Anna Karenina as a promoter of microbial diversity in the cosmopolitan agricultural pest Zeugodacus cucurbitae (Diptera, Tephritidae).

Gut microbial communities are critical in determining the evolutive success of fruit fly phytophagous pests (Diptera, Tephritidae), facilitating their adaptation to suboptimal environmental conditions and to plant allelochemical defences. An important source of variation for the microbial diversity of fruit flies is represented by the crop on which larvae are feeding. However, a "crop effect" is not always the main driver of microbial patterns, and it is often observed in combination with other and less obvious processes. In this work, we aim at verifying if environmental stress and, by extension, changing environmental conditions, can promote microbial diversity in Zeugodacus cucurbitae (Coquillett), a cosmopolitan pest of cucurbit crops. With this objective, 16S rRNA metabarcoding was used to test differences in the microbial profiles of wild fly populations in a large experimental setup in Eastern Central Tanzania. The analysis of 2,973 unique ASV, which were assigned to 22 bacterial phyla, 221 families and 590 putative genera, show that microbial α diversity (as estimated by Abundance Coverage Estimator, Faith's Phylogenetic Diversity, Shannon-Weiner and the Inverse Simpson indexes) as well as ß microbial diversity (as estimated by Compositional Data analysis of ASVs and of aggregated genera) significantly change as the species gets closer to its altitudinal limits, in farms where pesticides and agrochemicals are used. Most importantly, the multivariate dispersion of microbial patterns is significantly higher in these stressful environmental conditions thus indicating that Anna Karenina effects contribute to the microbial diversity of Z. cucurbitae. The crop effect was comparably weaker and detected as non-consistent changes across the experimental sites. We speculate that the impressive adaptive potential of polyphagous fruit flies is, at least in part, related to the Anna Karenina principle, which promotes stochastic changes in the microbial diversity of fly populations exposed to suboptimal environmental conditions.

Loop-mediated isothermal amplification of economically important Ceratitis species (Diptera: Tephritidae).

Ceratitis is an economically important genus of fruit flies that originated in Africa, has a wide host range, and causes serious economic losses due to its invasive damage. As a result, it is critical to identify them accurately and quickly in the world. Loop-mediated isothermal amplification (LAMP), as one of the representatives of isothermal amplification technology, has been widely used in the rapid nucleic acid detection of human pathogens and has shown its advantages in the identification of insect agricultural pests. In this study, using the mitochondrial cox1 and cob genes as target genes, the rapid molecular identification of the Ceratitis FARQ complex, C. cosyra, and C. capitata was realized based on LAMP. The experimental conditions optimization results showed that F3/B3:FIP/BIP = 1:8 was the optimal primer concentration ratio and 63 °C was the optimal reaction temperature. The sensitivity of the primers obtained in this study can reach up to 0.01 ng/µl DNA. A loop-mediated isothermal amplification identification technology system was established based on rapid, rough DNA extraction and visual detection of Ceratitis economically important fruit flies. The positive reaction system changed from pink to khaki by visual detection. The identification flow can be completed within 1 hour, including sample processing, DNA extraction, and LAMP visual detection.

Bactrocera dorsalis in the Indian Ocean: A tale of two invasions.

An increasing number of invasive fruit fly pests are colonizing new grounds. With this study, we aimed to uncover the invasion pathways of the oriental fruit fly, Bactrocera dorsalis into the islands of the Indian Ocean. By using genome-wide SNP data and a multipronged approach consisting of PCA, ancestry analysis, phylogenetic inference, and kinship networks, we were able to resolve two independent invasion pathways. A western invasion pathway involved the stepping-stone migration of B. dorsalis from the east African coast into the Comoros, along Mayotte and into Madagascar with a decreasing genetic diversity. The Mascarene islands (Reunion and Mauritius), on the contrary, were colonized directly from Asia and formed a distinct cluster. The low nucleotide diversity suggests that only a few genotypes invaded the Mascarenes. The presence of many long runs of homozygosity (ROH) in the introduced populations is indicative of population bottlenecks, with evidence of a more severe bottleneck for populations along the western migration pathway than on the Mascarene islands. More strict phytosanitary regulations are recommended in order to prevent the further spread of B. dorsalis.

Genomes of the cosmopolitan fruit pest Bactrocera dorsalis (Diptera: Tephritidae) reveal its global invasion history and thermal adaptation.

INTRODUCTION: The oriental fruit fly Bactrocera dorsalis is one of the most destructive agricultural pests worldwide, with highly debated species delimitation, origin, and global spread routes. OBJECTIVES: Our study intended to (i) resolve the taxonomic uncertainties between B. dorsalis and B. carambolae, (ii) reveal the population structure and global invasion routes of B. dorsalis across Asia, Africa, and Oceania, and (iii) identify genomic regions that are responsible for the thermal adaptation of B. dorsalis. METHODS: Based on a high-quality chromosome-level reference genome assembly, we explored the population relationship using a genome-scale single nucleotide polymorphism dataset generated from the resequencing data of 487 B. dorsalis genomes and 25 B. carambolae genomes. Genome-wide association studies and silencing using RNA interference were used to identify and verify the candidate genes associated with extreme thermal stress. RESULTS: We showed that B. dorsalis originates from the Southern India region with three independent invasion and spread routes worldwide: (i) from Northern India to Northern Southeast Asia, then to Southern Southeast Asia; (ii) from Northern India to Northern Southeast Asian, then to China and Hawaii; and (iii) from Southern India toward the African mainland, then to Madagascar, which is mainly facilitated by human activities including trade and immigration. Twenty-seven genes were identified by a genome-wide association study to be associated with 11 temperature bioclimatic variables. The Cyp6a9 gene may enhance the thermal adaptation of B. dorsalis and thus boost its invasion, which tended to be upregulated at a hardening temperature of 38 °C. Functional verification using RNA interference silencing against Cyp6a9, led to the specific decrease in Cyp6a9 expression, reducing the survival rate of dsRNA-feeding larvae exposed to extreme thermal stress of 45 °C after heat hardening treatments in B. dorsalis. CONCLUSION: This study provides insights into the evolutionary history and genetic basis of temperature adaptation in B. dorsalis.

Developing the Protocol Infrastructure for DNA Sequencing Natural History Collections.

Intentionally preserved biological material in natural history collections represents a vast repository of biodiversity. Advances in laboratory and sequencing technologies have made these specimens increasingly accessible for genomic analyses, offering a window into the genetic past of species and often permitting access to information that can no longer be sampled in the wild. Due to their age, preparation and storage conditions, DNA retrieved from museum and herbarium specimens is often poor in yield, heavily fragmented and biochemically modified. This not only poses methodological challenges in recovering nucleotide sequences, but also makes such investigations susceptible to environmental and laboratory contamination. In this paper, we review the practical challenges associated with making the recovery of DNA sequence data from museum collections more routine. We first review key operational principles and issues to address, to guide the decision-making process and dialogue between researchers and curators about when and how to sample museum specimens for genomic analyses. We then outline the range of steps that can be taken to reduce the likelihood of contamination including laboratory set-ups, workflows and working practices. We finish by presenting a series of case studies, each focusing on protocol practicalities for the application of different mainstream methodologies to museum specimens including: (i) shotgun sequencing of insect mitogenomes, (ii) whole genome sequencing of insects, (iii) genome skimming to recover plant plastid genomes from herbarium specimens, (iv) target capture of multi-locus nuclear sequences from herbarium specimens, (v) RAD-sequencing of bird specimens and (vi) shotgun sequencing of ancient bovid bone samples.

Large-scale and small-scale population genetic structure of the medically important gastropod species Bulinus truncatus (Gastropoda, Heterobranchia).

BACKGROUND: Gastropod snails remain strongly understudied, despite their important role in transmitting parasitic diseases. Knowledge of their distribution and population dynamics increases our understanding of the processes driving disease transmission. We report the first study to use high-throughput sequencing (HTS) to elucidate the population genetic structure of the hermaphroditic snail Bulinus truncatus (Gastropoda, Heterobranchia) on a regional (17-150 km) and inter-regional (1000-5400 km) scale. This snail species acts as an intermediate host of Schistosoma haematobium and Schistosoma bovis, which cause human and animal schistosomiasis respectively. METHODS: Bulinus truncatus snails were collected in Senegal, Cameroon, Egypt and France and identified through DNA barcoding. A single-end genotyping-by-sequencing (GBS) library, comprising 87 snail specimens from the respective countries, was built and sequenced on an Illumina HiSeq 2000 platform. Reads were mapped against S. bovis and S. haematobium reference genomes to identify schistosome infections, and single nucleotide polymorphisms (SNPs) were scored using the Stacks pipeline. These SNPs were used to estimate genetic diversity, assess population structure and construct phylogenetic trees of B. truncatus. RESULTS: A total of 10,750 SNPs were scored and used in downstream analyses. The phylogenetic analysis identified five clades, each consisting of snails from a single country but with two distinct clades within Senegal. Genetic diversity was low in all populations, reflecting high selfing rates, but varied between locations due to habitat variability. Significant genetic differentiation and isolation by distance patterns were observed at both spatial scales, indicating that gene flow is not strong enough to counteract the effects of population bottlenecks, high selfing rates and genetic drift. Remarkably, the population genetic differentiation on a regional scale (i.e. within Senegal) was as large as that between populations on an inter-regional scale. The blind GBS technique was able to pick up parasite DNA in snail tissue, demonstrating the potential of HTS techniques to further elucidate the role of snail species in parasite transmission. CONCLUSIONS: HTS techniques offer a valuable toolbox to further investigate the population genetic patterns of intermediate schistosome host snails and the role of snail species in parasite transmission.

Eating eggplants as a cucurbit feeder: Dietary shifts affect the gut microbiome of the melon fly Zeugodacus cucurbitae (Diptera, Tephritidae).

While contemporary changes in feeding preferences have been documented in phytophagous insects, the mechanisms behind these processes remain to be fully clarified. In this context, the insect gut microbiome plays a central role in adaptation to novel host plants. The cucurbit frugivorous fruit fly Zeugodacus cucurbitae (Diptera, Tephritidae) has occasionally been reported on "unconventional" host plants from different families, including Solanaceae. In this study, we focus on wild parental (F0 ) adults and semiwild first filial (F1 ) larvae of Z. cucurbitae from multiple sites in La Réunion and explore how the gut microbiome composition changes when this fly is feeding on a noncucurbit host (Solanum melongena). Our analyses show nonobvious gut microbiome responses following the F0 -F1 host shift and the importance of not just diet but also local effects, which heavily affected the diversity and composition of microbiomes. We identified the main bacterial genera responsible for differences between treatments. These data further stress the importance of a careful approach when drawing general conclusions based on laboratory populations or inadequately replicated field samples.

Phylogenomic resolution of the Ceratitis FARQ complex (Diptera: Tephritidae).

The Ceratitis FARQ complex (formerly FAR complex) includes four frugivorous tephritids, Ceratitis fasciventris, C. anonae, C. rosa and C. quilicii, the latter two causing important agricultural losses in Africa. Although FARQ species can be identified on the basis of subtle morphological differences, they cannot be resolved as monophyletic when trying phylogenetic tree reconstructions based on mitochondrial or nuclear gene fragments except for microsatellites. In this study, we used mitogenome and genome-wide SNPs to investigate the phylogenetic relationship within the complex as well as between all four Ceratitis subgenera. The analysis of 13 species supported the monophyly of the Ceratitis subgenera Ceratitis, Ceratalaspis, Pardalaspis, and recovered Pterandrus as paraphyletic but could not properly resolve species within the FARQ complex. Conversely, gene and species tree reconstructions based on 785,484 genome-wide SNPs could consistently resolve the FARQ taxa and provide insights into their phylogenetic relationships. Gene flow was detected by TreeMix analysis from C. quilicii to C. fasciventris, suggesting the existence of introgression events in the FARQ complex. Our results suggest that genome-wide SNPs represent a suitable tool for the molecular diagnosis of FARQ species and could possibly be used to develop rapid diagnostic methods or to trace the origins of intercepted samples.

Effects of male lure dispensers and trap types for monitoring of Ceratitis capitata and Bactrocera dorsalis (Diptera: Tephritidae).

BACKGROUND: Attractant-based trapping is used in the establishment of pest-free areas and areas of low pest prevalence for fruit flies (Diptera: Tephritidae). Male lures are commonly used attractants in fruit fly trapping. In this study, the effects of male lure dispensers, traps and combinations of dispensers and traps on monitoring of two fruit fly pests, Ceratitis capitata (Wiedemann) and Bactrocera dorsalis (Hendel), were investigated in South Africa. RESULTS: In the male-lure-based trapping systems evaluated, trimedlure (TML) for C. capitata and methyl eugenol (ME) for B. dorsalis, the type of dispenser affected catches for both species. Higher catches of B. dorsalis males were recorded in bucket traps baited with a dispenser containing 15 g ME compared with traps baited with dispensers containing either 4 g ME or 2 g ME. Catches of C. capitata males were higher with dispensers containing TML than those with TML plus extender (Capilure®). The type of trap used with TML also influenced catches of C. capitata with higher numbers recorded in yellow Delta trap compared with the Sensus bucket trap. CONCLUSIONS: Dispensers with higher ME loadings are more effective for monitoring of B. dorsalis. The yellow Delta trap baited with TML (without extender) would be an effective monitoring system for C. capitata. Fruit fly prevalence levels as determined by specific trapping systems should be related to their efficiency in terms of catches of the target pests. © 2020 Society of Chemical Industry.

Comparative Microbiomics of Tephritid Frugivorous Pests (Diptera: Tephritidae) From the Field: A Tale of High Variability Across and Within Species.

The family Tephritidae includes some of the most notorious insect pests of agricultural and horticultural crops in tropical and sub-tropical regions. Despite the interest in the study of their gut microbiome, our present knowledge is largely based on the analysis of laboratory strains. In this study, we present a first comparative analysis of the gut microbiome profiles of field populations of ten African and Mediterranean tephritid pests. For each species, third instar larvae were sampled from different locations and host fruits and compared using 16S rRNA amplicon sequencing and a multi-factorial sampling design. We observed considerable variation in gut microbiome diversity and composition both between and within fruit fly species. A "core" microbiome, shared across all targeted species, could only be identified at most at family level (Enterobacteriaceae). At genus level only a few bacterial genera (Klebsiella, Enterobacter, and Bacillus) were present in most, but not all, samples, with high variability in their relative abundance. Higher relative abundances were found for seven bacterial genera in five of the fruit fly species considered. These were Erwinia in Bactrocera oleae, Lactococcus in B. zonata, Providencia in Ceratitis flexuosa, Klebsiella, and Rahnella in C. podocarpi and Acetobacter and Serratia in C. rosa. With the possible exception of C. capitata and B. dorsalis (the two most polyphagous species considered) we could not detect obvious relationships between fruit fly dietary breadth and microbiome diversity or abundance patterns. Similarly, our results did not suggest straightforward differences between the microbiome profiles of species belonging to Ceratitis and the closely related Bactrocera/Zeugodacus. These results provide a first comparative analysis of the gut microbiomes of field populations of multiple economically relevant tephritids and provide base line information for future studies that will further investigate the possible functional role of the observed associations.

Mitogenomic characterization and systematic placement of the Congo blind barb Caecobarbus geertsii (Cypriniformes: Cyprinidae).

This study presents the first complete mitochondrial genome (mitogenome) of Caecobarbus geertsii, the Congo blind barb, a cave-dwelling, CITES-protected, cyprinid fish endemic to the Lower Congo basin (DRC). The length of the circular mitogenome is 16,565 base pairs. The 13 protein coding genes, 2 ribosomal RNA genes and 22 transfer RNA genes are similar in position and direction to those of other members of the family Cyprinidae. Phylogenetic analyses including 28 complete mitogenomes from representatives of the subfamily Smiliogastrinae (Cyprinidae), showed that Caecobarbus was nested within a clade including representatives of the genus Enteromius. The data presented in this study provide information on the molecular identification and classification of this threatened species. The results further suggest the need for a taxonomic revision of the genus Enteromius.

Worldwide Phylogeography of Ceratitis capitata (Diptera: Tephritidae) Using Mitochondrial DNA.

The Mediterranean fruit fly, Ceratitis capitata (Weidemann), is one of the most economically important tephritid species worldwide. It has spread across six geographic regions as a result of successful invasions and continues to cause substantial losses to agricultural communities. Our study examined 1,864 flies originating from 150 localities, using mitochondrial DNA sequencing methods. We tested for population structure and revealed the genetic diversity for 1,592 specimens gathered from 144 wild fly collections from 46 countries representing the entire geographic range for this species. We also include in this study 272 Sterile Insect Technique (SIT) specimens from four SIT facilities. We recovered 202 haplotypes from the current sampling and updated previously published work to reveal a total of 231 haplotypes for this pest. These data show population structure at and below the regional level for these collections, shedding light on the current demographics for this species. We observed four common haplotypes, seen among 62% of the samples sequenced that have worldwide distribution. Three haplotypes were seen in SIT flies, with one seen as the predominant haplotype. Our work showed that two of the haplotypes were private to SIT flies, not present among wild fly collections. However, a third haplotype common among wild fly collections was also seen in one SIT facility but at a low frequency based on the current sampling. We provide guidance on the interpretation of these methods for the source estimation of current and future infestations.

First mitochondrial genomes of five hoverfly species of the genus Eristalinus (Diptera: Syrphidae).

The hoverfly genus Eristalinus (Diptera, Syrphidae) contains many widespread pollinators. The majority of the species of Eristalinus occur in the Afrotropics and their molecular systematics still needs to be investigated. This study presents the first complete and annotated mitochondrial genomes for five species of Eristalinus. They were obtained by high-throughput sequencing of total genomic DNA. The total length of the mitogenomes varied between 15 757 and 16 245 base pairs. Gene composition, positions, and orientation were shared across species, and were identical to those observed for other Diptera. Phylogenetic analyses (maximum likelihood and Bayesian inference) based on the 13 protein coding and both rRNA genes suggested that the subgenus Eristalinus was paraphyletic with respect to the subgenus Eristalodes. An analysis of the phylogenetic informativeness of all protein coding and rRNA genes suggested that NADH dehydrogenase subunit 5 (nad5), cytochrome c oxidase subunit 1, nad4, nad2, cytochrome b, and 16S rRNA genes are the most promising mitochondrial molecular markers to result in supported phylogenetic hypotheses of the genus. In addition to the five complete mitogenomes currently available for hoverflies, the five mitogenomes published here will be useful for broader molecular phylogenetic analyses among hoverflies.

First genomic study on Lake Tanganyika sprat Stolothrissa tanganicae: a lack of population structure calls for integrated management of this important fisheries target species.

BACKGROUND: Clupeid fisheries in Lake Tanganyika (East Africa) provide food for millions of people in one of the world's poorest regions. Due to climate change and overfishing, the clupeid stocks of Lake Tanganyika are declining. We investigate the population structure of the Lake Tanganyika sprat Stolothrissa tanganicae, using for the first time a genomic approach on this species. This is an important step towards knowing if the species should be managed separately or as a single stock. Population structure is important for fisheries management, yet understudied for many African freshwater species. We hypothesize that distinct stocks of S. tanganicae could be present due to the large size of the lake (isolation by distance), limnological variation (adaptive evolution), or past separation of the lake (historical subdivision). On the other hand, high mobility of the species and lack of obvious migration barriers might have resulted in a homogenous population. RESULTS: We performed a population genetic study on wild-caught S. tanganicae through a combination of mitochondrial genotyping (96 individuals) and RAD sequencing (83 individuals). Samples were collected at five locations along a north-south axis of Lake Tanganyika. The mtDNA data had low global FST and, visualised in a haplotype network, did not show phylogeographic structure. RAD sequencing yielded a panel of 3504 SNPs, with low genetic differentiation (FST = 0.0054; 95% CI: 0.0046-0.0066). PCoA, fineRADstructure and global FST suggest a near-panmictic population. Two distinct groups are apparent in these analyses (FST = 0.1338 95% CI: 0.1239,0.1445), which do not correspond to sampling locations. Autocorrelation analysis showed a slight increase in genetic difference with increasing distance. No outlier loci were detected in the RADseq data. CONCLUSION: Our results show at most very weak geographical structuring of the stock and do not provide evidence for genetic adaptation to historical or environmental differences over a north-south axis. Based on these results, we advise to manage the stock as one population, integrating one management strategy over the four riparian countries. These results are a first comprehensive study on the population structure of these important fisheries target species, and can guide fisheries management.

Impact of Sample Preservation and Manipulation on Insect Gut Microbiome Profiling. A Test Case With Fruit Flies (Diptera, Tephritidae).

High-throughput sequencing (HTS) techniques are of great value for the investigation of microbial communities, and have been extensively used to study the gut microbiome. While most studies focus on the human gut, many others have investigated insects. However, because of the rapid spread of HTS techniques, a lot of variation exists in the protocols for sample preparation. In the present study, we investigated the impact of two widely adopted sample-processing procedures preceding library preparation, i.e., preservation of insect tissue in 70% ethanol (EtOH) and sample dissection. We used the fruit fly Ceratitis capitata (Diptera: Tephritidae) as a model organism and set up two experiments, one comparing the effects of sample manipulation and preservation across life stages and the other across fruit samples from different sources. The results of this study showed no major effects of dissection on the outcome of HTS. However, EtOH preservation did have effects on the recovered gut microbiome, the main effect being a significant reduction of the dominant genus, Providencia, in EtOH-preserved samples. Less abundant bacterial groups were also affected resulting in altered microbial profiles obtained from samples preserved in 70% EtOH. These results have important implications for the planning of future studies and when comparing studies that used different sample preparation protocols.

Tephritid Integrative Taxonomy: Where We Are Now, with a Focus on the Resolution of Three Tropical Fruit Fly Species Complexes.

Accurate species delimitation underpins good taxonomy. Formalization of integrative taxonomy in the past decade has provided a framework for using multidisciplinary data to make species delimitation hypotheses more rigorous. We address the current state of integrative taxonomy by using as a case study an international project targeted at resolving three important tephritid species complexes: Bactrocera dorsalis complex, Anastrepha fraterculus complex, and Ceratitis FAR (C. fasciventris, C. anonae, C. rosa) complex. The integrative taxonomic approach has helped deliver significant advances in resolving these complexes: It has been used to identify some taxa as belonging to the same biological species as well as to confirm hidden cryptic diversity under a single taxonomic name. Nevertheless, the general application of integrative taxonomy has not been without issue, revealing challenges that must be considered when undertaking an integrative taxonomy project. Scrutiny of this international case study provides a unique opportunity to document lessons learned for the benefit of not only tephritid taxonomists, but also the wider taxonomic community.

Taxonomy, Ecology, and Management of Native and Exotic Fruit Fly Species in Africa.

Horticulture is one of the most important agricultural subsectors in Africa, providing income, creating employment opportunities, and enhancing food and nutritional security. However, tephritid fruit flies are responsible for both direct and indirect losses, with alien invasive species often having the most severe ecological and economic impact. In the past 20 years, systematic analysis of tephritids has provided comparative information on taxonomy, synonymy, and character-state differentiation. New molecular techniques are now available for identifying species, reconstructing phylogenies, and studying population genetic structures. Research on biology, host range and shifts, thermotolerance, and demography has provided useful information for developing predictive and ecological niche models to guide management methods. In recent years, the responses of various species to attractants have been documented. Several suppression methods, including the release of coevolved parasitoid species targeting invasives, have been promoted within the context of integrated pest management, leading to improvement in the quality and quantity of fruits and vegetables produced. However, there is still the need for wide-scale availability of these technologies to smallholder growers across Africa.

DNA Barcoding to Improve the Taxonomy of the Afrotropical Hoverflies (Insecta: Diptera: Syrphidae).

The identification of Afrotropical hoverflies is very difficult because of limited recent taxonomic revisions and the lack of comprehensive identification keys. In order to assist in their identification, and to improve the taxonomy of this group, we constructed a reference dataset of 513 COI barcodes of 90 of the more common nominal species from Ghana, Togo, Benin and Nigeria (W Africa) and added ten publically available COI barcodes from nine nominal Afrotropical species to this (total: 523 COI barcodes; 98 nominal species; 26 genera). The identification accuracy of this dataset was evaluated with three methods (K2P distance-based, Neighbor-Joining (NJ) / Maximum Likelihood (ML) analysis, and using SpeciesIdentifier). Results of the three methods were highly congruent and showed a high identification success. Nine species pairs showed a low (< 0.03) mean interspecific K2P distance that resulted in several incorrect identifications. A high (> 0.03) maximum intraspecific K2P distance was observed in eight species and barcodes of these species not always formed single clusters in the NJ / ML analayses which may indicate the occurrence of cryptic species. Optimal K2P thresholds to differentiate intra- from interspecific K2P divergence were highly different among the three subfamilies (Eristalinae: 0.037, Syrphinae: 0.06, Microdontinae: 0.007-0.02), and among the different general suggesting that optimal thresholds are better defined at the genus level. In addition to providing an alternative identification tool, our study indicates that DNA barcoding improves the taxonomy of Afrotropical hoverflies by selecting (groups of) taxa that deserve further taxonomic study, and by attributing the unknown sex to species for which only one of the sexes is known.

Higher phylogeny of frugivorous flies (Diptera, Tephritidae, Dacini): localised partition conflicts and a novel generic classification.

The phylogenetic relationships within and among subtribes of the fruit fly tribe Dacini (Ceratitidina, Dacina, Gastrozonina) were investigated by sequencing four mitochondrial and one nuclear gene fragment. Bayesian, maximum likelihood and maximum parsimony analyses were implemented on two datasets. The first, aiming at obtaining the strongest phylogenetic signal (yet, having lower taxon coverage), consisted of 98 vouchers and 2338 concatenated base pairs (bp). The second, aiming at obtaining the largest taxonomic coverage (yet, providing lower resolution), included 159 vouchers and 1200 concatenated bp. Phylogenetic relationships inferred by different tree reconstruction methods were largely congruent and showed a general agreement between concatenated tree topologies. Yet, local conflicts in phylogenetic signals evidenced a number of critical sectors in the phylogeny of Dacini fruit flies. All three Dacini subtribes were recovered as monophyletic. Yet, within the subtribe Ceratitidina only Perilampsis and Capparimyia formed well-resolved monophyletic groups while Ceratitis and Trirhithrum did not. Carpophthoromyia was paraphyletic because it included Trirhithrum demeyeri and Ceratitis connexa. Complex phylogenetic relationships and localised conflict in phylogenetic signals were observed within subtribe Dacina with (a) Dacus, (b) Bactrocera (Zeugodacus) and (c) all other Bactrocera species forming separate clades. The subgenus Bactrocera (Zeugodacus) is therefore raised to generic rank (Zeugodacus Hendel stat. nov.). Additionally, Bactrocera subgenera grouped under the Zeugodacus group should be considered under new generic combinations. Although there are indications that Zeugodacus and Dacus are sister groups, the exact relationship between Zeugodacus stat. nov., Dacus and Bactrocera still needs to be properly resolved.

An integrative approach to unravel the Ceratitis FAR (Diptera, Tephritidae) cryptic species complex: a review.

This paper reviews all information gathered from different disciplines and studies to resolve the species status within the Ceratitis FAR (Ceratitis fasciventris, Ceratitis anonae, Ceratitis rosa) complex, a group of polyphagous fruit fly pest species (Diptera, Tephritidae) from Africa. It includes information on larval and adult morphology, wing morphometrics, cuticular hydrocarbons, pheromones, microsatellites, developmental physiology and geographic distribution. The general consensus is that the FAR complex comprises Ceratitis anonae, two species within Ceratitis rosa (so-called R1 and R2) and two putatitve species under Ceratitis fasciventris. The information regarding the latter is, however, too limited to draw final conclusions on specific status. Evidence for this recognition is discussed with reference to publications providing further details.