A checklist of the hoverflies (Diptera: Syrphidae) of Austria.

Hoverflies are a conspicuous and popular family within the Diptera. The larvae as well as the adults are able to colonize a wide range of habitats, and many species play important roles as pollinators, in pest management and nowadays in applied nature conservation issues. Despite of this, the state of knowledge on the hoverflies of Austria is deficient: Available literature is outdated in systematics and the species inventory is obviously incomplete. These facts led us to study the Austrian hoverfly fauna in more detail. Syrphidae records from an extensive literature search, additional unpublished data from museum collections and data from own fieldwork were compiled to build a comprehensive checklist of Austrian hoverflies. The species distributions are given by federal state in order to retain a precise overview of this Diptera group. The framework of this research is based on 20520 records and in total, 271957 individuals. The checklist includes 430 confirmed species. An additional 25 hoverfly species are discussed but not included in the list. These are species for which it is unclear if the locality lies within the present borders of Austria, or whose voucher specimens were not available for re-examination. In total 17 hoverfly species are new to Austria: Anasimyia contracta Claussen Torp, 1980; Brachyopa grunewaldensis Kassebeer 2000; Brachyopa obscura Thompson Torp, 1982; Brachyopa silviae Doczkal Dziock, 2004; Cheilosia orthotricha Vuji Claussen, 1994; Eristalis picea (Falln, 1817); Melangyna ericarum (Collin, 1946); Melangyna lucifera Nielsen, 1980; Melangyna pavlovskyi Violovitsh, 1956; Melanogaster curvistylus Vuji Stuke, 1998; Merodon moenium (Wiedemann, 1822); Paragus absidatus Goeldlin, 1971; Paragus bradescui Stanescu, 1981; Platycheirus laskai (Nielsen, 1999); Sphegina verecunda Collin, 1937; Temnostoma angustistriatum Krivosheina, 2002 and Temnostoma meridionale Krivosheina Mamayev, 1962. In addition, 278 first records for several Austrian federal states are published. With 331 species, Styria currently hosts the largest number of documented species per federal state, followed by Lower Austria (307 spp.), Upper Austria (269 spp.), Carinthia (259 spp.), Vorarlberg (234 spp.), Burgenland (201 spp.), North Tyrol (172 spp.), Vienna (169 spp.), Salzburg (155 spp.) and Eastern Tyrol (154 spp.).

Peering into the Darkness: DNA Barcoding Reveals Surprisingly High Diversity of Unknown Species of Diptera (Insecta) in Germany.

Determining the size of the German insect fauna requires better knowledge of several megadiverse families of Diptera and Hymenoptera that are taxonomically challenging. This study takes the first step in assessing these "dark taxa" families and provides species estimates for four challenging groups of Diptera (Cecidomyiidae, Chironomidae, Phoridae, and Sciaridae). These estimates are based on more than 48,000 DNA barcodes (COI) from Diptera collected by Malaise traps that were deployed in southern Germany. We assessed the fraction of German species belonging to 11 fly families with well-studied taxonomy in these samples. The resultant ratios were then used to estimate the species richness of the four "dark taxa" families (DT families hereafter). Our results suggest a surprisingly high proportion of undetected biodiversity in a supposedly well-investigated country: at least 1800-2200 species await discovery in Germany in these four families. As this estimate is based on collections from one region of Germany, the species count will likely increase with expanded geographic sampling.

Fluctuating insect diversity, abundance and biomass across agricultural landscapes.

Habitat destruction and deterioration of habitat quality caused a severe decline of biodiversity, such as insect diversity. In this study, we analyze insect diversity and biomass across agro-environments. We collected flying insects with 20 malaise traps across a landscape mosaic consisting of organic (eight traps) and conventional (four traps) farmland, as well as across agricultural land that has been recently converted from conventional to organic farming (eight traps). Sampling was conducted over 2 years, in 2019 and 2020, with in total 340 sampling events. We measured the dry weight of the captured organisms and identified species diversity by analyzing Operational Taxonomic Units (OTUs) and Barcode Index Numbers (BINs) via metabarcoding. The results obtained show temporal dynamics. The number of OTUs were always higher than the number of BINs. OTUs and BINs were moderately to highly correlated, while the number of OTUs and BINs were only moderately positively correlated with dry biomass. OTUs and BINs as well as biomass were highest in the recently transformed farmland if compared with pure organic and conventional farmland sites, which showed no significant differences in respect of insect diversity. OTU and BIN numbers but not the OTU/BIN ratio significantly decreased with increasing distance from the nearest forest fringe. The numbers of OTUs, BINs and the OTU/BIN proportion, as well as OTU and BIN/biomass proportions varied strongly over seasons, irrespective of agricultural practice. Based on our findings, we suggest to combine data on insect species richness and biomass measured over a period of time, to derive a largely complete and meaningful assessment of biodiversity for a specific region.

DNA metabarcoding for biodiversity monitoring in a national park: Screening for invasive and pest species.

DNA metabarcoding was utilized for a large-scale, multiyear assessment of biodiversity in Malaise trap collections from the Bavarian Forest National Park (Germany, Bavaria). Principal component analysis of read count-based biodiversities revealed clustering in concordance with whether collection sites were located inside or outside of the National Park. Jaccard distance matrices of the presences of barcode index numbers (BINs) at collection sites in the two survey years (2016 and 2018) were significantly correlated. Overall similar patterns in the presence of total arthropod BINs, as well as BINs belonging to four major arthropod orders across the study area, were observed in both survey years, and are also comparable with results of a previous study based on DNA barcoding of Sanger-sequenced specimens. A custom reference sequence library was assembled from publicly available data to screen for pest or invasive arthropods among the specimens or from the preservative ethanol. A single 98.6% match to the invasive bark beetle Ips duplicatus was detected in an ethanol sample. This species has not previously been detected in the National Park.

Toward a standardized quantitative and qualitative insect monitoring scheme.

The number of insect species and insect abundances decreased severely during the past decades over major parts of Central Europe. Previous studies documented declines of species richness, abundances, shifts in species composition, and decreasing biomass of flying insects. In this study, we present a standardized approach to quantitatively and qualitatively assess insect diversity, biomass, and the abundance of taxa, in parallel. We applied two methods: Malaise traps, and automated and active light trapping. Sampling was conducted from April to October 2018 in southern Germany, at four sites representing conventional and organic farming. Bulk samples obtained from Malaise traps were further analyzed using DNA metabarcoding. Larger moths (Macroheterocera) collected with light trapping were further classified according to their degree of endangerment. Our methods provide valuable quantitative and qualitative data. Our results indicate more biomass and higher species richness, as well as twice the number of Red List lepidopterans in organic farmland than in conventional farmland. This combination of sampling methods with subsequent DNA metabarcoding and assignments of individuals according depending on ecological characteristics and the degree of endangerment allows to evaluate the status of landscapes and represents a suitable setup for large-scale long-term insect monitoring across Central Europe, and elsewhere.

A DNA barcode library for 5,200 German flies and midges (Insecta: Diptera) and its implications for metabarcoding-based biomonitoring.

This study summarizes results of a DNA barcoding campaign on German Diptera, involving analysis of 45,040 specimens. The resultant DNA barcode library includes records for 2,453 named species comprising a total of 5,200 barcode index numbers (BINs), including 2,700 COI haplotype clusters without species-level assignment, so called "dark taxa." Overall, 88 out of 117 families (75%) recorded from Germany were covered, representing more than 50% of the 9,544 known species of German Diptera. Until now, most of these families, especially the most diverse, have been taxonomically inaccessible. By contrast, within a few years this study provided an intermediate taxonomic system for half of the German Dipteran fauna, which will provide a useful foundation for subsequent detailed, integrative taxonomic studies. Using DNA extracts derived from bulk collections made by Malaise traps, we further demonstrate that species delineation using BINs and operational taxonomic units (OTUs) constitutes an effective method for biodiversity studies using DNA metabarcoding. As the reference libraries continue to grow, and gaps in the species catalogue are filled, BIN lists assembled by metabarcoding will provide greater taxonomic resolution. The present study has three main goals: (a) to provide a DNA barcode library for 5,200 BINs of Diptera; (b) to demonstrate, based on the example of bulk extractions from a Malaise trap experiment, that DNA barcode clusters, labelled with globally unique identifiers (such as OTUs and/or BINs), provide a pragmatic, accurate solution to the "taxonomic impediment"; and (c) to demonstrate that interim names based on BINs and OTUs obtained through metabarcoding provide an effective method for studies on species-rich groups that are usually neglected in biodiversity research projects because of their unresolved taxonomy.

Species Identification in Malaise Trap Samples by DNA Barcoding Based on NGS Technologies and a Scoring Matrix.

The German Barcoding initiatives BFB and GBOL have generated a reference library of more than 16,000 metazoan species, which is now ready for applications concerning next generation molecular biodiversity assessments. To streamline the barcoding process, we have developed a meta-barcoding pipeline: We pre-sorted a single malaise trap sample (obtained during one week in August 2014, southern Germany) into 12 arthropod orders and extracted DNA from pooled individuals of each order separately, in order to facilitate DNA extraction and avoid time consuming single specimen selection. Aliquots of each ordinal-level DNA extract were combined to roughly simulate a DNA extract from a non-sorted malaise sample. Each DNA extract was amplified using four primer sets targeting the CO1-5' fragment. The resulting PCR products (150-400bp) were sequenced separately on an Illumina Mi-SEQ platform, resulting in 1.5 million sequences and 5,500 clusters (coverage ≥10; CD-HIT-EST, 98%). Using a total of 120,000 DNA barcodes of identified, Central European Hymenoptera, Coleoptera, Diptera, and Lepidoptera downloaded from BOLD we established a reference sequence database for a local CUSTOM BLAST. This allowed us to identify 529 Barcode Index Numbers (BINs) from our sequence clusters derived from pooled Malaise trap samples. We introduce a scoring matrix based on the sequence match percentages of each amplicon in order to gain plausibility for each detected BIN, leading to 390 high score BINs in the sorted samples; whereas 268 of these high score BINs (69%) could be identified in the combined sample. The results indicate that a time consuming presorting process will yield approximately 30% more high score BINs compared to the non-sorted sample in our case. These promising results indicate that a fast, efficient and reliable analysis of next generation data from malaise trap samples can be achieved using this pipeline.

Phylogenetic relationships of the genus Cheilosia and the tribe Rhingiini (Diptera, Syrphidae) based on morphological and molecular characters.

The phylogenetic relationships of the tribe Rhingiini and the genus Cheilosia (Diptera, Syrphidae) were investigated using morphological and molecular characters. The genus Cheilosia is one of the most diverse lineages of hoverflies (Syrphidae). The mitochondrial protein coding gene cytochrome c oxidase subunit I (COI), and the D2-3 region of the nuclear 28S rRNA gene were chosen for sequencing, and morphological characters were scored for both adults and immature stages. The combined dataset included 56 ingroup taxa. The datasets were analyzed separately and in conjunction, using both static and dynamic alignment under the parsimony criterion. The aim of the study was to assess the phylogenetic relationships of the tribe Rhingiini, and to explore if the subgenera of Cheilosia were supported as monophyletic clades. Results showed that the monophyly of subtribes of Rhingiini remained ambiguous, especially due to unstable phylogenetic placements of the genera Portevinia and Rhingia. We recovered most subgenera of Cheilosia as monophyletic clades. Dynamic alignment, using the optimization alignment program POY, always recovered more parsimonious topologies under all parameter weighting schemes, than did parsimony analyses using static alignment and analyzed with NONA.