BackgroundAnchored hybrid enrichment is a form of next-generation sequencing that uses oligonucleotide probes to target conserved regions of the genome flanked by less conserved regions in order to acquire data useful for phylogenetic inference from a broad range of taxa. Once a probe kit is developed, anchored hybrid enrichment is superior to traditional PCR-based Sanger sequencing in terms of both the amount of genomic data that can be recovered and effective cost. Due to their incredibly diverse nature, importance as pollinators, and historical instability with regard to subfamilial and tribal classification, Syrphidae (flower flies or hoverflies) are an ideal candidate for anchored hybrid enrichment-based phylogenetics, especially since recent molecular phylogenies of the syrphids using only a few markers have resulted in highly unresolved topologies. Over 6200 syrphids are currently known and uncovering their phylogeny will help us to understand how these species have diversified, providing insight into an array of ecological processes, from the development of adult mimicry, the origin of adult migration, to pollination patterns and the evolution of larval resource utilization.ResultsWe present the first use of anchored hybrid enrichment in insect phylogenetics on a dataset containing 30 flower fly species from across all four subfamilies and 11 tribes out of 15. To produce a phylogenetic hypothesis, 559 loci were sampled to produce a final dataset containing 217,702 sites. We recovered a well resolved topology with bootstrap support values that were almost universally >95 %. The subfamily Eristalinae is recovered as paraphyletic, with the strongest support for this hypothesis to date. The ant predators in the Microdontinae are sister to all other syrphids. Syrphinae and Pipizinae are monophyletic and sister to each other. Larval predation on soft-bodied hemipterans evolved only once in this family.ConclusionsAnchored hybrid enrichment was successful in producing a robustly supported phylogenetic hypothesis for the syrphids. Subfamilial reconstruction is concordant with recent phylogenetic hypotheses, but with much higher support values. With the newly designed probe kit this analysis could be rapidly expanded with further sampling, opening the door to more comprehensive analyses targeting problem areas in syrphid phylogenetics and ecology.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-016-0714-0) contains supplementary material, which is available to authorized users.

Intraspecific phylogeographical patterns largely depend on the life history traits of a species. Especially species with a high degree of cold tolerance, limited requirements towards habitat preferences, and relatively low active dispersal capacities may have responded in a different way to the Pleistocene climatological fluctuations than the majority of taxa studied so far. To evaluate this possibility, we studied Arion fuscus (Muller, 1774), a common and widespread European terrestrial slug, from 88 locations (N = 964). Sequence variation was assessed for fragments of the mitochondrial 16S rDNA and COI genes by means of single-strand conformation polymorphisms (SSCP) and subsequent DNA sequencing. Additionally, eight allozyme loci were scored in 843 individuals. Phylogenetic analysis revealed the presence of two major evolutionary lineages, one in the Balkan region and another in the Alps and the rest of Europe. The sequence divergence between the two lineages was limited (3.3%), but gene flow between the regions was absent, suggesting that the two regions have been isolated since the late Pliocene or early Pleistocene. Allozyme differentiation among geographical regions and mitochondrial DNA (mtDNA) lineages was low. The geographical patterns observed in our data showed that (i) haplotype and nucleotide diversities are very low in northern Europe, suggesting that single haplotypes rapidly colonized large areas; (ii) recently expanded haplotype clades have restricted distribution ranges, suggesting that current gene flow is low; and (iii) genetic diversity in the Alps is much higher than in other regions and estimated past gene flow from the Eastern Alps to other regions was high, suggesting that this was a refugial zone during the Pleistocene. This full-range phylogeography suggests the existence of an alternative refugial zone, situated north of the refugial areas currently recognized in most other taxa.

The importance and abundance of cryptic species among invertebrate taxa is well documented. Nowadays, taxonomic, phylogenetic and conservation biological studies frequently use molecular markers to delineate cryptic taxa. Such studies, however, often face the problem of the differential resolution of the molecular markers and techniques involved. This issue is explored in the present study of cryptic taxa within the terrestrial slug complex Arion subfuscus/fuscus in continental north-west Europe. To this end, morphological, allozyme and mitochondrial 16S rDNA sequence data have been jointly evaluated. Using allozyme data and gonad type, two distinct groups were consistently delineated, even under sympatric conditions. The 16S rDNA data strongly supported both those groups and even suggested the presence of three distinct taxa within one of them. However, in view of: (1) the allopatric distribution of three OTUs, (2) the lack of allozyme or morphological differentiation, and (3) the extremely high degree of intraspecific mtDNA variation reported in pulmonate gastropods, they are, for the time being, not regarded as valid species under the biological species concept. By means of 16S rDNA and allozyme data, the position of type and topotype material of A. subfuscus s.s. and A. fuscus relative to the newly defined OTUs was determined, thus clarifying the nomenclature of this species complex. Additionally, gonad type proved to be a useful character for distinguishing the two species in north-west Europe.

We propose a general working strategy to deal with incomplete reference libraries in the DNA barcoding identification of species. Considering that (1) queries with a large genetic distance with their best DNA barcode match are more likely to be misidentified and (2) imposing a distance threshold profitably reduces identification errors, we modelled relationships between identification performances and distance thresholds in four DNA barcode libraries of Diptera (n = 4270), Lepidoptera (n = 7577), Hymenoptera (n = 2067) and Tephritidae (n = 602 DNA barcodes). In all cases, more restrictive distance thresholds produced a gradual increase in the proportion of true negatives, a gradual decrease of false positives and more abrupt variations in the proportions of true positives and false negatives. More restrictive distance thresholds improved precision, yet negatively affected accuracy due to the higher proportions of queries discarded (viz. having a distance query-best match above the threshold). Using a simple linear regression we calculated an ad hoc distance threshold for the tephritid library producing an estimated relative identification error <0.05. According to the expectations, when we used this threshold for the identification of 188 independently collected tephritids, less than 5% of queries with a distance query-best match below the threshold were misidentified. Ad hoc thresholds can be calculated for each particular reference library of DNA barcodes and should be used as cut-off mark defining whether we can proceed identifying the query with a known estimated error probability (e.g. 5%) or whether we should discard the query and consider alternative/complementary identification methods.

No abstract

A popular hypothesis to explain the high degree of host specialisation observed among mites and insects is the existence of host-associated fitness trade-offs. According to this theory, adaptation to a host results in a relatively poorer performance on alternative hosts due to the antagonistic pleiotropic action of one or more genes. Evidence in favour of the genetic trade-off hypothesis is however scarce. Recent ecological work has shown that the optimisation of adult performance drives the evolution of host choice in at least some phytophagous insects. Yet, most ecological and evolutionary studies on host choice assume that females maximise their fitness by optimising offspring performance. In this paper, we investigate whether a general lack of attention for the role of adult performance in host choice may have diminished the chance of detecting genetic trade-offs. We reviewed the literature on genetic trade-offs and showed that most studies neglected host specific variation in adult performance. Moreover, studies that considered both adult and offspring performance had a higher chance of detecting genetic fitness trade-offs. Our results also suggested that studies on asexual reproducing species tend to detect trade-offs more often than studies on obligate sexual reproducing species. We argue that future studies on genetic trade-offs should take all fitness parameters into account in order to be conclusive. This approach may reveal (i) that genetic trade-offs are more common than hitherto reported and/or (ii) that genetic trade-offs are more common, or more easily detected among asexual reproducing species like mites and aphids.

Extremely high levels of intraspecific mtDNA differences in pulmonate gastropods have been reported repeatedly and several hypotheses to explain them have been postulated. We studied the phylogeny and phylogeography of 51 populations (n = 843) of the highly polymorphic terrestrial slug Arion subfuscus (Draparnaud, 1805) across its native distribution range in Western Europe. By combining the analysis of single stranded conformation polymorphisms (SSCP) and nucleotide sequencing, we obtained individual sequence data for a fragment of the mitochondrial 16S rDNA and a fragment of the nuclear ITS1. Additionally, five polymorphic allozyme loci were scored. Based on the 16S rDNA phylogeny, five monophyletic haplotype groups with sequence divergences of 9–21% were found. Despite this deep mitochondrial divergence, the haplotype groups were not monophyletic for the nuclear ITS1 fragment and haplotype group‐specific allozyme alleles were not found. Although there is evidence for an accelerated mtDNA clock, the divergence among the haplotype groups is older than the Pleistocene and their current allopatric ranges probably reflect allopatric divergence and glacial survival in separate refugia from which different post‐glacial colonization routes were established. A range‐overlap of two mtDNA groups (S1 and S2, 21% sequence divergence) stretched from Central France and Belgium up to the North of the British Isles. The nuclear data suggest that this secondary contact resulted in hybridization between the allopatrically diverged groups. Therefore, it seems that, at least for two of the groups, the deep mtDNA divergence was only partially accompanied by the formation of reproductive isolation.

Delimiting species in facultatively selfing taxa is a challenging problem of which the terrestrial pulmonate snail genus Rumina is a good example. These snails have a mixed breeding system and show a high degree of shell and color variation. Three nominal species (R. decollata, R. saharica and R. paivae) and two color morphs within R. decollata (dark and light) are currently recognized. The present study aims at evaluating to what extent these entities reflect evolutionary diverging taxonomic units, rather than fixed polymorphisms due to sustained selfing. Therefore, a phylogenetic analysis of nuclear (ITS1, ITS2) and mitochondrial DNA (COI, CytB, 12S rDNA, 16S rDNA) sequences was performed. Putative species in Rumina, inferred from the mitochondrial DNA phylogeny, were compared with those proposed on the basis of the COI gene by (1) DNA barcoding gap analysis, (2) Automatic Barcode Gap Discovery, (3) the species delimitation plug-in of the Geneious software, (4) the Genealogical Sorting Index, and (5) the General Mixed Yule Coalescent model. It is shown that these methods produce a variety of different species hypotheses and as such one may wonder to what extent species delimitation methods are really useful. With respect to Rumina, the data suggest at least seven species, one corresponding to R. saharica and six that are currently grouped under the name R. decollata. The species-level status of R. paivae is rejected.