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Only three saproxylic species of Pyrochroinae (Coleoptera: Pyrochroidae) are distributed in Europe, two of which belonging to Pyrochroa: P. coccinea and P. serraticornis. However, P. serraticornis is polytypic, for the presence of the endemic subspecies P. s. kiesenwetteri in southern Italy. Using both molecular and morphological data, we explored the phylogeny of the European Pyrochroa species. A multilocus (COI, CAD, 28S) phylogenetic analysis helped highlight different evolutionary histories for the two examined species. First, P. coccinea, distributed throughout Europe, showed a high differentiation among Italian and European populations. Furthermore, three different taxonomic entities were identified within P. serraticornis, among which the cryptic species Pyrochroa bifoveata sp. n. from central Europe is described and illustrated. A comprehensive identification key to the European Pyrochroinae is also provided. Our results also suggested an historical survival of P. coccinea and P. s. kiesenwetteri in glacial refugia in Italy, and a subsequent post-glacial spread of the former species throughout the Peninsula. In contrast, the current distribution of P. s. serraticornis likely originated from a post-glacial colonization of western European relict populations, while the survival of P. bifoveata plausibly occurred in more eastern glacial refugia (e.g. Carpathian or Balkan regions). Similarly, the European populations of P. coccinea could have originated from relict populations in glacial refugia out from the Italian Peninsula. More comprehensive data on the taxonomy, ecology and biogeography of Pyrochroa are needed to learn more about these species and to help preserve the European saproxylic fauna.

Polymerase chain reaction (PCR)-based genotyping of mutations in the voltage-sensitive sodium channel (vssc) associated with resistance to pyrethroid insecticides is widely used and represents a potential early warning and monitoring system for insecticide resistance arising in mosquito populations, which are vectors of different human pathogens. In the secondary vector Aedes albopictus&mdash, an Asian species that has invaded and colonized the whole world, including temperate regions&mdash, sequencing of domain II of the vssc gene is still needed to detect the V1016G mutation associated with pyrethroid resistance. In this study we developed and tested a novel allele-specific PCR (AS-PCR) assay to genotype the V1016G mutation in this species and applied it to the analysis of wild populations from Italy. The results confirm the high accuracy of the novel AS-PCR and highlight frequencies of the V1016G allele as &gt, 5% in most sampling sites, with peaks of 20&ndash, 45% in coastal touristic sites where pyrethroid treatments are extensively implemented, mostly for mosquito nuisance reduction. The high frequency of this mutation observed in Italian Ae. albopictus populations should serve as a warning bell, advocating for increased monitoring and management of a phenomenon which risks neutralizing the only weapon today available to counteract (risks of) arbovirus outbreaks.

Background Meloidae (blister beetles) are known to synthetize cantharidin (CA), a toxic and defensive terpene mainly stored in male accessory glands (MAG) and emitted outward through reflex-bleeding. Recent progresses in understanding CA biosynthesis and production organ(s) in Meloidae have been made, but the way in which self-protection is achieved from the hazardous accumulation and release of CA in blister beetles has been experimentally neglected. To provide hints on this pending question, a comparative de novo assembly transcriptomic approach was performed by targeting two tissues where CA is largely accumulated and regularly circulates in Meloidae: the male reproductive tract (MRT) and the haemolymph. Differential gene expression profiles in these tissues were examined in two blister beetle species, Lydus trimaculatus (Fabricius, 1775) (tribe Lyttini) and Mylabris variabilis (Pallas, 1781) (tribe Mylabrini). Upregulated transcripts were compared between the two species to identify conserved genes possibly involved in CA detoxification and transport. Results Based on our results, we hypothesize that, to avoid auto-intoxication, ABC, MFS or other solute transporters might sequester purported glycosylated CA precursors into MAG, and lipocalins could bind CA and mitigate its reactivity when released into the haemolymph during the autohaemorrhaging response. We also found an over-representation in haemolymph of protein-domains related to coagulation and integument repairing mechanisms that likely reflects the need to limit fluid loss during reflex-bleeding. Conclusions The de novo assembled transcriptomes of L. trimaculatus and M. variabilis here provided represent valuable genetic resources to further explore the mechanisms employed to cope with toxicity of CA in blister beetle tissues. These, if revealed, might help conceiving safe and effective drug-delivery approaches to enhance the use of CA in medicine.

Hycleus is a hyper-diverse genus of blister beetles including ~500 species widely distributed in the Old World, currently divided into three "sections" and into 45 "phenetic" species groups according to morphological characters. Recently the monophyly of Hycleus was questioned pointing out its paraphyly with respect to the genera Ceroctis and Paractenodia. In this study, we built a time-calibrated phylogenetic tree based on DNA sequence data from mitochondrial and nuclear genes obtained from 125 species, to understand the phylogenetic relationships among the species of this genus, to infer the biogeographic processes behind their diversification, and to assess their taxonomy and classification. Our results identified four main lineages one of which included the species belonging to Ceroctis and Paractenodia; therefore, both taxa are now referred to Hycleus as new synonyms. The three described sections of Hycleus resulted polyphyletic and are rejected, whereas several species groups represented well supported clades. Hycleus likely originated in Africa during the Early Miocene (~20 Mya), and subsequently spread in Europe and western Asia. Later, in the Late Miocene (~6 Mya) a Saharo-Sindian group branched off from the Palaearctic lineage, whereas the Oriental Region was colonized following a dispersal event through the Arabian Peninsula from the Afrotropical Region (~5 Mya).

Recent molecular studies revealed high level of endemism and numerous cryptic species within opisthobranchs, with Mediterranean taxa clearly understudied. Here we used genetic data from both mitochondrial and nuclear gene fragments as well as morphological data from taxonomically relevant characters to investigate the phylogenetic relationships and systematics of Mediterranean taxa of the Flabellinidae and Piseinotecidae families. Phylogenetic analyses based on Bayesian and Maximum-Likelihood methods indicate that Flabellinidae and Pisenotecidae taxa and species within the genera Flabellina, Calmella and Piseinotecus do not form monophyletic clades. These results are supported by our morphological analyses which allowed the re-evaluation of the triseriate radula condition in Pisenotecidae and Calmella taxa and their inclusion in the genus Flabellina as Flabellina gaditana comb. nov. (synonym of F. confusa), Flabellina gabinierei comb. nov. and Flabellina cavolini comb. nov. Species delimitation and barcoding gap analyses allowed uncovering cryptic species within Flabellina gracilis (Alder and Hancock, 1844), F. trophina (Bergh, 1890), F. verrucosa (M. Sars, 1829) and F. ischitana Hirano and Thompson, 1990, the latter with an Atlantic form which is under description. This study corroborates the relevance of combining molecular and morphological data from multiple populations and species in the assessment of nudibranch diversity and classification.

Habitat loss and fragmentation are threatening amphibians by increasing population isolation. However, artificial waterbodies created for livestock may contrast this phenomenon by providing surrogate habitats for amphibians. Here, we performed a genetic study on an amphibian species, Lissotriton vulgaris meridionalis, in a rural area in central Italy where natural wetlands are disappearing and drinking troughs for cattle watering are widespread. Specifically, we tested a panel of microsatellite markers to identify a suitable tool for addressing conservation genetic issues of this species that is undergoing severe local decline. Twelve of the 20 tested loci produced reliable amplifications and were polymorphic. Three distinct units with a low level of gene flow were distinguished and the population genetic structuring overlapped with geographic distribution. Such loci will be useful to assess the genetic diversity of the species across multiscale levels for its management and conservation.

Morimus funereus is a large longhorn beetle included in the European Habitats Directive and in previous releases of the IUCN red list. It represents a flagship species of old-growth forest saproxylic communities in E and SE Europe. The morphologically based taxonomy of W Palaearctic Morimus is rather unstable due to high phenetic intrapopulational and geographic variability and different authors have attempted to recognise one to five different taxa of specific/subspecific rank. No previous molecular data are available for the genus Morimus. Here, for the first time, a molecular approach based on COI and ITS2 gene sequences was applied in European and Anatolian Morimus specimens. The genetic variability among Euro-Anatolian Morimus populations and the geographical structure suggest that they can not be ascribed to the currently accepted five W Palaearctic Morimus species and may actually represent a single, genetically and morphologically variable biological species (M. asper), highlighting the necessity of an extended taxonomical revision. In light of these results, a phylogeographical hypothesis of postglacial colonisation of the central Mediterranean area has been developed and the consequences of this new taxonomic arrangement regarding conservation strategies for “Morimus funereus” and allied taxa in Europe and Turkey are discussed.

Resolving complexes of closely related and cryptic insect species can be challenging, especially when dealing with rare and protected taxa that are difficult to collect for genetic and morphological analyses. Until recently, populations of the genus Osmoderma (Scarabaeidae), widespread in Europe, were treated as a single species O. eremita (Scopoli, 1763) in spite of observed geographic variation in morphology. A previous survey using sequence data from the mtDNA cytochrome C oxidase I gene (COI) revealed the occurrence of at least two distinct lineages within this species complex: O. eremita in the west and O. barnabita Motschulsky, 1845, in the east. Interestingly, beetles confined to Sicily have been described as a distinct species, O. cristinae Sparacio, 1994, based on morphological traits. Only few Sicilian specimens were included in the former genetic analysis, and the results led to a still questionable taxonomic rank for these populations. To explore the robustness of the previous taxonomic arrangement for O. cristinae, a combination of genetic, morphological and pheromonal analyses was used. A 617-bp fragment of the COI gene, aligned with O. cristinae and O. eremita sequences already available in GenBank, showed a clear genetic divergence between the two species (interspecific mean distance = 6.6%). Moreover, results from AFLP markers sustained the distinction of the two species. In addition, geometric morphometric analyses of the shape of male genitalia revealed a clear differentiation between the two species. Via scent analysis and field trapping, we demonstrated the production of the sex pheromone (R)-(+)-γ-decalactone by males of O. cristinae, the attraction by conspecific individuals (mostly females) to this compound, and a lack of antagonistic effect of (S)-(-)-γ-decalactone. The fact that O. eremita and O. eremita use the same compound for mate finding suggests that this sex pheromone has not undergone a differentiation and probably the allopatry of these two species compensates for the absence of a mechanism to avoid cross-attraction. Our genetic and morphological data support the divergence of the two species and confirm the species status for O. cristinae, while sex pheromones are confirmed to be invariant among different species of the genus Osmoderma.

Speciation with gene flow may be aided by reduced recombination helping to build linkage between genes involved in the early stages of reproductive isolation. Reduced recombination on chromosome X has been implicated in speciation within the Anopheles gambiae complex, species of which represent the major Afrotropical malaria vectors. The most recently diverged, morphologically indistinguishable, species pair, A. gambiae and Anopheles coluzzii, ubiquitously displays a ‘genomic island of divergence’ spanning over 4 Mb from chromosome X centromere, which represents a particularly promising candidate region for reproductive isolation genes, in addition to containing the diagnostic markers used to distinguish the species. Very low recombination makes the island intractable for experimental recombination studies, but an extreme hybrid zone in Guinea Bissau offers the opportunity for natural investigation of X-island recombination. SNP analysis of chromosome X hemizygous males revealed: (i) strong divergence in the X-island despite a lack of autosomal divergence; (ii) individuals with multiple-recombinant genotypes, including likely double crossovers and localized gene conversion; (iii) recombination-driven discontinuity both within and between the molecular species markers, suggesting that the utility of the diagnostics is undermined under high hybridization. The largely, but incompletely protected nature of the X centromeric genomic island is consistent with a primary candidate area for accumulation of adaptive variants driving speciation with gene flow, while permitting some selective shuffling and removal of genetic variation.

"Far-West" Africa is known to be a secondary contact zone between the two major malaria vectors Anopheles coluzzii and A. gambiae. We investigated gene-flow and potentially adaptive introgression between these species along a west-to-east transect in Guinea Bissau, the putative core of this hybrid zone. To evaluate the extent and direction of gene flow, we genotyped site 702 in Intron-1 of the para Voltage-Gated Sodium Channel gene, a species-diagnostic nucleotide position throughout most of A. coluzzii and A. gambiae sympatric range. We also analyzed polymorphism in the thioester-binding domain (TED) of the innate immunity-linked thioester-containing protein 1 (TEP1) to investigate whether elevated hybridization might facilitate the exchange of variants linked to adaptive immunity and Plasmodium refractoriness. Our results confirm asymmetric introgression of genetic material from A. coluzzii to A. gambiae and disruption of linkage between the centromeric "genomic islands" of inter-specific divergence. We report that A. gambiae from the Guinean hybrid zone possesses an introgressed TEP1 resistant allelic class, found exclusively in A. coluzzii elsewhere and apparently swept to fixation in West Africa (i.e. Mali and Burkina Faso). However, no detectable fixation of this allele was found in Guinea Bissau, which may suggest that ecological pressures driving segregation between the two species in larval habitats in this region may be different from those experienced in northern and more arid parts of the species' range. Finally, our results also suggest a genetic subdivision between coastal and inland A. gambiae Guinean populations and provide clues on the importance of ecological factors in intra-specific differentiation processes.

Background Genomic differentiation between Anopheles gambiae and Anopheles coluzzii - the major malaria vectors in sub-Saharan Africa - is localized into large “islands” toward the centromeres of chromosome-X and the two autosomes. Linkage disequilibrium between these genomic islands was first detected between species-specific polymorphisms within ribosomal DNA genes (IGS-rDNA) on the X-chromosome and a single variant at position 702 of intron 1 (Int-1702) of the para Voltage-Gated Sodium Channel (VGSC) gene on chromosome arm 2 L. Intron-1 sequence data from West and Central Africa revealed two clearly distinct and species-specific haplogroups, each characterized by very low polymorphism, which has been attributed to a selective sweep. The aim of this study was to analyse Int-1 sequence diversity in A. gambiae and A. coluzzii populations from the Far-West of their range, in order to assess whether this selective-sweep signature could persist in a zone of high interspecific hybridization. Methods A 531 bp region of VGSC Int-1 was sequenced in 21 A. coluzzii, 31 A. gambiae, and 12 hybrids from The Gambia and Guinea Bissau, located within the Far-West geographical region, and in 53 A. gambiae s.l. samples from the rest of the range. Results Far-West samples exhibit dramatic Int-1 polymorphism, far higher within each country than observed throughout the rest of the species range. Moreover, patterning of haplotypes within A. coluzzii confirms previous evidence of a macro-geographic subdivision into a West and a Central African genetic cluster, and reveals a possible genetic distinction of A. coluzzii populations from the Far-West. Conclusions The results suggest a relaxation of selective pressures acting across the VGSC gene region in the hybrid zone. Genetic differentiation in the Far-West could be attributable to a founder effect within A. coluzzii, with subsequent extensive gene flow with secondarily-colonizing A. gambiae, potentially yielding a novel insight on the dynamic processes impacting genetic divergence of these key malaria vectors. Electronic supplementary material The online version of this article (doi:10.1186/s12936-014-0522-1) contains supplementary material, which is available to authorized users.

Background Anopheles gambiae M and S molecular forms, the major malaria vectors in the Afro-tropical region, are ongoing a process of ecological diversification and adaptive lineage splitting, which is affecting malaria transmission and vector control strategies in West Africa. These two incipient species are defined on the basis of single nucleotide differences in the IGS and ITS regions of multicopy rDNA located on the X-chromosome. A number of PCR and PCR-RFLP approaches based on form-specific SNPs in the IGS region are used for M and S identification. Moreover, a PCR-method to detect the M-specific insertion of a short interspersed transposable element (SINE200) has recently been introduced as an alternative identification approach. However, a large-scale comparative analysis of four widely used PCR or PCR-RFLP genotyping methods for M and S identification was never carried out to evaluate whether they could be used interchangeably, as commonly assumed. Results The genotyping of more than 400 A. gambiae specimens from nine African countries, and the sequencing of the IGS-amplicon of 115 of them, highlighted discrepancies among results obtained by the different approaches due to different kinds of biases, which may result in an overestimation of MS putative hybrids, as follows: i) incorrect match of M and S specific primers used in the allele specific-PCR approach; ii) presence of polymorphisms in the recognition sequence of restriction enzymes used in the PCR-RFLP approaches; iii) incomplete cleavage during the restriction reactions; iv) presence of different copy numbers of M and S-specific IGS-arrays in single individuals in areas of secondary contact between the two forms. Conclusions The results reveal that the PCR and PCR-RFLP approaches most commonly utilized to identify A. gambiae M and S forms are not fully interchangeable as usually assumed, and highlight limits of the actual definition of the two molecular forms, which might not fully correspond to the two A. gambiae incipient species in their entire geographical range. These limits are discussed and operational suggestions on the choice of the most convenient method for large-scale M- and S-form identification are provided, also taking into consideration technical aspects related to the epidemiological characteristics of different study areas.

A molecular analysis was carried out on the European hermit beetles (the Osmoderma eremita species complex) to explore their genetic diversification and the robustness of previous morphologically based taxonomic arrangements. Complete sequences of mtDNA cytochrome C oxidase I gene were obtained from 26 individuals. Mean levels of interspecific sequence divergence ranged from 0.044 to 0.186. The results indicate a clear-cut distinction between two clades. The first one includes the W-European O. eremita Scopoli 1763; and the two Italian endemic taxa Osmoderma italicum Sparacio, 2000 and Osmoderma cristinae Sparacio, 1994; from southern peninsular Italy and Sicily, respectively. The second one includes the widespread E-European Osmoderma barnabita; and the southern Balcanic Osmoderma lassallei Baraud and Tauzin, 1991 from Greece and European Turkey. Within the two clades, molecular and morphological data well support a specific rank for O. lassallei and O. barnabita on one side, and for O. eremita and O. cristinae on the other side, while the taxonomic position of O. italicum, more closely related to O. eremita, is still uncertain. Current geographical distribution, interspecific genetic diversification, and very low levels of intraspecific genetic divergence in western European populations of O. eremita sensu stricto are hypothesized to be the result of multiple speciation events (mainly occurred in refugial forest areas of the Italian and Balkan peninsulas and Sicily before and during the Pleistocene glacial peaks), followed by fast post-glacial northward and westward expansion of some species. Key words: cytochrome C oxidase I gene – molecular taxonomy – sibling species – threatened species – Osmoderma – Coleoptera

A comparative analysis of ITS2 sequences and secondary structures in 89 species of pollen beetles of the subfamily Meligethinae (Coleoptera, Nitidulidae) was performed. The ITS2 folding pattern was highly conserved and comparable with the general model proposed for eukaryotes. Simple sequence repeats (SSRs) were responsible for most of the observed nucleotide variability (approximately 1-3%) and length variation (359-459bp). When plotted on secondary structures, SSRs mapped in expansion segments positioned at the apices of three ITS2 helices ('A', 'B' and 'D1') and appeared to have evolved under mechanisms of compensatory slippage. Homologies among SSRs nucleotides could not be unambiguously assigned, and thus were not useful to resolve phylogeny. However, slippage-derived motifs provided some preliminary genetic support for newly proposed taxonomic arrangements of several genera and subgenera of Meligethinae, corroborating existing morphological and ecological datasets.

Background SINEs (Short INterspersed Elements) are homoplasy-free and co-dominant genetic markers which are considered to represent useful tools for population genetic studies, and could help clarifying the speciation processes ongoing within the major malaria vector in Africa, Anopheles gambiae s.s. Here, we report the results of the analysis of the insertion polymorphism of a nearly 200 bp-long SINE (SINE200) within genome areas of high differentiation (i.e. "speciation islands") of M and S A. gambiae molecular forms. Methods A SINE-PCR approach was carried out on thirteen SINE200 insertions in M and S females collected along the whole range of distribution of A. gambiae s.s. in sub-Saharan Africa. Ten specimens each for Anopheles arabiensis, Anopheles melas, Anopheles quadriannulatus A and 15 M/S hybrids from laboratory crosses were also analysed. Results Eight loci were successfully amplified and were found to be specific for A. gambiae s.s.: 5 on 2L chromosome and one on X chromosome resulted monomorphic, while two loci positioned respectively on 2R (i.e. S200 2R12D) and X (i.e. S200 X6.1) chromosomes were found to be polymorphic. S200 2R12D was homozygote for the insertion in most S-form samples, while intermediate levels of polymorphism were shown in M-form, resulting in an overall high degree of genetic differentiation between molecular forms (Fst = 0.46 p < 0.001) and within M-form (Fst = 0.46 p < 0.001). The insertion of S200 X6.1 was found to be fixed in all M- and absent in all S-specimens. This led to develop a novel easy-to-use PCR approach to straightforwardly identify A. gambiae molecular forms. This novel approach allows to overcome the constraints associated with markers on the rDNA region commonly used for M and S identification. In fact, it is based on a single copy and irreversible SINE200 insertion and, thus, is not subjected to peculiar evolutionary patterns affecting rDNA markers, e.g. incomplete homogenization of the arrays through concerted evolution and/or mixtures of M and S IGS-sequences among the arrays of single chromatids. Conclusion The approach utilized allowed to develop new easy-to-use co-dominant markers for the analysis of genetic differentiation between M and S-forms and opens new perspectives in the study of the speciation process ongoing within A. gambiae.

The organization of the mitochondrial DNA (mtDNA) control region (CR) of the pollen beetle Meligethes thalassophilus is described. This mtDNA CR represents the longest sequenced for beetles so far, since the entire nucleotide sequence ranges from approximately 5000 to approximately 5500 bp. The CR of M. thalassophilus is organized in three distinct domains: a conserved domain near the tRNAIle gene, a variable domain flanking the 12S rRNA gene, and a relatively large central tandem array made up of a variable number of approximately 170 bp repeats that is responsible for the intraspecific length variation observed. Like other CRs found in insects, the M. thalassophilus CR contains two long homopolymeric runs that may be involved in mtDNA replication. Furthermore, conserved stem-and-loop structures in the repetitive domain were identified and their possible role in generating length variation is examined. Intraspecific comparison of the tandem repeat elements of M. thalassophilus suggests mechanisms of concerted evolution leading to homogenization of the repetitive region. The utility of such an array of tandem repeats as a genetic marker for assessing population-level variability and evolutionary relationships among populations is discussed. Finally, the technical difficulties found in isolating the mtDNA CR in beetles are remarked upon.

The main Afrotropical malaria vector, Anopheles gambiae sensu stricto, is undergoing a process of sympatric ecological diversification leading to at least two incipient species (the M and S molecular forms) showing heterogeneous levels of divergence across the genome. The physically unlinked centromeric regions on all three chromosomes of these closely related taxa contain fixed nucleotide differences which have been found in nearly complete linkage disequilibrium in geographic areas of no or low M-S hybridization. Assays diagnostic for SNP and structural differences between M and S forms in the three centromeric regions were applied in samples from the western extreme of their range of sympatry, the only area where high frequencies of putative M/S hybrids have been reported. The results reveal a level of admixture not observed in the rest of the range. In particular, we found: i) heterozygous genotypes at each marker, although at frequencies lower than expected under panmixia; ii) virtually all possible genotypic combinations between markers on different chromosomes, although genetic association was nevertheless detected; iii) discordant M and S genotypes at two X-linked markers near the centromere, suggestive of introgression and inter-locus recombination. These results could be indicative either of a secondary contact zone between M and S, or of the maintenance of ancestral polymorphisms. This issue and the perspectives opened by these results in the study of the M and S incipient speciation process are discussed. publishersversion published