Enzymatic Activity of Soil on the Occurrence of the Endangered Beetle Cheilotoma musciformis (Coleoptera: Chrysomelidae) in Xerothermic Grasslands.

This work attempts to find the reasons for the rather limited range of occurrence of Cheilotoma musciformis in Poland, based on soil properties, which affects both the plant cover and the entomofauna. The aim of the study was to assess the influence of soil enzyme activity on the occurrence of Ch. musciformis in xerothermic grasslands in Southern Poland. The sites inhabited by the beetle were most often extensively grazed by farm animals or had recently been cleared of bushes. The control plots were in wasteland. The soils of most sites with Ch. musciformis were characterized by significantly higher activity of the tested enzymes and higher content of total organic carbon and total nitrogen, as well as lower pHKCl compared to the control sites. The higher enzymatic activity of soils in sites with the beetle than in the control sites may indicate the dependence of the occurrence of this beetle on the presence of patches of extensively grazed xerothermic grasslands. Grazing influences the behavior of preferred host plant species. Therefore, when planning active protection of xerothermic grasslands inhabited by Ch. musciformis, changes in the biochemical properties of the soil and vegetation structure should be taken into account.

The transformation of river ecosystems caused by mining affects bird breeding in indigenous riparian habitats.

Our study considered the excavation of sand and gravel, which modifies the landscapes of riparian ecosystems. It promotes the creation of water bodies with surrounding vegetation, but it also results in the loss of natural habitats. We investigated the species richness, composition, and abundance of aquatic and terrestrial breeding birds and their interaction with three habitat types: managed and abandoned flooded pits, and oxbow lakes. We surveyed 117 sites in medium-sized river valleys in the foreground of the Bohemian Massif (Czech Republic), and in the Carpathian Mountains (Poland, Slovakia) in 2022. Flooded pits were suitable for open-water and colonial birds. Managed flooded pits were also suitable for early successional land birds, but they did not provide habitat for birds that use marshes and wet meadows, or riparian woodlands. The majority of species preferred to breed in oxbow lakes with riparian forests and these areas hosted the highest number of threatened species. We concluded that high levels of disturbance in riparian ecosystems promoted some birds (e.g. colonial or breeding in early-successional habitats), but it negatively affected the overall bird diversity, and it led to a species composition shift with the elimination of taxa associated with indigenous riparian habitats. The importance of flooded pits increases with subsequent plant succession. Our results indicate that gravel or sand mine pits, although beneficial for some taxa, are not substitutes for natural habitats in riparian ecosystems, as they do not support birds breeding in indigenous riparian habitats. Natural oxbow lakes with riparian forests are habitats that need to be preserved to effectively promote local biodiversity.

Limited congruence in phylogeographic patterns observed for riverine predacious beetles sharing distribution along the mountain rivers.

Riverine predacious beetles (RPB) (Carabidae, Staphylinidae) are highly diverse and numerous elements of riverine ecosystems. Their historical and contemporary distribution and diversity are highly dependent on natural flow regimes and topography of watercourses. Despite broad knowledge of their ecology, data on population genetic diversity and connectivity are lacking. This study aimed to fill this gap in order to solve two principal hypotheses assuming (i) congruence of phylogeographic patterns observed for RPB indicating that they share a common history and the ecological adaptations to the dynamic environment, (ii) genetic structuration of populations according to river basins. The Carpathian populations of four ground beetles and three rove beetles were examined using cytochrome oxidase and arginine kinase sequencing. There are substantial differences in RPB demographic history and current genetic diversity. Star-like phylogeny of Bembidion and complex haplotype networks of Paederus/Paederidus, with some haplotypes being drainage-specific and others found in distant populations, indicate a general lack of isolation by distance. Signs of recent demographic expansion were detected for most RPB with the latest population collapse for some rove beetles. To some extent, migration of examined species has to be limited by watersheds. Observed phylogeographic patterns are essential for correctly understanding RPB meta-population functioning.

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.

Disentangling phylogenetic relations and biogeographic history within the Cucujus haematodes species group (Coleoptera: Cucujidae).

Recent progress in the taxonomy of flat bark beetles (Cucujidae), specifically, in the genus Cucujus, has revealed great diversity in subtropical Asia, but the seemingly well-known temperate and boreal taxa need further attention because of their conservation status. Here, we used an integrative approach using morphology, DNA, and species distribution modelling to disentangle phylogenetic relations, verify the number of species, and understand the historical biogeography of Palearctic and Nearctic Cucujus beetles, particularly the C. haematodes species group. Species distinctiveness was supported for C. cinnaberinus, but present-day C. haematodes turned out to be a species complex made up of separate lineages in the western, middle and eastern parts of its Palearctic range. Cucujus muelleri was a member of that complex, being sister to Asian C. haematodes. Moreover, C. haematodes caucasicus was found to be phylogenetically closely related to Italian C. tulliae, and both to be sister to European C. haematodes. North American C. clavipes clavipes and C. c. puniceus resulted to be enough divergent to be considered different species. Interestingly, western American C. puniceus turned out to be closely related to the C. haematodes complex, whereas eastern American C. clavipes constituted a separate lineage, being distantly related to both C. puniceus and C. cinnaberinus. These patterns suggest former trans-continental connections among the ancestors of extant flat bark beetle species. Moreover, a divergent lineage of C. cinnaberinus was found in Calabria, which should be regarded at the very least as a subspecies. The ancestor of C. hameatodes group originated in mid-Miocene, and next, ca. 6.2 Mya, a line leading to C. cinnaberinus had split. Speciation of the American lineages occurred during Pliocene (4.4 Mya for C. clavipes and 3.3 Mya for C. puniceus). Species classified as C. haematodes, C. tulliae and C. muelleri, as well as distinct lineages within C. cinnaberinus split during mid Pleistocene (ca. 1.5 Mya). A comparison of species climatic requirements and their present distribution allowed to identify glacial refugia in south-eastern areas of North America (C. clavipes), south-western areas of North America (C. puniceus), and the Mediterranean and Caspian Sea Basins (European Cucujus species), or south-eastern areas of Asia and the foothills of the central Asian mountains (eastern C. haematodes). Subsequent climatic changes in the Holocene forced these beetles to move their ranges northwards along the coasts of the Pacific (C. puniceus) or Atlantic (C. clavipes), north-eastwards to central, northern, and eastern Europe (C. cinnaberinus and European C. haematodes) or Siberia (Asian C. haematodes). The combined use of molecular, morphological and climatic data allows a comprehensive understanding of the phylogenetic relations and past distributions of Cucujus beetles, highlighting the complexity of C. haematodes species group evolution.

The Origin of Isolated Populations of the Mountain Weevil, Liparus glabrirostris-The Flagship Species for Riparian Habitats.

Liparus glabrirostris is one of the largest European weevils, and it has been recently proposed as the flagship species for threatened riparian habitats in the mountains. For effective conservation of its populations (and associated habitats), it is crucial to understand its history, contemporary distribution, genetic diversity and predict changes in the range, including its highly isolated populations on the Baltic coast. Here, we examined numerous populations of L. glabrirostris across almost the entire species range using phylogeography and species niche modeling (SNM) approaches. Analyses of mtDNA and nucDNA markers revealed the existence of 2 major mitochondrial lineages generally separated between 1) the Alpine region and 2) the Bohemian Massif, the Carpathians, and the Baltic coast areas. Genetic diversity in nuclear genes was more complicated with no clear division between populations. The origin of Baltic populations was derived from the Carpathians, but there were probably multiple expansion events to the north. SNM suggested the existence of glacial refugia for L. glabrirostris, mainly in the Alps and the Southern Carpathians. Current predictions of species range were found to be generally congruent with zoogeographic data; however, the Baltic coast was not really supported as a suitable area for L. glabrirostris. An important prediction of future distribution (2050-2070 CE) suggests a shrinkage of the L. glabrirostris range and extinction of some of its populations (particularly those isolated on lower altitudes). Based on the aforementioned data, proposals for the protection of this species are proposed, including the designation of several evolutionary units of conservation importance.

How Hosts Taxonomy, Trophy, and Endosymbionts Shape Microbiome Diversity in Beetles.

Bacterial communities play a crucial role in the biology, ecology, and evolution of multicellular organisms. In this research, the microbiome of 24 selected beetle species representing five families (Carabidae, Staphylinidae, Curculionidae, Chrysomelidae, Scarabaeidae) and three trophic guilds (carnivorous, herbivorous, detrivorous) was examined using 16S rDNA sequencing on the Illumina platform. The aim of the study was to compare diversity within and among species on various levels of organization, including evaluation of the impact of endosymbiotic bacteria. Collected data showed that beetles possess various bacterial communities and that microbiota of individuals of particular species hosts are intermixed. The most diverse microbiota were found in Carabidae and Scarabaeidae; the least diverse, in Staphylinidae. On higher organization levels, the diversity of bacteria was more dissimilar between families, while the most distinct with respect to their microbiomes were trophic guilds. Moreover, eight taxa of endosymbiotic bacteria were detected including common genera such as Wolbachia, Rickettsia, and Spiroplasma, as well as the rarely detected Cardinium, Arsenophonus, Buchnera, Sulcia, Regiella, and Serratia. There were no correlations among the abundance of the most common Wolbachia and Rickettsia; a finding that does not support the hypothesis that these bacteria occur interchangeably. The abundance of endosymbionts only weakly and negatively correlates with diversity of the whole microbiome in beetles. Overall, microbiome diversity was found to be more dependent on host phylogeny than on the abundance of endosymbionts. This is the first study in which bacteria diversity is compared between numerous species of beetles in a standardized manner.

Using host species traits to understand the Wolbachia infection distribution across terrestrial beetles.

Knowledge of Wolbachia prevalence with respect to its hosts is restricted mainly to taxonomic/phylogenetic context. In contrast, relations between infection and most host's ecological and biological traits are poorly understood. This study aimed to elaborate on relations between bacteria and its beetle hosts in taxonomic and the ecological contexts. In particular, the goal is to verify which ecological and biological traits of beetles could cause them to be prone to be infected. Verification of Wolbachia infection status across 297 beetle taxa showed that approximately 27% of taxa are infected by supergroups A and B. Only minor support for coevolution between bacteria and its beetle hosts was observed in some genera of beetles, but in general coevolution between beetles and Wolbachia was rejected. Some traits of beetles were found to be unrelated to Wolbachia prevalence (type of range and thermal preferences); some traits were related with ambiguous effects (habitats, distribution, mobility and body size); some were substantially related (reproduction mode and trophy). The aforementioned summary does not show obvious patterns of Wolbachia prevalence and diversity in relation to host taxonomy, biology, and ecology. As both Wolbachia and Coleoptera are diverse groups, this lack of clear patterns is probably a reflection of nature, which is characterised by highly diversified and probably unstable relations.

Current state of knowledge on Wolbachia infection among Coleoptera: a systematic review.

BACKGROUND: Despite great progress in studies on Wolbachia infection in insects, the knowledge about its relations with beetle species, populations and individuals, and the effects of bacteria on these hosts, is still unsatisfactory. In this review we summarize the current state of knowledge about Wolbachia occurrence and interactions with Coleopteran hosts. METHODS: An intensive search of the available literature resulted in the selection of 86 publications that describe the relevant details about Wolbachia presence among beetles. These publications were then examined with respect to the distribution and taxonomy of infected hosts and diversity of Wolbachia found in beetles. Sequences of Wolbachia genes (16S rDNA, ftsZ) were used for the phylogenetic analyses. RESULTS: The collected publications revealed that Wolbachia has been confirmed in 204 beetle species and that the estimated average prevalence of this bacteria across beetle species is 38.3% and varies greatly across families and genera (0-88% infected members) and is much lower (c. 13%) in geographic studies. The majority of the examined and infected beetles were from Europe and East Asia. The most intensively studied have been two groups of herbivorous beetles: Curculionidae and Chrysomelidae. Coleoptera harbor Wolbachia belonging to three supergroups: F found in only three species, and A and B found in similar numbers of beetles (including some doubly infected); however the latter two were most prevalent in different families. A total of 59% of species with precise data were found to be totally infected. Single infections were found in 69% of species and others were doubly- or multiply-infected. Wolbachia caused numerous effects on its beetle hosts, including selective sweep with host mtDNA (found in 3% of species), cytoplasmic incompatibility (detected in c. 6% of beetles) and other effects related to reproduction or development (like male-killing, possible parthenogenesis or haplodiploidy induction, and egg development). Phylogenetic reconstructions for Wolbachia genes rejected cospeciation between these bacteria and Coleoptera, with minor exceptions found in some Hydraenidae, Curculionidae and Chrysomelidae. In contrast, horizontal transmission of bacteria has been suspected or proven in numerous cases (e.g., among beetles sharing habitats and/or host plants). DISCUSSION: The present knowledge about Wolbachia infection across beetle species and populations is very uneven. Even the basic data about infection status in species and frequency of infected species across genera and families is very superficial, as only c. 0.15% of all beetle species have been tested so far. Future studies on Wolbachia diversity in Coleoptera should still be based on the Multi-locus Sequence Typing system, and next-generation sequencing technologies will be important for uncovering Wolbachia relations with host evolution and ecology, as well as with other, co-occurring endosymbiotic bacteria.

Species delimitation within the Bothryorrhynchapion weevils: Multiple evidence from genetics, morphology and ecological associations.

Curculionidae is a hyperdiverse group of beetles, whose taxonomy and phylogenetics are still poorly understood, especially at the genus level. The latest work on the evolution of Apionini showed a noticeable "mess" in the subtribe Oxystomatina, where most of the morphology-based genera were found to be polyphyletic or paraphyletic. These discrepancies between classical taxonomy and molecular phylogenetics implied the need for further taxonomic revision of these groups. Here, we used sets of morphological, molecular and ecological characters to verify the taxonomic statuses and disentangle the phylogenetic relations among the Bothryorrhynchapion apionids, which are classified as a subgenus of Cyanapion. Morphological data including morphometrics, and multilocus molecular analyses confirmed the monophyly of the Bothryorrhynchapion and species statuses of five species. The morphological analyses showed that Cyanapion (Bothryorrhynchapion) protractum (Sharp, 1891) from the southeast Palaearctic is a synonym of C. (B.) gyllenhalii (Kirby). Moreover, ecological features (host plant use and presence/absence of the endosymbiotic bacteria Wolbachia) helped to unravel the relations among the examined weevils. The speciation of Bothryorrhynchapion apionids was probably affected by allopatric distribution, shifts in the preferred host plants (Vicia sp. or Lathyrus sp.) of sympatric taxa, and infection by different strains of Wolbachia. The paper presents the first comprehensive description of the species' morphology, biology and ecology, and includes a key to the species.

European species of the aphid genus Eulachnus Del Guercio, 1909 (Hemiptera: Aphididae: Lachninae): revision and molecular phylogeny.

The aphid genus Eulachnus in Europe is revised to include 12 species, using an integrative taxonomy approach, based on morphometric, molecular and biological traits. Fundatrix, apterous and alate viviparous female of a new species-Eulachnus stekolshchikovi Kanturski sp. nov. are described. Neotypes are designated for E. agilis, E. brevipilosus and E. nigricola. Lectotypes are designated for E. alticola, E. cembrae, E. rileyi and E. tuberculostemmatus. New synonyms are proposed: E. abameleki syn. nov. (= Cinara pini), E. cretaceus syn. nov. (= E. agilis), E. tauricus syn. nov. (= E. rileyi), E. pallidus syn. nov. (= E. tuberculostemmatus). Eulachnus mingazzinii (near Cinara piniphila) and E. nigrofasciatus (near C. brauni) are regarded as incertae sedis. Full species status is given for E. garganicus stat. nov. and E. ibericus stat. nov. Apterous viviparous female of E. cembrae; apterous and alate viviparous females of remaining species are redescribed. Sexual morphs of E. agilis, E. alticola, E. cembrae, E. intermedius, E. nigricola, E. rileyi and oviparous female of E. tuberculostemmatus are fully redescribed and figured for the first time. Fundatrices of E. agilis, E. brevipilosus, E. cembrae, E. rileyi and E. tuberculostemmatus, sexuales of E. brevipilosus and the alate male of E. tuberculostemmatus are described and figured. A new host plant-Pinus cembra for E. brevipilosus is reported, and this species is recorded for the first time from Czech Republic. Eulachnus tuberculostemmatus is reported for the first time from Croatia. Phylogenetic studies, based on the COI and ITS2 molecular markers, are provided to visualize and discuss the relationships within the European species. COI barcodes are provided for seven species.

The relationships within the Chaitophorinae and Drepanosiphinae (Hemiptera, Aphididae) inferred from molecular-based phylogeny and comprehensive morphological data.

The Chaitophorinae is a bionomically diverse Holarctic subfamily of Aphididae. The current classification includes two tribes: the Chaitophorini associated with deciduous trees and shrubs, and Siphini that feed on monocotyledonous plants. We present the first phylogenetic hypothesis for the subfamily, based on molecular and morphological datasets. Molecular analyses were based on the mitochondrial gene cytochrome oxidase subunit I (COI) and the nuclear gene elongation factor-1α (EF-1α). Phylogenetic inferences were obtained individually on each of genes and joined alignments using Bayesian inference (BI) and Maximum likelihood (ML). In phylogenetic trees reconstructed on the basis of nuclear and mitochondrial genes as well as a morphological dataset, the monophyly of Siphini and the genus Chaitophorus was supported. Periphyllus forms independent lineages from Chaitophorus and Siphini. Within this genus two clades comprising European and Asiatic species, respectively, were indicated. Concerning relationships within the subfamily, EF-1α and joined COI and EF-1α genes analysis strongly supports the hypothesis that Chaitophorini do not form a monophyletic clade. Periphyllus is a sister group to a clade containing Chaitophorus and Siphini. The Asiatic unit of Periphyllus also includes Trichaitophorus koyaensis. The analysis of morphological dataset under equally weighted parsimony also supports the view that Chaitophorini is an artificial taxon, as Lambersaphis pruinosae and Pseudopterocomma hughi, both traditionally included in the Chaitophorini, formed independent lineages. COI analyses support consistent groups within the subfamily, but relationships between groups are poorly resolved. These analyses were extended to include the species of closely related and phylogenetically unstudied subfamily Drepanosiphinae, which produced congruent results. Genera Drepanosiphum and Depanaphis are monophyletic and sister. The position of Yamatocallis tokyoensis differs in the molecular and morphological analyses, i.e. it is either an independent lineage (EF-1α, COI, joined COI and EF-1α genes) or is nested inside this unit (morphology). Our data also support separation of Chaitophorinae from Drepanosiphinae.

Reconstructed historical distribution and phylogeography unravels non-steppic origin of Caucasotachea vindobonensis (Gastropoda: Helicidae).

Existing data on the phylogeography of European taxa of steppic provenance suggests that species were widely distributed during glacial periods but underwent range contraction and fragmentation during interglacials into "warm-stage refugia." Among the steppe-related invertebrates that have been examined, the majority has been insects, but data on the phylogeography of snails is wholly missing. To begin to fill this gap, phylogeographic and niche modeling studies on the presumed steppic snail Caucasotachea vindobonensis were conducted. Surprisingly, reconstruction of ancestral areas suggests that extant C. vindobonensis probably originated in the Balkans and survived there during the Late Pleistocene glaciations, with a more recent colonization of the Carpatho-Pannonian and the Ponto-Caspian regions. In the Holocene, C. vindobonensis colonized between the Sudetes and the Carpathians to the north, where its recent and current distribution may have been facilitated by anthropogenic translocations. Together, these data suggest a possible non-steppic origin of C. vindobonensis. Further investigation may reveal the extent to which the steppic snail assemblages consist partly of Holocene newcomers.

The Impact of Environmental Conditions on Efficiency of Host Plant DNA Barcoding for Polyphagous Beetles.

Recently, several papers were published dealing with host plant identification for selected species of insects, including beetles. These studies took advantage of the DNA barcoding approach and generally showed that it is possible to identify diet composition from plant DNA present in insect guts. However, none of these studies considered how the impact of environmental conditions affected the likelihood of insect feeding and, therefore, the presence of host plant DNA that could be amplified and sequenced. In the present study, individuals of the polyphagous weevil Centricnemus leucogrammus (Germar, 1824) (Curculionidae: Entiminae) were used to test the hypothesis that harsh environmental conditions limited its feeding activity. The diet of 50 specimens collected during favourable conditions in the middle of the species reproductive period was compared against the diet of 50 specimens collected during harsh environmental conditions. Results clearly showed that almost no weevils fed during rainy and cold conditions and only a minority of individuals (20%) fed during the drought condition (on drought-resistant plants). It is important to consider such factors in any studies dealing with host plant identification and feeding behaviour. Results of ecological studies could lead to erroneous conclusions, e.g., underestimation of number and composition of host plants in the diet of studies species.

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 DNA metabarcoding study of a polyphagous beetle dietary diversity: the utility of barcodes and sequencing techniques.

Recently developed techniques of DNA barcoding and next-generation sequencing (NGS) overcome previous limitations of evaluation of animal diet composition and together are promising method in molecular ecology. The objective was to compare standard ABI Sanger sequencing with new high throughput sequencing (Illumina MiSeq) technique and the two selected plant barcodes (rbcL gene and trnL intron) in terms of the identification of host plant composition for the selected beetle species--the Centricnemus leucogrammus weevil. A comparison of two sequencing techniques showed that NGS (in this case Illumina) gave more exhaustive results than the Sanger method. Moreover, it was proven that a two-locus barcoding systems (rbcL and trnL) is sufficient for host plant identification from DNA isolated from insect bodies, at least at the genus level. A comparison of host plant composition among distant populations revealed that the studied species did not feed uniformly across its range. This probably reveals an ecological adaptation of geographically and genetically isolated populations. These findings, beside broadening basic knowledge on the use of barcoding and sequencing techniques for host plant identifications in insect populations, can have implications for conservation studies and strategies for rare and endangered species.

Development and characterization of microsatellite loci in the Centricnemus leucogrammus weevil.

Centricnemus leucogrammus is a weevil characteristic of European xerothermic habitats and steppes. The species was probably more widespread during the Pleistocene glaciations, while its current distribution is limited to "warm-stage refugia." It may be regarded as a typical representative of flightless xerothermophilous beetles. Previous studies concentrated on its genetic variation using mitochondrial genes. Here, we identified, tested and characterized 24 polymorphic microsatellite loci with the use of 454 sequencing of microsatellite enriched genomic libraries. The new set of loci will be used in studies on the population structure of this weevil and may provide valuable information for its conservation.

Genetic constitution of parthenogenetic form of Polydrusus inustus (Coleoptera: Curculionidae)--hints of hybrid origin and recombinations.

Asexual reproduction among insects may arise spontaneously, as a result of infectious agents or hybridization between phylogenetically distinct organisms. Polyploidy, which may be the result of auto- or allopoliploidization (also associated with hybridization), is often connected with parthenogenesis. However, all studied parthenogens of weevils were apomictic, rare meiotic stages have been observed in a few of them what could suggests in their genomes besides the mutational changeability also recombination may occurs. We studied the level of heterozygosity in Internal Transcribed Spacer 2 sequences, and congruence ofmitochondrial (cytochrome B) and nuclear genealogies among individuals of parthenogenetic form of Polydrusus inustus. High frequency (87.0%) of heterozygotes in ITS2 sequences may indicate a hybrid origin of the parthenogen. It is possible that this parthenogenetic form arose via a hybridization event between phylogenetically distinct lineages of bisexual P. inustus, known only from Georgia and Turkey, or between this species and one of the closely relatives from the subgenus Scythodrusus. Although result of partition- homogeneity test did not show significant incongruence of mitochondrial and nuclear genealogies, in the network was detected one loop. Most probable explanations for existence of that loop in parthenogenetic form and for origin of rare homozygotes, which were detected in ITS2 sequences, is gene conversion. It is supported by recently cytogenetic studies indicating rare meiotic stages during the oogenesis of triploids of P. inustus.

Karyology of parthenogenetic weevils (Coleoptera, Curculionidae): do meiotic prophase stages occur?

We investigated the cytological mechanism of parthenogenesis by analyzing the chromosomes in five weevil species. All examined species are polyploids, four of which: Otiorhynchus ovatus, Simo variegates, Cathormiocerus aristatus, and Tropiphorus elevatus possess three haploid sets of chromosomes (3n=33), whereas the fifth, Trachyphloeus parallelus, is tetraploid with 44 chromosomes (4n=44). The plates contained 27-31 chromosomes in triploid species and 38, 39, 41 and even 44 in tetraploid T. parallelus. In all species single clusters of metaphase plates with a haploid number of n=11 were visible. Some oogonial cells showed nuclei configurations resembling the stages of diplotene and diakinesis. The spiralized chromosomes in these nuclei may have been connected by chiasmata resulting in rods figures and ring-shaped bivalents. Occurrence of the remnants of meiosis could suggest some degree of recombination in parthenogenetic lineages of weevils.