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The introduction of non‐native species across the world represents a major global challenge. Retracing invasion origin is an important first step in understanding the invasion process, often requiring detailed sampling within the native range. Insect species frequently host Wolbachia, a widespread endosymbiotic bacterium that manipulates host reproduction to increase infected female fitness. Here, we draw on the spatial variation in infection frequencies of an actively spreading Wolbachia strain wCer2 to investigate the invasion origin of the European cherry fruit fly, Rhagoletis cerasi. This pest of cherries was introduced from Europe to North America within the last decade. First, we screen the introduced fly population for the presence of Wolbachia. The introduced populations lack the wCer2 strain and the strongly associated mitochondrial haplotype, suggesting strain absence due to founder effects with invading individuals originating from wCer2‐uninfected native population(s). To narrow down geographic regions of invasion origin, we perform spatial interpolation of the wCer2 infection frequency across the native range and predict the infection frequency in unsampled regions. For this, we use an extensive dataset of R. cerasi infection covering 238 populations across Europe over 25 years, complemented with 14 additional populations analyzed for this study. We find that R. cerasi was unlikely introduced from wCer2‐infected populations in Central and Western Europe. We propose wCer2‐uninfected populations from Eastern Europe and the Mediterranean region as the most likely candidates for the invasion origin. This work utilizes Wolbachia as an indirect instrument to provide insights into the invasion source of R. cerasi in North America, revealing yet another application for this multifaceted heritable endosymbiont. Given the prevalence of biological invasions, rapidly uncovering invasion origins gives fundamental insights into how invasive species adapt to new environments. [ABSTRACT FROM AUTHOR]

The relationship between phytophagous insects and plants is a central aspect of food webs and ecosystem functioning. The introduction of new species into an environment can have significant impacts on the food web of a native ecosystem. In many cases, there is a lack of knowledge on the biology and feeding behavior of invasive species prior their introduction and in the invaded regions. Gut content analyses of insects have provided valuable information on the host spectrum of insects. However, current approaches are time‐consuming and costly. Here, we describe a new molecular gut content analysis (GCA) approach using the Oxford Nanopore (ONT) Flongle sequencing platform to characterize the plant DNA present in the gut of the highly polyphagous insect species Halyomorpha halys. We demonstrate that this technique efficiently amplifies and correctly identifies plant DNA in a mock community. We performed a feeding experiment to determine the sensitivity of this approach and to assess how long the plant DNA can be detected. All plants used in the feeding experiment were correctly identified and detected after 56 days. Surprisingly, we also detected various plant genera that were not included in the feeding experiment and thus were likely ingested months before the experiment. Our study suggests that the GCA using the ONT Flongle sequencing platform represents a rapid and cost‐efficient diagnosis of the dietary preferences, host range, and the diversity of consumed plant species of pest insects with high precision. [ABSTRACT FROM AUTHOR]

Psyllids are phloem‐feeding insects that can transmit plant pathogens such as phytoplasmas, intracellular bacteria causing numerous plant diseases worldwide. Their microbiomes are essential for insect physiology and may also influence the capacity of vectors to transmit pathogens. Using 16S rRNA gene metabarcoding, we compared the microbiomes of three sympatric psyllid species associated with pear trees in Central Europe. All three species are able to transmit 'Candidatus Phytoplasma pyri', albeit with different efficiencies. Our results revealed potential relationships between insect biology and microbiome composition that varied during psyllid ontogeny and between generations in Cacopsylla pyri and C. pyricola, as well as between localities in C. pyri. In contrast, no variations related to psyllid life cycle and geography were detected in C. pyrisuga. In addition to the primary endosymbiont Carsonella ruddii, we detected another highly abundant endosymbiont (unclassified Enterobacteriaceae). C. pyri and C. pyricola shared the same taxon of Enterobacteriaceae which is related to endosymbionts harboured by other psyllid species from various families. In contrast, C. pyrisuga carried a different Enterobacteriaceae taxon related to the genus Sodalis. Our study provides new insights into host–symbiont interactions in psyllids and highlights the importance of host biology and geography in shaping microbiome structure. [ABSTRACT FROM AUTHOR]

Phytoplasmas are obligatory intracellular bacteria that colonize the phloem of many plant species and cause hundreds of plant diseases worldwide. In nature, phytoplasmas are primarily transmitted by hemipteran vectors. While all phloem‐feeding insects could in principle transmit phytoplasmas, only a limited number of species have been confirmed as vectors. Knowledge about factors that might determine the vector capacity is currently scarce. Here, we characterized the microbiomes of vector and non‐vector species of apple proliferation (AP) phytoplasma 'Candidatus Phytoplasma mali' to investigate their potential role in the vector capacity of the host. We performed high‐throughput 16S rRNA metabarcoding of the two principal AP‐vectors Cacopsylla picta and Cacopsylla melanoneura and eight Cacopsylla species, which are not AP‐vectors but co‐occur in apple orchards. The microbiomes of all species are dominated by Carsonella, the primary endosymbiont of psyllids and a second uncharacterized Enterobacteriaceae endosymbiont. Each Cacopsylla species harboured a species‐specific phylotype of both symbionts. Moreover, we investigated differences between the microbiomes of AP‐vector versus non‐vector species and identified the predominant endosymbionts but also Wolbachia and several minor taxa as potential indicator species. Our study highlights the importance of considering the microbiome in future investigations of potential factors influencing host vector competence. We investigated the potential role of symbiotic bacteria in the acquisition and transmission of phytoplasma. By comparing the two main psyillid vector species of Apple proliferation (AP) phytoplasma and eight co‐occurring species, which are not able to vector AP‐phytoplasma, we found differences in the microbial communities of AP‐vector and non‐vector species, which appear to be driven by the predominant symbionts in both vector species and Wolbachia and several minor taxa in the non‐vector species. In contrast, infection with AP‐phytoplasma did not affect microbiome composition in both vector species. Our study provides new insights into the endosymbiont diversity of Cacopsylla spp. and highlights the importance of considering the microbiome when investigating potential factors influencing host vector competence. [ABSTRACT FROM AUTHOR]

Endosymbiont‐induced cytoplasmic incompatibility (CI) may play an important role in arthropod speciation. However, whether CI consistently becomes associated or coupled with other host‐related forms of reproductive isolation (RI) to impede the transfer of endosymbionts between hybridizing populations and further the divergence process remains an open question. Here, we show that varying degrees of pre‐ and postmating RI exist among allopatric populations of two interbreeding cherry‐infesting tephritid fruit flies (Rhagoletis cingulata and R. indifferens) across North America. These flies display allochronic and sexual isolation among populations, as well as unidirectional reductions in egg hatch in hybrid crosses involving southwestern USA males. All populations are infected by a Wolbachia strain, wCin2, whereas a second strain, wCin3, only co‐infects flies from the southwest USA and Mexico. Strain wCin3 is associated with a unique mitochondrial DNA haplotype and unidirectional postmating RI, implicating the strain as the cause of CI. When coupled with nonendosymbiont RI barriers, we estimate the strength of CI associated with wCin3 would not prevent the strain from introgressing from infected southwestern to uninfected populations elsewhere in the USA if populations were to come into secondary contact and hybridize. In contrast, cytoplasmic–nuclear coupling may impede the transfer of wCin3 if Mexican and USA populations were to come into contact. We discuss our results in the context of the general paucity of examples demonstrating stable Wolbachia hybrid zones and whether the spread of Wolbachia among taxa can be constrained in natural hybrid zones long enough for the endosymbiont to participate in speciation. [ABSTRACT FROM AUTHOR]

Wolbachia is a maternally inherited obligate endosymbiont that can induce a wide spectrum of effects in its host, ranging from mutualism to reproductive parasitism. At the genomic level, recombination within and between strains, transposable elements, and horizontal transfer of strains between host species make Wolbachia an evolutionarily dynamic bacterial system. The invasive cherry fruit fly Rhagoletis cingulata arrived in Europe from North America ~40 years ago, where it now co‐occurs with the native cherry pest R. cerasi. This shared distribution has been proposed to have led to the horizontal transfer of different Wolbachia strains between the two species. To better understand transmission dynamics, we performed a comparative genome study of the strain wCin2 in its native United States and invasive European populations of R. cingulata with wCer2 in European R. cerasi. Previous multilocus sequence genotyping (MLST) of six genes implied that the source of wCer2 in R. cerasi was wCin2 from R. cingulata. However, we report genomic evidence discounting the recent horizontal transfer hypothesis for the origin of wCer2. Despite near identical sequences for the MLST markers, substantial sequence differences for other loci were found between wCer2 and wCin2, as well as structural rearrangements, and differences in prophage, repetitive element, gene content, and cytoplasmic incompatibility inducing genes. Our study highlights the need for whole‐genome sequencing rather than relying on MLST markers for resolving Wolbachia strains and assessing their evolutionary dynamics. [ABSTRACT FROM AUTHOR]

An important criterion for understanding speciation is the geographic context of population divergence. Three major modes of allopatric, parapatric, and sympatric speciation define the extent of spatial overlap and gene flow between diverging populations. However, mixed modes of speciation are also possible, whereby populations experience periods of allopatry, parapatry, and/or sympatry at different times as they diverge. Here, we report clinal patterns of variation for 21 nuclear‐encoded microsatellites and a wing spot phenotype for cherry‐infesting Rhagoletis (Diptera: Tephritidae) across North America consistent with these flies having initially diverged in parapatry followed by a period of allopatric differentiation in the early Holocene. However, mitochondrial DNA (mtDNA) displays a different pattern; cherry flies at the ends of the clines in the eastern USA and Pacific Northwest share identical haplotypes, while centrally located populations in the southwestern USA and Mexico possess a different haplotype. We hypothesize that the mitochondrial difference could be due to lineage sorting but more likely reflects a selective sweep of a favorable mtDNA variant or the spread of an endosymbiont. The estimated divergence time for mtDNA suggests possible past allopatry, secondary contact, and subsequent isolation between USA and Mexican fly populations initiated before the Wisconsin glaciation. Thus, the current genetics of cherry flies may involve different mixed modes of divergence occurring in different portions of the fly's range. We discuss the need for additional DNA sequencing and quantification of prezygotic and postzygotic reproductive isolation to verify the multiple mixed‐mode hypothesis for cherry flies and draw parallels from other systems to assess the generality that speciation may commonly involve complex biogeographies of varying combinations of allopatric, parapatric, and sympatric divergence. [ABSTRACT FROM AUTHOR]

Elucidating the mechanisms and conditions facilitating the formation of biodiversity are central topics in evolutionary biology. A growing number of studies imply that divergent ecological selection may often play a critical role in speciation by counteracting the homogenising effects of gene flow. Several examples involve phytophagous insects, where divergent selection pressures associated with host plant shifts may generate reproductive isolation, promoting speciation. Here, we use ddRADseq to assess the population structure and to test for host‐related genomic differentiation in the European cherry fruit fly, Rhagoletis cerasi (L., 1758) (Diptera: Tephritidae). This tephritid is distributed throughout Europe and western Asia, and has adapted to two different genera of host plants, Prunus spp. (cherries) and Lonicera spp. (honeysuckle). Our data imply that geographic distance and geomorphic barriers serve as the primary factors shaping genetic population structure across the species range. Locally, however, flies genetically cluster according to host plant, with consistent allele frequency differences displayed by a subset of loci between Prunus and Lonicera flies across four sites surveyed in Germany and Norway. These 17 loci display significantly higher FST values between host plants than others. They also showed high levels of linkage disequilibrium within and between Prunus and Lonicera flies, supporting host‐related selection and reduced gene flow. Our findings support the existence of sympatric host races in R. cerasi embedded within broader patterns of geographic variation in the fly, similar to the related apple maggot, Rhagoletis pomonella, in North America. [ABSTRACT FROM AUTHOR]

The shift of the fruit fly Rhagoletis pomonella (Walsh) in the mid‐1800s from downy hawthorn, Crataegus mollis (Torrey & Asa Gray) Scheele, to introduced domesticated apple, Malus domestica (Borkhausen), in the eastern USA is a model for ecological divergence with gene flow. A similar system may exist in the northwestern USA and British Columbia, Canada, where Rhagoletis indifferens Curran (Diptera: Tephritidae) attacks the native bitter cherry Prunus emarginata (Douglas ex Hooker) Eaton (Rosaceae). Populations of R. indifferens have shifted and became economic pests on domesticated sweet cherry, Prunus avium (L.) L., shortly after sweet cherries were introduced to the region in the mid‐1800s. The fruiting phenologies of the two cherries differ in a similar manner as apples and hawthorns, with domesticated sweet cherries typically ripening in June and July, and bitter cherries in July and August. Here we report, however, little evidence for genetic differentiation between bitter vs. sweet cherry populations of R. indifferens or for pronounced genetic associations between allele frequencies and adult eclosion time, as has been documented for apple and downy hawthorn flies. The current findings support a previous more geographically limited survey of R. indifferens in the province of British Columbia, Canada, and an analysis of its sister species, R. cingulata, in the state of Michigan, USA, implying a lack of host‐related differentiation for flies infesting different cherry host plants. Possible causes for why host races are readily genetically detected for R. pomonella but not for R. indifferens are discussed. We report little evidence for genetic differentiation between bitter vs. sweet cherry populations of Rhagoletis indifferens (Diptera: Tephritidae) or for pronounced genetic associations between allele frequencies and adult eclosion time, as has been documented for apple and downy hawthorn‐infesting host races of Rhagoletis pomonella flies. Possible causes for why host races are readily genetically detected for R. pomonella but not for R. indifferens are discussed. [ABSTRACT FROM AUTHOR]

Haplodiploidy is a sex determination system in which fertilized diploid eggs develop into females and unfertilized haploid eggs develop into males. The evolutionary explanations for this phenomenon include the possibility that haplodiploidy can be reinforced by infection with endosymbiotic bacteria, such as Wolbachia. The subfamily Scolytinae contains species with haplodiploid and diploid sex determination systems. Thus, we studied the association with Wolbachia in 12 diploid and 11 haplodiploid scolytine beetles by analyzing wsp and multilocus sequence typing (MLST) of five loci in this endosymbiont. Wolbachia genotypes were compared with mitochondrial (COI) and nuclear (EF) genotypes in the scolytines. Eight of the 23 scolytine species were infected with Wolbachia, with haplodiploids at significantly higher rates than diploid species. Cloning and sequencing detected multiple infections with up to six Wolbachia strains in individual species. Phylogenetic analyses of wsp and five MLST genes revealed different Wolbachia strains in scolytines. Comparisons between the beetle and Wolbachia phylogenies revealed that closely related beetles were infected with genetically different Wolbachia strains. These results suggest the horizontal transmission of multiple Wolbachia strains between scolytines. We discuss these results in terms of the evolution of different sex determination systems in scolytine beetles. [ABSTRACT FROM AUTHOR]

Wolbachia is a maternally inherited and ubiquitous endosymbiont of insects. It can hijack host reproduction by manipulations such as cytoplasmic incompatibility ( CI) to enhance vertical transmission. Horizontal transmission of Wolbachia can also result in the colonization of new mitochondrial lineages. In this study, we present a 15-year-long survey of Wolbachia in the cherry fruit fly Rhagoletis cerasi across Europe and the spatiotemporal distribution of two prevalent strains, wCer1 and wCer2, and associated mitochondrial haplotypes in Germany. Across most of Europe, populations consisted of either 100% singly ( wCer1) infected individuals with haplotype HT1, or 100% doubly ( wCer1&2) infected individuals with haplotype HT2, differentiated only by a single nucleotide polymorphism. In central Germany, singly infected populations were surrounded by transitional populations, consisting of both singly and doubly infected individuals, sandwiched between populations fixed for wCer1&2. Populations with fixed infection status showed perfect association of infection and mitochondria, suggesting a recent CI-driven selective sweep of wCer2 linked with HT2. Spatial analysis revealed a range expansion for wCer2 and a large transition zone in which wCer2 splashes appeared to coalesce into doubly infected populations. Unexpectedly, the transition zone contained a large proportion (22%) of wCer1&2 individuals with HT1, suggesting frequent intraspecific horizontal transmission. However, this horizontal transmission did not break the strict association between infection types and haplotypes in populations outside the transition zone, suggesting that this horizontally acquired Wolbachia infection may be transient. Our study provides new insights into the rarely studied Wolbachia invasion dynamics in field populations. [ABSTRACT FROM AUTHOR]

Geography is often a key factor facilitating population divergence and speciation. In this regard, the geographic distributions of flies in the genus Rhagoletis ( Diptera: Tephritidae) in temperate North America have been affected by cycles of Pleistocene glaciation and interglacial periods. Fluctuations in climatic conditions may have had their most dramatic effects on geographically isolating Rhagoletis flies in the central highland region of Mexico. During past periods of allopatry, a degree of post-zygotic reproductive isolation appears to have evolved between hawthorn-infesting populations of Rhagoletis pomonella ( Walsh) in the central Eje Volcanico Trans Mexicano ( EVTM) and those from the Sierra Madre Oriental Mountains ( SMO) of Mexico, as well as hawthorn flies from the eastern USA. Here, we investigate the generality of this finding in the genus Rhagoletis by testing for reproductive isolation among populations of Rhagoletis cingulata ( Loew) ( Diptera: Tephritidae) collected from infested domesticated sweet cherry ( Prunus avium L.) in the USA and black cherry [ Prunus serotina Ehrh. (both Rosaceae)] from the SMO and EVTM. We report evidence for marked post-mating reproductive isolation among certain R. cingulata populations. The high levels of reproductive isolation were observed between R. cingulata flies from populations in the USA and SMO differed from the pattern seen for R. pomonella, primarily involving the EVTM. In addition, egg hatch was significantly reduced for crosses between SMO males and EVTM females, but not greatly in the opposite direction. We discuss potential causes for the different patterns of post-mating reproductive isolation among Rhagoletis flies. [ABSTRACT FROM AUTHOR]

The widespread occurrence of Wolbachia in arthropods and nematodes suggests that this intracellular, maternally inherited endosymbiont has the ability to cross species boundaries. However, direct evidence for such a horizontal transmission of Wolbachia in nature is scarce. Here, we compare the well-characterized Wolbachia infection of the European cherry fruit fly, Rhagoletis cerasi, with that of the North American eastern cherry fruit fly, Rhagoletis cingulata, recently introduced to Europe. Molecular genetic analysis of Wolbachia based on multilocus sequence typing and the Wolbachia surface protein wsp showed that all R. cingulata individuals are infected with wCin2 identical to wCer2 in R. cerasi. In contrast, wCin1, a strain identical to wCer1 in R. cerasi, was present in several European populations of R. cingulata, but not in any individual from the United States. Surveys of R. cingulata from Germany and Hungary indicated that in some populations, the frequency of wCin1 increased significantly in just a few years with at least two independent horizontal transmission events. This is corroborated by the analysis of the mitochondrial cytochrome oxidase II gene that showed association of wCin1 with two distinct haplotypes in Germany, one of which is also infected with wCin1 in Hungary. In summary, our study provides strong evidence for a very recent inter-specific Wolbachia transmission with a subsequent spatial spread in field populations. [ABSTRACT FROM AUTHOR]

Ips typographus and Pityogenes chalcographus are two sympatric Palearctic bark beetle species with wide distribution ranges. As both species are comparable in biology, life history, and habitat, including sharing the same host, Picea abies, they provide excellent models for applying a comparative approach in which to identify common historical patterns of population differentiation and the influence of species-specific ecological characteristics. We analysed patterns of genetic diversity, genetic structure and demographic history of ten I. typographus and P. chalcographus populations co-distributed across Europe using both COI and ITS2 markers. Rather than similarities, our results revealed striking differences. Ips typographus was characterised by low genetic diversity, shallow population structure and strong evidence that all extant haplogroups arose via a single Holocene population expansion event. In contrast, genetic variation and structuring were high in P. chalcographus indicating a longer and more complex evolutionary history. This was estimated to be five times older than I. typographus, beginning during the last Pleistocene glacial maximum over 100 000 years ago. Although the expansions of P. chalcographus haplogroups also date to the Holocene or just prior to its onset, we show that these occurred from at least three geographically separated glacial refugia. Overall, these results suggest that the much longer evolutionary history of P. chalcographus greatly influenced the levels of phylogeographic subdivision among lineages and may have led to the evolution of different life-history traits which in turn have affected genetic structure and resulted in an advantage over the more aggressive I. typographus. [ABSTRACT FROM AUTHOR]

The American cherry fruit fly is an invasive pest species in Europe, of serious concern in tart cherry production as well as for the potential to hybridize with the European cherry fruit fly, Rhagoletis cerasi L. ( Diptera: Tephritidae), which might induce new pest dynamics. In the first European reports, the question arose whether only the eastern American cherry fruit fly, Rhagoletis cingulata ( Loew) ( Diptera: Tephritidae), is present, or also the closely related western American cherry fruit fly, Rhagoletis indifferens Curran. In this study, we investigate the species status of European populations by comparing these with populations of both American species from their native ranges, the invasion dynamics in German (first report in 1993) and Hungarian (first report in 2006) populations, and we test for signals of hybridization with the European cherry fruit fly. Although mt DNA sequence genealogy could not separate the two American species, cross-species amplification of 14 microsatellite loci separated them with high probabilities (0.99-1.0) and provided evidence for R. cingulata in Europe. German and Hungarian R. cingulata populations differed significantly in microsatellite allele frequencies, mt DNA haplotype and wing pattern distributions, and both were genetically depauperate relative to North American populations. The diversity suggests independent founding events in Germany and Hungary. Within each country, R. cingulata displayed little or no structure in any trait, which agrees with rapid local range expansions. In cross-species amplifications, signals of hybridization between R. cerasi and R. cingulata were found in 2% of R. cingulata individuals and in 3% of R. cerasi. All putative hybrids had R. cerasi mt DNA indicating that the original between-species mating involved R. cerasi females and R. cingulata males. [ABSTRACT FROM AUTHOR]

Phylogeographic studies call for attention as nuclear copies of mitochondrial DNA (NUMT) may generate erroneous results. Here, we report the presence of NUMTs differing only by 1-3 bp from authentic mitochondrial haplotypes, consequently named cryptic NUMTs. In contrast to traditional NUMTs, for which reliable tools for detection are established, cryptic NUMTs question the validity of phylogeographic analyses based solely on mitochondrial DNA, like the one presented here on the European bark beetle Ips typographus. Caution is called as cryptic NUMTs might be responsible for haplotype richness found in several species, and the necessity of refined methods for NUMT detection is highlighted. [ABSTRACT FROM AUTHOR]

Rhagoletis pomonella Walsh (Diptera: Tephritidae) is a model species for sympatric speciation through host race formation on apple and hawthorn. The bacterial endosymbiont Wolbachia, a manipulator of arthropod reproduction, has been considered to contribute to speciation in several species. A potential role of Wolbachia in sympatric speciation of R. pomonella remains to be tested despite an earlier detection by PCR. In this study, we isolated Wolbachia from R. pomonella individuals from both host species using multi-locus sequence typing (MLST) and the surface protein wsp. By cloning and sequencing of 311 plasmids, we found sequence types of at least four wPom strains. A complete MLST profile was obtained only for wPom1, whereas MLST loci of the other putative strains were difficult to assign because of multiple infections and low sample numbers. wPom1 occurs in both host races, whereas different sequence types were found at low frequencies only in apple-infesting R. pomonella. This warrants further investigation as it cannot be excluded that Wolbachia plays a part in this model of sympatric speciation. [ABSTRACT FROM AUTHOR]