A DNA barcode reference library for the Tipulidae (Insecta, Diptera) of Germany.

Tipulidae, commonly known as true crane flies, represent one of the most species-rich dipteran families, boasting approximately 4,500 known species globally. Their larvae serve as vital decomposers across diverse ecosystems, prompting their frequent and close observation in biomonitoring programs. However, traditional morphological identification methods are laborious and time-consuming, underscoring the need for a comprehensive DNA barcode reference library to speed up species determination. In this study, we present the outcomes of the German Barcode of Life initiative focused on Tipulidae. Our DNA barcode library comprises 824 high-quality cytochrome c oxidase I (COI) barcodes encompassing 76 crane fly species, counting for ca. 54% of the German tipulid fauna. Our results significantly increased the number of European tipulid species available in the Barcode of Life Data System (BOLD) by 14%. Additionally, the number of barcodes from European tipulid specimens more than doubled, with an increase of 118%, bolstering the DNA resource for future identification inquiries. Employing diverse species delimitation algorithms - including the multi-rate Poisson tree processes model (mPTP), Barcode Index Number assignments (BIN), Assemble Species by Automatic Partitioning (ASAP), and the TaxCI R-script - we successfully match 76-86% of the morphologically identified species. Further validation through neighbor-joining tree topology analysis and comparison with 712 additional European tipulid barcodes yield a remarkable 89% success rate for the species identification of German tipulids based on COI barcodes. This comprehensive DNA barcode dataset not only enhances species identification accuracy but also serves as a pivotal resource for ecological and biomonitoring studies, fostering a deeper understanding of crane fly diversity and distribution across terrestrial landscapes.

Preserving morphology while extracting DNA: a non-destructive field-to-museum protocol for slide-mounted specimens.

Our study aimed to develop an optimised laboratory protocol ensuring the preservation of morphological structures and extraction of high-quality DNA sequences from Psychodidae (Insecta, Diptera) specimens. With 310 analysed specimens, we investigated the impact of distinct laboratory treatments by employing two shaking categories (constant and interrupted) with five different incubation periods (16, 12, 8, 4 and 2 hours) during the DNA extraction process. Notably, 80.65% of the specimens exhibited morphological changes during DNA extraction. Our results indicated no statistical difference between constant and interrupted shaking for the total of morphological structures lost. However, within each shaking category, the loss of structures was influenced significantly by the incubation period. Prolonged incubation correlated with increased structural losses, whereas shorter incubation periods caused minor alterations in structures lost. In addition, our results showed a significant difference between constant and interrupted shaking treatments for DNA concentration. Likewise, the incubation period showed differences within each shaking category. Successful COI sequencing was achieved in 89.6% of specimens, with negligible differences in DNA fragment lengths across treatments. Our findings underscore the importance of an optimised protocol and its potential in systematic research involving nematoceran dipteran specimens by balancing morphological integrity and DNA extraction efficiency.

More complex than you think: Taxonomic and temporal patterns of plant-pollinator networks of caraway (Carum carvi L.).

Caraway (Carum carvi L.) is a crop species that is gaining in importance in Europe, especially as a condiment and medicinal plant. Here, we present the plant-pollinator network of caraway in a central European agricultural landscape, focusing on two diverse potential pollinator taxa, Diptera: Brachycera (= true flies) and Hymenoptera (sawflies, bees, and wasps). We specifically studied qualitative differences in interactions between the two insect taxa as well as the intraday and intraseasonal variability of the network. Insect and pollen plant species determination was done via morphological identification and DNA (meta)barcoding. In total, 121 species representing 33 families of Hymenoptera and Brachycera were found to carry caraway pollen. These taxa included many nonhoneybee and nonhoverfly species, showing a wide taxonomic breadth of potential pollinators and a higher network complexity than previously anticipated. There are distinct qualitative differences between Brachycera and Hymenoptera networks, suggesting complementary roles of both taxa in the pollination of native and crop plants. Strong intraday differences in potential pollinator diversity make it necessary to collect insects and pollen at different times of the day to compile complete plant-pollinator networks. Intraseasonal analyses of the plant-pollinator network of caraway show the potential of caraway as an important food source for insect species with an activity peak in late summer.

The phylogeny and evolutionary ecology of hoverflies (Diptera: Syrphidae) inferred from mitochondrial genomes.

Hoverflies (Diptera: Syrphidae) are a diverse group of pollinators and a major research focus in ecology, but their phylogenetic relationships remain incompletely known. Using a genome skimming approach we generated mitochondrial genomes for 91 species, capturing a wide taxonomic diversity of the family. To reduce the required amount of input DNA and overall cost of the library construction, sequencing and assembly was conducted on mixtures of specimens, which raises the problem of chimera formation of mitogenomes. We present a novel chimera detection test based on gene tree incongruence, but identified only a single mitogenome of chimeric origin. Together with existing data for a final set of 127 taxa, phylogenetic analysis on nucleotide and amino acid sequences using Maximum Likelihood and Bayesian Inference revealed a basal split of Microdontinae from all other syrphids. The remainder consists of several deep clades assigned to the subfamily Eristalinae in the current classification, including a clade comprising the subfamily Syrphinae (plus Pipizinae). These findings call for a re-definition of subfamilies, but basal nodes had insufficient support to fully justify such action. Molecular-clock dating placed the origin of the Syrphidae crown group in the mid-Cretaceous while the Eristalinae-Syrphinae clade likely originated near the K/Pg boundary. Transformation of larval life history characters on the tree suggests that Syrphidae initially had sap feeding larvae, which diversified greatly in diet and habitat association during the Eocene and Oligocene, coinciding with the diversification of angiosperms and the evolution of various insect groups used as larval host, prey, or mimicry models. Mitogenomes proved to be a powerful phylogenetic marker for studies of Syrphidae at subfamily and tribe levels, allowing dense taxon sampling that provided insight into the great ecological diversity and rapid evolution of larval life history traits of the hoverflies.

Alepia viatrix sp. nov. (Diptera: Psychodidae), a new species of a Neotropical genus found on the Azores Archipelago (Portugal).

A new species of the Neotropical genus Alepia Enderlein, 1937 is described from the Azores Archipelago based on morphological characters and DNA barcodes from male and female specimens. Images of the new species as well as a discussion of the origin of this species are also provided. Moreover, we include an identification key for the adult male Psychodidae species recorded on the Azores Archipelago and comment on each species present on these islands. This is the first record of the genus Alepia from Azores.

Description of the Third-Stage Larva and Puparium of Platycheirus (Carposcalis) chalconota (Philippi) (Diptera: Syrphidae) with New Information About the Trophic Interactions and Larval Habitats.

In this study, the third-stage larva and puparium of the copper-backed fly, Platycheirus (Carposcalis) chalconota (Philippi, 1865) are fully described using stereomicroscopy and scanning electron microscopy from material collected in peach orchards of central-west Argentina. The immature stages of P. chalconota were compared with the third-stage larva and puparium of the best-studied species in the genus Platycheirus scutatus (Meigen, 1822); as well as with the only known Carposcalis immature stages description available for the Neotropical region namely Platycheirus stegnus (Say, 1829). New data on trophic interactions and larval habitats are given: P. chalconota larvae were found feeding on the aphid's species Uroleucon sonchi (Linnaeus, 1767) (large sow thistle aphid) and Hyperomyzus lactucae (Linnaeus, 1758) (blackcurrant-sow thistle aphid) in low vegetation as Sonchus oleraceus (common sow thistle); the species was also found feeding on Myzus persicae (Sulzer, 1776) (green peach aphid) in high vegetation as Prunus persicae (L.) Stokes (Peach trees). The third larval stage and puparium of P. chalconota are described and illustrated for the first time improving substantially the knowledge about the immature stages and natural history of Neotropical Platycheirus, constituting also a baseline for future comparative morphological studies. Despite the efficiency of P. chalconota in the biological control of pests has not been assessed, we think that relevant data presented here can be used in pest management of peach orchards and suggest P. chalconota as an excellent candidate for future studies on the life cycle, prey consumption, efficiency, artificial rearing, and its potential importance as pollinators of Peach crops.

Is it time to describe new species without diagnoses?A comment on Sharkey et al. (2021).

New methods in taxonomy and systematics can influence the overall practice of formally naming and describing biodiversity. DNA barcoding has been controversial since its emergence, but now, large scale species descriptions exclusively based on barcodes have created what can be called a 'new quality of performance. Its limitations are discussed from different perspectives: nomenclature, general pragmatism, and problems of DNA-based species delimitation in the light of the central aim of achieving a robust and stable nomenclature of organisms, essential for all applications of biodiversity research. This issue needs to be addressed to prevent restraining the progress of taxonomy and its ability to contribute to modern science.

Checklist of hover flies (Diptera, Syrphidae) of the Republic of Georgia.

A checklist of the Syrphidae species of the Republic of Georgia is presented. New hover fly (Diptera: Syrphidae) records from Georgia are provided as a result of field work conducted in 2018. At the same time, published syrphid records for the country are here reviewed and updated. A total of 357 species of hoverflies are now documented from Georgia, 40 of which are reported for the first time. Moreover, DNA barcodes were sequenced for 238 specimens, representing 74 species from this country.

A world review of reported myiases caused by flower flies (Diptera: Syrphidae), including the first case of human myiasis from Palpada scutellaris (Fabricius, 1805).

Rat-tailed larvae of the syrphid species Palpada scutellaris (Fabricius, 1805) are documented causing an enteric human myiasis in Costa Rica. This is the first time that the genus Palpada is recorded as a human myiasis agent. We report a 68-year-old woman with intestinal pain and bloody diarrhea with several live Palpada larvae present in the stool. Using molecular techniques (DNA barcodes) and both electronic and optical microscopy to study the external morphology, the preimaginal stages of the fly were unambiguously identified. An identification key to all syrphid genera actually known as agents of human and animal myiases is provided for larvae, puparia, and adults. Moreover, a critical world review of more than 100 references of Syrphidae as myiasis agents is also given, with emphasis on the species with rat-tailed larvae.

Eristalinus arvorum (Fabricius, 1787) (Diptera: Syrphidae) in Human Skull: A New Fly Species of Forensic Importance.

A body of an unknown adult female was found within a shallow burial ground in Malaysia whereas the skull was exposed and visible on the ground. During autopsy examination, nine insect larvae were recovered from the interior of the human skull and subsequently preserved in 70% ethanol. The larvae were greyish in appearance, each with a posterior elongated breathing tube. A week after the autopsy, more larvae were collected at the burial site, and some of them were reared into adults. Adult specimens and larvae from the skull and from the burial site were sequenced to obtain DNA barcodes. Results showed all adult flies reared from the burial site, as well as the larvae collected from the skull were identified as Eristalinus arvorum (Fabricius, 1787) (Diptera: Syrphidae). Here, we report the colonization of E. arvorum larvae on a human corpse for the first time.

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.

Unraveling Ocyptamus and the Baccha legacy (Diptera: Syrphidae): redefinition of groups and new species descriptions.

The systematics of Ocyptamus sensu lato has been reviewed, its species groups redefined, and some taxa resurrected. These and other related taxa are diagnosed to aid in distinguishing between them. The status of the following genera is revised: Calostigma Shannon stat. rev., Hermesomyia Vockeroth stat. rev., Hybobathus Enderlein stat. rev., Mimocalla Hull stat. rev., Orphnabaccha Hull stat. rev., Pipunculosyrphus Hull stat. rev., Pseudoscaeva Vockeroth stat. rev., and Styxia Hull stat. rev. New species of the Neotropical genus Ocyptamus are described from Costa Rica and Venezuela: Ocyptamus maximus Thompson sp. nov., Ocyptamus myiophagus Thompson sp. nov., and Ocyptamus megafemur Thompson sp. nov. In addition, an identification key for the genera and species groups of Ocyptamus sensu lato is provided, as well as a redescription of Syrphus sargoides Macquart.Lectotypes are designated for Doros disjuncta Sack, Baccha placiva Williston, Baccha pulla Sack, Ocyptamus funebris Macquart, Syrphus sargoides, and Syrphus immaculatus Macquart. Moreover, Syrphus immaculatus Macquart is considered to be a species of the genus Toxomerus, and new species synonyms are also given: Baccha atypica Curran is a junior synonym of Syrphus conjunctus Wiedemann; Baccha flata Hull and Doros disjuncta Sack are jun. syn. of Syrphus sargoides Macquart; Baccha bromleyi Curran is a jun. syn. of Ocyptamus funebris Macquart; Baccha sepia Hull, Baccha danaida Hull, Baccha violacea Hull, and Baccha ursula Hull are all synonyms of Ocyptamus pullus (Sack); Baccha nerissa Hull is a jun. syn. of Pelecinobaccha hiantha (Hull); and Baccha panamensis Curran is a jun. syn. of Pelecinobaccha transatlantica (Schiner).

Checklist of the flower flies of Ecuador (Diptera, Syrphidae).

Syrphidae is one of the most speciose families of true flies, with more than 6,100 described species and worldwide distribution. They are important for humans acting as crucial pollinators, biological control agents, decomposers, and bioindicators. One third of its diversity is found in the Neotropical Region, but the taxonomic knowledge for this region is incomplete. Thus, taxonomic revisions and species checklists of Syrphidae in the Neotropics are the highest priority for biodiversity studies. Therefore, we present the first checklist of Syrphidae for Ecuador based on literature records, and provide as well the original reference for the first time species citations for the country. A total of 201 species were recorded for Ecuador, with more than 600 records from 24 provinces and 237 localities. Tungurahua, Pastaza, and Galápagos were the best sampled provinces. Although the reported Ecuadorian syrphid fauna only comprises 11.2 % of the described Neotropical species, Ecuador has the third highest flower fly diversity density after Costa Rica and Suriname. These data indicate the high species diversity for this country in such small geographic area.

The nectar spur is not only a simple specialization for long-proboscid pollinators.

Since the time of Darwin, biologists have considered the floral nectar spur to be an adaptation representing a high degree of plant specialization. Nevertheless, some researchers suggest that nature is more complex and that even morphologically specialized plants attract a wide spectrum of visitors. We observed visitors on Impatiens burtonii (Balsaminaceae) and measured the depth of the proboscis insertion into the spur, the distance of the nectar surface from the spur entrance and the visitor's effectiveness. The hoverfly Melanostoma sp., with the shortest proboscis, was most active early in the morning and fed on pollen and nectar near the spur entrance. The honeybee Apis mellifera and the hoverfly Rhingia mecyana were the most frequent visitors before and after noon, respectively. Although R. mecyana, the only visitor able to reach the end of the spur, was the most frequent, it did not deposit the largest number of pollen grains per visit. Nectar spurs may function as complex structures allowing pollination by both short- and long-proboscid visitors and separating their spatial and temporal niches. Spurred plants should be considered as more generalized and exposed to more diverse selection pressures than previously believed.

Phylogenetic relationships of the tribe Toxotrypanini (Diptera: Tephritidae) based on molecular characters.

Current hypotheses of relationship among the species of the fruit fly genera Anastrepha and Toxotrypana are tested using sequence data from six DNA regions: the mitochondrial regions 16S, CAD, and COI, and the nuclear regions EF1a, PER, and PGD. DNA sequences were obtained from 146 species of Anastrepha, representing 19 of the 21 species groups as well as five of the six clades of the robusta group, and four species of Toxotrypana in addition to species of Hexachaeta, Pseudophorellia, Alujamyia, and 13 other tephritid genera used as outgroups. The results indicate that Hexachaeta is more closely related to the Molynocoelia group than to Toxotrypana and Anastrepha, and it is removed from the tribe Toxotrypanini. The group Anastrepha+Toxotrypana and the genus Toxotrypana are strongly supported as monophyletic, consistent with previous studies, but Toxotrypana arises within Anastrepha, confirming that Anastrepha as currently defined is paraphyletic. The placement of Toxotrypana within Anastrepha is clearly defined for the first time with high support, as the sister group to the cryptostrepha clade of the robusta group of Anastrepha. Within Anastrepha, the daciformis, dentata, leptozona, raveni, and striata species groups are highly supported clades. The serpentina group is recognized with lower support, and the fraterculus and pseudoparallela groups are supported with minor alterations. The robusta group is resolved as polyphyletic, but four of the six species clades within it are recovered monophyletic (one clade is not represented and another is represented by one species). The punctata and panamensis groups are resolved together in a clade. At least some species of the mucronota group are related, however this group requires further study. The benjamini, grandis, and spatulata groups appear to be polyphyletic. Relationships among the species groups are generally poorly resolved, with the following exceptions: (1) the lineage including Toxotrypana, the cryptostrepha clade, and the tripunctata group; (2) the sister group relationship of the daciformis+dentata groups; (3) a clade comprising the punctata and panamensis groups; and (4) the large clade comprising the pseudoparallela+spatulata+ramosa+grandis+serpentina+striata+fraterculus groups.

Anchored enrichment dataset for true flies (order Diptera) reveals insights into the phylogeny of flower flies (family Syrphidae).

BACKGROUND: Anchored 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. RESULTS: We 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. CONCLUSIONS: Anchored 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.

Where Is My Food? Brazilian Flower Fly Steals Prey from Carnivorous Sundews in a Newly Discovered Plant-Animal Interaction.

A new interaction between insects and carnivorous plants is reported from Brazil. Larvae of the predatory flower fly Toxomerus basalis (Diptera: Syrphidae: Syrphinae) have been found scavenging on the sticky leaves of several carnivorous sundew species (Drosera, Droseraceae) in Minas Gerais and São Paulo states, SE Brazil. This syrphid apparently spends its whole larval stage feeding on prey trapped by Drosera leaves. The nature of this plant-animal relationship is discussed, as well as the Drosera species involved, and locations where T. basalis was observed. 180 years after the discovery of this flower fly species, its biology now has been revealed. This is (1) the first record of kleptoparasitism in the Syrphidae, (2) a new larval feeding mode for this family, and (3) the first report of a dipteran that shows a kleptoparasitic relationship with a carnivorous plant with adhesive flypaper traps. The first descriptions of the third instar larva and puparium of T. basalis based on Scanning Electron Microscope analysis are provided.

Mosquito species (Diptera, Culicidae) in three ecosystems from the Colombian Andes: identification through DNA barcoding and adult morphology.

Colombia, one of the world's megadiverse countries, has a highly diverse mosquito fauna and a high prevalence of mosquito-borne diseases. In order to provide relevant information about the diversity and taxonomy of mosquito species in Colombia and to test the usefulness of DNA barcodes, mosquito species collected at different elevations in the departments of Antioquia and Caldas were identified combining adult morphology and barcode sequences. A total of 22 mosquito species from eight genera were identified using these combined techniques. We generated 77 barcode sequences with 16 species submitted as new country records for public databases. We examined the usefulness of DNA barcodes to discriminate mosquito species from the Neotropics by compiling 1,292 sequences from a total of 133 species and using the tree-based methods of neighbor-joining and maximum likelihood. Both methodologies provided similar results by resolving 105 species of mosquitoes separated into distinct clusters. This study shows the importance of combining classic morphological methodologies with molecular tools to accurately identify mosquitoes from Colombia.

Australian Allograpta Osten Sacken (Diptera, Syrphidae).

Allograptaterraenovae sp. n. and Allograptanotiale sp. n. are described from Australia. Notes on the Australian species of Allograpta and an identification key to them are also given. The lectotype of Allograptajavana Wiedemann is designated, and the species Syrphuspallidus Bigot is synonymized under Allograptaaustralensis (Schiner).

A revision of the genus Pelecocera Meigen with the description of the male of pelecocera persiana Kuznetzov from Iran (Diptera: Syrphidae).

The genus Pelecocera Meigen (Diptera: Syrphidae) is revised. Type material of most species was studied to describe, illustrate and delimit the male of Pelecocera persiana Kuznetzov, recently discovered from Iran. This is the first known specimen of this species since 1914, when the type female was collected. The diagnostic characters of P. persiana are provided, along with an identification key for Pelecocera species. The lectotype of Pelecocera latifrons Loew is designated.