of two large bushcricket genera of the tribe Barbitistini (Orthoptera: Tettigoniidae: Phaneropterinae).

BACKGROUND: Poecilimon and Isophya are the largest genera of the tribe Barbitistini and among the most systematically complicated and evolutionarily intriguing groups of Palearctic tettigoniids. We examined the genomic organization of 79 taxa with a stable chromosome number using classical (C-banding, silver and fluorochrome staining) and molecular (fluorescence in situ hybridization with 18S rDNA and (TTAGG)n telomeric probes) cytogenetic techniques. These tools were employed to establish genetic organization and differences or similarities between genera or species within the same genus and determine if cytogenetic markers can be used for identifying some taxonomic groups of species. RESULTS: Differences between the karyotypes of the studied genera include some general changes in the morphology of the X chromosome in Isophya (in contrast to Poecilimon). The number of major rDNA clusters per haploid genome divided Poecilimon into two main almost equal groups (with either one or two clusters), while two rDNA clusters predominated in Isophya. In both genera, rDNA loci were preferentially located in the paracentromeric region of the autosomes and rarely in the sex chromosomes. Our results demonstrate a coincidence between the location of rDNA loci and active NORs and GC-rich heterochromatin regions. The C/DAPI/CMA3 bands observed in most Poecilimon chromosomes suggest the presence of more families of repetitive DNA sequences as compared to the heterochromatin patterns in Isophya. CONCLUSIONS: The results show both differences and similarities in genome organization among species of the same genus and between genera. Previous views on the systematics and phylogenetic grouping of certain lineages are discussed in light of the present cytogenetic results. In some cases, variation of chromosome markers was observed to correspond with variation in other evolutionary traits, which is related to the processes of ongoing speciation and hybridization in zones of secondary contact. It was concluded that the physical mapping of rDNA sequences and heterochromatin may be used as an additional marker for understanding interspecific relationships in these groups and their routes of speciation.

Variation in sperm formation patterns in jumping plant-lice (Hemiptera: Psylloidea): a light microscopic study.

The patterns of sperm formation in sternorrhynchous jumping plant-lice are reviewed. To date information is available for 143 species belonging to 54 genera, 17 subfamilies and seven of the eight psylloid families (only Phacopteronidae is not represented). For the majority of the taxa (116 species, 39 genera, 10 subfamilies and the families Calophyidae, Carsidaridae and Liviidae) the data presented here is new and is based exclusively on studies using light microscopy. Five distinct patterns of chromatin reorganisation during metamorphosis of spermatids into spermatozoa are recognised, described and named here types I-V. Types I and III were previously known, types II, IV and V are described here for the first time for jumping plant-lice. The most widespread is type I which is found in every family representing the plesiomorphic condition of spermiogenesis in Psylloidea. Types III and V are autapomorphic for the Aphalarinae and Spondyliaspidinae, respectively. Type IV was found only in two genera of Euphyllurinae and may represent a synapomorphy. Type II occurs in several unrelated subfamilies and is interpreted as a homoplasy. The patterns of sperm formation are stable within most of the examined subfamilies and can be used, to a certain extent, to elucidate phylogenetic relationships within Psylloidea.

Cytogenetic analysis of Otiorhynchus bisulcatus (Fabricius, 1781) and O.(Zadrehus) atroapterus (De Geer, 1775) (Coleoptera, Curculionidae, Entiminae) using C bands, NORs, and DAPI/CMA3 staining.

The structure of the karyotypes of two Otiorhynchus species belonging to separate subgenera, viz. Otiorhynchus s.str. bisulcatus and O. (Zadrehus) atroapterus, is compared and described for the first time. Both species have the same chromosome number (2n = 22), sex chromosome system of an achiasmate parachute type (Xy(p)), symmetric karyotype with the prevalence of metacentrics, similar meiotic behaviour, localization of NORs and positive DAPI signals. The main differences involve the morphology of autosomes and the X chromosome in the C-banding pattern and DAPI/CMA3 signals as well as in the presence of additional B chromosomes.

Mediterranean species of the spittlebug genus Philaenus: modes of chromosome evolution.

The evolution of karyotypes and sex determination system of Philaenus Stål (Auchenorrhyncha: Aphrophoridae) species is studied here in detail. The most plausible scenario of chromosomal rearrangements accompanying phylogenetic differentiation in Philaenus is advanced. It is postulated that the ancestral karyotype of Philaenus was 2n = 24 + X0. Karyotype changes occurred several times independently in the genus. The karyotype of 2n = 22 + X0 (P. spumarius and P. tesselatus) originated from 2n = 24 + X0 by fusion between two autosomal pairs. The neo-XY system (P. arslani, P. loukasi, P. signatus, P. maghresignus, and P. tarifa) also originated from the 24 + X0 karyotype by means of independent fusions between autosomes and the original X chromosome. The neo-X(1)X(2)Y system (P. italosignus) evolved from the 2n = 22 + neo-XY karyotype by an additional fusion between the Y chromosome and one more autosomal pair. The neo-X(n)Y system of P. italosignus is the first reported case of an evolutionarily fixed multiple sex chromosome system in Auchenorrhyncha.

Karyotype of the bird blowfly, Protocalliphora falcozi Séguy, 1928 (Diptera, Calliphoridae).

The karyotype structure of Protocalliphora falcozi is described for the first time. The diploid complement comprises 2n = 12, n(male) = 5+XY. Male mitotic plates include four pairs of long (metacentric) and one pair of medium-sized (submetacentric) autosomes. The submetacentric sex chromosomes X and Y are the smallest elements of the set. An achiasmatic meiosis was determined. The heterochromosomes do not form a heterovalent because they occur separately during the first meiotic division. The examined karyotype shows a pericentromeric position of constitutive heterochromatin in all autosomes. The longer arm of the X chromosome is heterochromatic, while the Y is entirely euchromatic. The NORs are active at mitotic prophase and at early meiotic stages. Small, bright signals were observed in the centromeric regions of autosomes after DAPI staining.

The distribution and population sex ratio of Cacopsylla myrtilli (W. Wagner, 1947) (Hemiptera: Psylloidea).

In Cacopsylla myrtilli (W. Wagner, 1947) bisexual populations, all-female populations and populations heavily biased towards females have been described. In the present paper all the available data on the distribution and population sex ratio of C. myrtilli are summarized. New data obtained by the authors are also presented. First records for Russia are described from Siberia and the southern part of the Far East.

Taxonomy of the genus Isophya (Orthoptera, Phaneropteridae, Barbitistinae): comparison of karyological and morphological data.

The genus Isophya represents one of the largest orthopteran genera with about 45 species occurring in Europe. All over its range the genus includes groups of sibling species, the recognition of which is hampered by their morphological similarity. In part, some species were grouped according to their morphology: the Isophya straubei-group, the I. amplipennis-group and the I. major-group. Nevertheless, many species have not been studied using new methods. This is particularly true for 15 taxa described from the Balkans, more or less resembling I. modesta, known mostly from old, unsatisfactorily informative descriptions. Chromosomal analysis of 25 species/subspecies of the genus Isophya showed the karyotypic evolution among species. The karyotype of 18 species/subspecies was investigated for the first time. The most remarkable changes in the sex chromosomes were the ancient acrocentric X chromosome inversions in 19 species and an X chromosome/autosome mutual tandem translocation, from which the sex determination system neo-XY originated in I. hemiptera. Karyotype differentiation has been less rapid in autosomes than in sex chromosomes, in this case interspecific autosomal differentiation has involved the distribution and quantity of C-heterochromatin as well as the number of NORs. Cytogenetic analysis revealed some changes reflecting the level of genomic organization, and the results suggest some association of related taxa from biogeographic regions.

First evidence of polyploidy in Psylloidea (Homoptera, Sternorrhyncha): a parthenogenetic population of Cacopsylla myrtilli (W. Wagner, 1947) from northeast Finland is apomictic and triploid.

This paper reports results of the first cytogenetic study of parthenogenetic psyllids, carried out on an asexual population of the holarctic species Cacopsylla myrtilli W. Wagner from northeast Finland. Preparations of mature eggs extracted from females revealed 39 univalent chromosomes in prophase and metaphase cells. Hence, female meiosis is of apomictic type and replaced by a modified mitosis. The karyotype consists of 3n = 39 (36 + XXX). Clearly, the population is triploid, the haploid number being n = 12 + X as characteristic of the genus Cacopsylla as a whole. As typical for Psylloidea, the chromosomes are holokinetic, only slightly varying in size and without any visible markers, rendering impossible the precise identification of triplets of homologous chromosomes in the triploid complement. The distribution of bisexual and parthenogenetic populations of C. myrtilli throughout the world is briefly given, and a possible origin of the triploid parthenogenetic population is discussed.