Wolfgang Arthofer

Hidden Wolbachia diversity in field populations of the European cherry fruit fly, Rhagoletis cerasi (Diptera, Tephritidae).

The European cherry fruit fly Rhagoletis cerasi has been a field model for cytoplasmic incompatibility since the mid 1970s. Two Wolbachia strains were detected in this tephritid species and wCer2 was described as the CI inducing agent dividing European populations into two unidirectional incompatible groups, i.e. southern females produce viable offspring with northern males, whereas the reciprocal cross results in incompatibility. We detected three new Wolbachia strains by sequencing a multitude of plasmids derived from Wolbachia surface protein gene (wsp) polymerase chain reaction (PCR) products. Strain‐specific primers were developed allowing individual diagnosis without need for cloning. Hybridization of specific PCR products with a wsp oligonucleotide enhanced the detection limit significantly and revealed the presence of low‐titre infections in some strains, in different ontogenetic stages and in adults of different age. We then performed a survey of strain prevalence and infection frequency in eight European regions. wCer1 was fixed in all populations, whereas wCer2 was detected only in the South. wCer3 frequency was the lowest without a clear distribution pattern. The abundance of wCer4 was homogenous across Europe. Like wCer2, wCer5 showed significant differences in spatial distribution. Our new findings of previously undetected and recombinant Wolbachia strains in R.u2003cerasi reveal a major caveat to the research community not to overlook hidden Wolbachia diversity in field populations. Low‐titres and geographical variability in Wolbachia diversity are expected to influence the outcome of Wolbachia population dynamics and Wolbachia‐based insect population control and may create invasion barriers for expanding and artificially introduced Wolbachia strains.

Permanent Genetic Resources added to Molecular Ecology Resources Database 1 April 2010-31 May 2010

This article documents the addition of 396 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Anthocidaris crassispina, Aphis glycines, Argyrosomus regius, Astrocaryum sciophilum, Dasypus novemcinctus, Delomys sublineatus, Dermatemys mawii, Fundulus heteroclitus, Homalaspis plana, Jumellea rossii, Khaya senegalensis, Mugil cephalus, Neoceratitis cyanescens, Phalacrocorax aristotelis, Phytophthora infestans, Piper cordulatum, Pterocarpus indicus, Rana dalmatina, Rosa pulverulenta, Saxifraga oppositifolia, Scomber colias, Semecarpus kathalekanensis, Stichopus monotuberculatus, Striga hermonthica, Tarentola boettgeri and Thermophis baileyi. These loci were cross‐tested on the following species: Aphis gossypii, Sooretamys angouya, Euryoryzomys russatus, Fundulus notatus, Fundulus olivaceus, Fundulus catenatus, Fundulus majalis, Jumellea fragrans, Jumellea triquetra Jumellea recta, Jumellea stenophylla, Liza richardsonii, Piper marginatum, Piper aequale, Piper darienensis, Piper dilatatum, Rana temporaria, Rana iberica, Rana pyrenaica, Semecarpus anacardium, Semecarpus auriculata, Semecarpus travancorica, Spondias acuminata, Holigarna grahamii, Holigarna beddomii, Mangifera indica, Anacardium occidentale, Tarentola delalandii, Tarentola caboverdianus and Thermophis zhaoermii.

Evidence for low-titre infections in insect symbiosis: Wolbachia in the bark beetle Pityogenes chalcographus (Coleoptera, Scolytinae).

Wolbachia are obligatory endosymbiotic alpha-proteobacteria found in many insect species. They are maternally transmitted and often exhibit reproductive phenotypes like cytoplasmic incompatibility. Pityogenes chalcographus is a bark beetle causing severe damage in spruce stands. Its European populations are divided into several mitochondrial clades separated by partial crossing barriers. In this study, we tested a large sample set covering the natural range of the beetle in Europe for the presence of Wolbachia and associations between infection pattern and mitotypes using a highly sensitive nested PCR technique. 35.5% of the individuals were infected with the endosymbiont and two distinct strains were identified. Both strains occur in low titre not accessible by conventional detection methods. The infections are present all over Europe, unlikely to cause the partial crossing barriers in this host and uncoupled from mitochondrial clades. This pattern is indicative for populations evolving towards endosymbiont loss and for repeated intraspecific horizontal transfer of Wolbachia. Alternatively, the low-titre infections found in P. chalcographus are yet another example for Wolbachia that can persist in host species at low densities and frequencies.

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 1–3u2003bp 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.

tinyFLP and tinyCAT: software for automatic peak selection and scoring of AFLP data tables

Amplified fragment length polymorphism (AFLP) is a well‐established method for DNA fingerprinting. In the last years, fragment separation gradually changed from using gel to capillary electrophoresis, resulting in large, digital data sets. Automatic scoring softwares available to date are proprietary and expensive. tinyFLP/tinyCAT is a compact, open‐source software for automatic scoring of AFLP data. It reads from PeakScanner© generated tables, optimizes the conversion of peak data into a binary allelic matrix and removes operator‐mediated ambiguities from the scoring process. The output is formatted for direct use in MrBayes and other phylogenetic applications.

Abandoning Aggression but Maintaining Self-Nonself Discrimination as a First Stage in Ant Supercolony Formation

An ant supercolony is a very large entity with very many queens. Although normal colonies of small extent and few queens remain distinct, a supercolony is integrated harmoniously over a large area [1, 2]. The lack of aggression is advantageous: Aggression is costly, involving direct and indirect losses and recognition errors [3, 4]. Indeed, supercolonial ants are among the ecologically most successful organisms [5-7]. But how supercolonies arise remains mysterious [1, 2, 8]. Suggestions include that reduced within-colony relatedness or reduced self-nonself discrimination would foster supercolony formation [1, 2, 5, 7, 9-12]. However, one risks confusing correlation and causality in deducing the evolution from distinct colonies to supercolonies when observing established supercolonies. It might help to follow up observations of another lack of aggression, that between single-queened colonies in some ant species. We show that the single-queened Lasius austriacus lacks aggression between colonies and occasionally integrates workers across colonies but maintains high within-colony relatedness and self-nonself discrimination. Provided that the ecological framework permits, reduced aggression might prove adaptive for any ant colony irrespective of within-colony relatedness. Abandoning aggression while maintaining discrimination might be a first stage in supercolony formation. This adds to the emphasis of ecology as central to the evolution of cooperation in general [13].

Rapid and cost-effective screening of newly identified microsatellite loci by high-resolution melting analysis

This study describes a new method for identifying microsatellite loci that will reliably amplify and show high degree of polymorphism in a given species. Microsatellites are the most powerful codominant markers available today, but the development of novel loci remains a labour-intensive and expensive process. In de novo isolation, approaches using next generation sequencing (NGS) are gradually replacing ones using Escherichia coli libraries, resulting in unparalleled numbers of candidate loci available. We present a systematic review of published microsatellite primer notes and show that, on average, about half of all candidate loci are lost due to insufficient PCR amplification, monomorphism or multicopy status in the genome, no matter what isolation strategy is used. Thus, the screening of candidate loci remains a major step in marker development. Re-assessing capillary-electrophoresis genotyped loci via high-resolution melting analysis (HRM), we evaluate the usefulness of HRM for this step. We demonstrate its applicability in a genotyping case study and introduce a fast, HRM-based workflow for the screening of microsatellite loci. This workflow may readily be applied to NGS-based marker development and has the potential to cut the costs of traditional testing by half to three quarters.

Chloroplast DNA‐based studies in molecular ecology may be compromised by nuclear‐encoded plastid sequence

Ongoing genetic transfer from mitochondria and plastids into the nucleus is a well‐documented fact. While in metazoan molecular ecology the need for surveillance against pseudogene‐mediated artefacts when analysing mtDNA sequences is commonly accepted, no comparable measurements have been established for plastid‐based studies. We highlight the impact and management of nuclear mitochondrial insertions, argue that nuclear plastid sequences represent an underestimated but major factor in plant molecular ecology, and discuss potential avenues of remedy in chloroplast studies.

How diverse is Mitopus morio? Integrative taxonomy detects cryptic species in a small-scale sample of a widespread harvestman

Mitopus morio is a widespread harvestman species occurring in most of Europe and in moderate and cold‐moderate zones of Asia and North America. The species is characterized by extreme variability in body size and leg length. As leg length is correlated with habitat temperature, M. morio has been considered as an example of Allens rule. Recently, observations for a single location in Tyrol, Austria, indicated the absence of mating between short‐ and long‐legged individuals. This study examines for signs of putative cryptic species in M. morio using an integrative approach that combines mating trials, amplified fragment length polymorphism whole‐genome scans, mitochondrial sequences and morphometrics. The mating trials did not corroborate the initial hypothesis of a reproductive barrier associated with leg size. Both types of genetic data revealed the existence of three distinct groups, in line with the mating results but largely unrelated to leg morphology and geographical origin of specimens. Morphometric characters supporting the findings of the other disciplines were identified using a supervised approach. We infer from all data together the existence of strongly diverged cryptic lineages among the analysed individuals, cautiously interpret them as three sympatric species and conclude that in these harvestmen Allens rule applies at different levels. Due to the unexpected amount of differentiation found within a geographical scale very small compared with the distribution of M. morio, we suggest a thorough revision of the genus prior to formal taxonomic changes. Our case study underlines the general applicability of the integrative taxonomic protocol used and highlights the relevance of several rationales implemented in the protocol.