Hannes Schuler

Hit or miss in phylogeographic analyses: the case of the cryptic NUMTs

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.

Evidence for a recent horizontal transmission and spatial spread of Wolbachia from endemic Rhagoletis cerasi (Diptera: Tephritidae) to invasive Rhagoletis cingulata in Europe.

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.

Divergent evolutionary histories of two sympatric spruce bark beetle species

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.

The hitchhiker's guide to Europe: the infection dynamics of an ongoing Wolbachia invasion and mitochondrial selective sweep in Rhagoletis cerasi

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.

Allele Intersection Analysis: A Novel Tool for Multi Locus Sequence Assignment in Multiply Infected Hosts

Wolbachia are wide-spread, endogenous α-Proteobacteria of arthropods and filarial nematodes. 15–75% of all insect species are infected with these endosymbionts that alter their hosts reproduction to facilitate their spread. In recent years, many insect species infected with multiple Wolbachia strains have been identified. As the endosymbionts are not cultivable outside living cells, strain typing relies on molecular methods. A Multi Locus Sequence Typing (MLST) system was established for standardizing Wolbachia strain identification. However, MLST requires hosts to harbour individual and not multiple strains of supergroups without recombination. This study revisits the applicability of the current MLST protocols and introduces Allele Intersection Analysis (AIA) as a novel approach. AIA utilizes natural variations in infection patterns and allows correct strain assignment of MLST alleles in multiply infected host species without the need of artificial strain segregation. AIA identifies pairs of multiply infected individuals that share Wolbachia and differ in only one strain. In such pairs, the shared MLST sequences can be used to assign alleles to distinct strains. Furthermore, AIA is a powerful tool to detect recombination events. The underlying principle of AIA may easily be adopted for MLST approaches in other uncultivable bacterial genera that occur as multiple strain infections and the concept may find application in metagenomic high-throughput parallel sequencing projects.

Multiple Wolbachia infections in Rhagoletis pomonella

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.

Invasion genetics of American cherry fruit fly in Europe and signals of hybridization with the European cherry fruit fly

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 mtDNA 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, mtDNA 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 mtDNA indicating that the original between‐species mating involved R. cerasi females and R. cingulata males.

Wolbachia endosymbionts in haplodiploid and diploid scolytine beetles (Coleoptera: Curculionidae: Scolytinae).

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.

Wolbachia in Parasitoids Attacking Native European and Introduced Eastern Cherry Fruit Flies in Europe

Abstract The eastern cherry fruit fly, Rhagoletis cingulata Loew (Diptera: Tephritidae), is an economically important pest of cherries in North America. In 1983 it was first reported in Europe where it shares its ecological niche with the native European cherry fruit fly, Rhagoletis cerasi L. (Diptera: Tephritidae). Their coexistence in Europe led to the recent horizontal transmission of the Wolbachia strain wCer1 from R. cerasi to R. cingulata. Horizontal Wolbachia transmission is mediated by either sharing of ecological niches or by interacting species such as parasitoids. Here we describe for the first time that two braconid wasps, Psyttalia rhagoleticola Sachtleben (Hymenoptera: Braconidae) and Utetes magnus Fischer (Hymenoptera: Braconidae), naturally parasitizing R. cerasi, use the invasive R. cingulata in Europe as a new host. In contrast, no parasitoids that parasitize R. cingulata in its native American range were detected in the introduced European range. Diagnostic Wolbachia PCR screening and sequence analyses demonstrated that all P. rhagoleticola individuals were infected with the newly described Wolbachia strain wRha while all U. magnus individuals were uninfected. wRha is different from wCer1 but had an Wolbachia surface protein (wsp) gene sequence that was identical to wCer2 of R. cerasi and wCin2 of R. cingulata. However, multi locus sequence typing revealed differences in all loci between wRha and the tephritids strains. The horizontal transmission of wCer1 between the two tephritid species did not result in fixed heritable infections in the parasitoids. However, the parasitoids may have acted as a transient wCer1 vector.

Reproductive Manipulators in the Bark Beetle Pityogenes chalcographus (Coleoptera: Curculionidae)—The Role of Cardinium, Rickettsia, Spiroplasma, and Wolbachia

Abstract Heritable bacterial endosymbionts can alter the biology of numerous arthropods. They can influence the reproductive outcome of infected hosts, thus affecting the ecology and evolution of various arthropod species. The spruce bark beetle Pityogenes chalcographus (L.) (Coleoptera: Curculionidae: Scolytinae) was reported to express partial, unidirectional crossing incompatibilities among certain European populations. Knowledge on the background of these findings is lacking; however, bacterial endosymbionts have been assumed to manipulate the reproduction of this beetle. Previous work reported low-density and low-frequency Wolbachia infections of P. chalcographus but found it unlikely that this infection results in reproductive alterations. The aim of this study was to test the hypothesis of an endosymbiont-driven incompatibility, other than Wolbachia, reflected by an infection pattern on a wide geographic scale. We performed a polymerase chain reaction (PCR) screening of 226 individuals from 18 European populations for the presence of the endosymbionts Cardinium, Rickettsia, and Spiroplasma, and additionally screened these individuals for Wolbachia. Positive PCR products were sequenced to characterize these bacteria. Our study shows a low prevalence of these four endosymbionts in P. chalcographus. We detected a yet undescribed Spiroplasma strain in a single individual from Greece. This is the first time that this endosymbiont has been found in a bark beetle. Further, Wolbachia was detected in three beetles from two Scandinavian populations and two new Wolbachia strains were described. None of the individuals analyzed were infected with Cardinium and Rickettsia. The low prevalence of bacteria found here does not support the hypothesis of an endosymbiont-driven reproductive incompatibility in P. chalcographus.