Oscinella Becker, 1909

4.2.6 | Genus Oscinella Becker, 1909 View in CoL

There are 23 species of Oscinella Becker in the Palearctic region (von Tschirnhaus pers. comm.). Although this genus is fairly abundant across Europe, encompasses agricultural pests within the O. frit complex ( Nartshuk & Andersson, 2013) and are addressed in thousands of publications, a taxonomic revision of its Palearctic species is still pending. Nielsen (2014) established four criteria to identify species of O. frit complex from Denmark.

In our dataset, 13 species of Oscinella were recognised, all originally grouped into a single molecular cluster (Figure 6). As expected, this cluster posed several challenges, not only for the O. frit species complex described in the literature ( Nartshuk & Andersson, 2013). Seven of the 13 species were separated reclustering at a 3% threshold ( O. maura (Fállen) , O.pusila (Meigen) , O. vindicata (Meigen) , O. frit var. 1, O. frit var. 2, O. frit var. 5 and O. cf. cariciphila Collin ). However, the genetic boundaries between the other six species were less clear, with O. angularis and O. nitidissima (Meigen) sharing the same haplotype.

For the remaining four species of Oscinella , O.frit var. 3 and O. frit var. 6 were split at a 0.96% threshold. The only haplotype assigned to O. cf. nigerrima (Macquart) and the five haplotypes of O. frit var. 4 were studied in detail. Despite the male terminalia morphology being a useful tool for species recognition within Chloropidae , small variations in the surstylus width and pilosity, cercus shape and surface and phallic complex among the five haplotypes verified were not conclusive for establishing reliable species boundaries within O. frit var. 4. Certainly, additional genomic markers allied to three-dimensional imaging techniques of complex morphological features can provide additional evidence for resolving further the Oscinella frit species conundrum. The diagnostic features of the Oscinella species are outlined in Table 3.

The above criteria related to the gena width, arista ratio and tibia colour proposed by Nielsen (2014) were less informative than the femoral organ and male terminalia to identify the species from the O. frit complex. Additionally, the species O. frit var. 2 have the fore femur notably enlarged as a diagnostic feature. Although the species O. frit . var. 5 and var. 6 share the femoral organ covered with spine-like setulae, there are differences in the ocellar triangle length and setulae coverage. Furthermore, they did not appear as directly related in the haplotype network; therefore, they were considered as separate species.

4.3 | Distribution

4.3.1 | Biogeographic regions

According to Nartshuk and Andersson (2013), adults of Chloropidae are mostly found from June to September in Fennoscandia, this influenced the time frame chosen for the data sampling. The sampled localities with highest abundance of chloropids from June to August of 2004 were Tyresta (site 2; 705 specimens; bog), Marma skjutfält (site 6; 545 specimens; dry meadow with birch), Drakamöllan (site 38; 522 specimens; common bent heath), Gamla Skogsby (site 22; 493 specimens; meadow with shrub vegetation), Nötmyran (site 11; 415 specimens; hay meadow), representing grassland ecosystems (Figure 2) across Boreal, Boreonemoral and Nemoral biogeographic regions (Figure 1). Similarly, the localities with the highest number of species verified were: Nötmyran (site 11; 29 species), Marma skjutfält (site 6; 21 species), Gamla Skogsby (site 22; 21 species), Tyresta (site 2; 18 species) and Drakamöllan (site 38; 18 species). These results agree with Nartshuk and Andersson's (2013) statement that meadows abundant in grasses, sedges and reeds, which serve as the primary host plants for the majority of species, appear to have the greatest abundance and diversity of chloropids.

With a large gap in abundance, the 10 remaining localities accounted for less than 100 specimens each (Figure 2) with only the locality Kronmyran (site 53; 45 specimens; bog) representing a grassland environment in the northeast Boreal region. Therefore, our data suggest that the higher abundance of chloropids seems to be related to Swedish grassland ecosystems located across biogeographic regions.

4.3.2

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Geographic range

The distribution of the verified clusters revealed that 29 species (almost half of the species diversity) were reported from just a single locality each. Conversely, Tricimba cincta was found to be the most widespread species, recorded in 13 of the sampled localities. Following closely were Oscinella frit var. 4 in eight localities, Oscinella frit var. 5 in seven localities and Hapleginella laevifrons in six localities. Although our results on the abundance and species diversity of Chloropidae improve the distribution of this lineage in Sweden, our findings should be interpreted with caution due to temporal and spatial limitations of our sampling.

Previous identifications of Chloropidae collected by the SMTP project available from the GBIF database include a total of 231 occurrences of 53 described species and 25 genera (Karlsson, Ronquist, & Holston, 2020; https://doi. org/10.15468/dl.ucns52). Similar to our results, Tricimba cincta was the most widespread species, recorded in 25 localities, followed by Oscinella frit recorded in 18 localities throughout Sweden. Although Hapleginella laevifrons has only four occurrences in GBIF, the sites are located in the Boreal, Boreonemoral and Nemoral biogeographic regions. The variation in feeding habits of these widespread Chloropidae species, as the exceptionally diverse feeding strategies of Tricimba cincta larvae, ranging from cereals to meat baits, and the extensive host range of over 60 grass species recorded for Oscinella frit larvae, may provide an explanation for the results found in our study and the data available in the GBIF database. Furthermore, Hapleginella laevifrons larvae act as secondary invaders in the cones of various coniferous trees ( Nartshuk & Andersson, 2013).

4.3.3 | New records

Our findings include two rare species that were not previously documented in the GBIF database, and our records have contributed to a better understanding of their distribution range. Lasiosina albipila (Loew) is newly recorded in the Province of Södermanland, and Chlorops cf. finitimus Becker (if conspecific with C. finitimus ) is new to Hälsingland.

4.4 | Genetic data

Through the precise taxonomic assignment of 1371 specimens, this study revealed for the first time the COI barcodes of 22 described species of Chloropidae : Calamoncosis glyceriae Nartshuk , C. halterata Nartshuk & Andersson , Chlorops calceatus Meigen , C.planifrons Loew , C. scutellaris (Zetterstedt) , Conioscinella frontella (Fállen) , C. mimula Collin , C. sordidella (Zetterstedt) , Dicraeus tjederi Nartshuk & Andersson , Elachiptera tuberculifera (Corti) , Incertella albipalpis , I.antennata (Collin) , I. kerteszi , I. nigrifrons , Oscinella angularis Collin , O. cariciphila Collin , O. vindicata , O. pusila , Rhopalopterum fasciola (Meigen) , Trachysiphonella scutellata (Roser) , Tricimba cinta and T. lineela (Fállen) .

The identity of the clusters was compared using BLAST against the NCBI-nt database. Sequences for Cetema elongatum (Meigen) , Pseudopachychaeta ruficeps (Zetterstedt) , Siphonella oscinina , Thaumatomyia notata and Aphanotrigonum nigripes (Zetterstedt) were already available in public databases and confirmed by our results.

Several potential complications were revealed by the blast results. Eight species had>97% match with Nearctic species of grass flies and two species exhibited>95% match with Nearctic and Oriental species (Table 4).

For these six cases (Table 4), the examination of the database species images revealed that the Nearctic and Oriental species are not conspecific with the correspondent Swedish taxon. The erroneous matches in the BLAST results for Trachysiphonella and Incertella reveal issues related to the problematic taxonomy of Oscinellinae genera. Trachysiphonella lacks tribal classification, and it is not found in the Nearctic region, while Dasyopa belongs to the Liparaini tribe and it is restricted to the Americas ( Nartshuk, 2012; Riccardi pers. comm.). Although both genera have yellow pruinose species with wide gena, their suprageneric relationships, as of most Oscinellinae genera, remain unexplored using explicit phylogenetic methods.

The genus Incertella belongs to the tribe Incertellini and Rhopalopterum to the tribe Oscinisomatini , with both genera occurring in the Nearctic and Palearctic regions ( Nartshuk, 2012). Of the five Incertella species identified, three displayed extremely low interspecific genetic distance (0.32% threshold). Such closely related species of Incertella could also account for the incorrect match with Rhopalopterum , as there are longstanding problems with the generic delimitation and species composition of these two genera. Moreover, the taxonomy of both Oscinellinae genera has not been thoroughly addressed on a global scale.

Most of the species in our dataset were best recovered at a 3% clustering threshold. Nevertheless, the intraspecific genetic diversity ranged from 5% (e.g., Siphonella oscinina and Trachysiphonella scutellata ) to 0% (between Oscinella angularis and O. nitidissima ) revealing the limitations of COI minibarcodes to correctly assign species and confirming integrative taxonomy as essential.

Additionally, we compared our DNA barcode sequences to the 1955 chloropid barcodes from 15 European countries that were found in the BOLD Systems public database that had at least a 300 bp overlap with our minibarcodes (Supplementary material S4). The BOLD sequences were aligned and BLAST searches were conducted using our SMTP database as a query and>96% similarity as our threshold. Only eight of the SMTP species had matching sequences in BOLD from three countries (Table 5), with the striking majority of sequences being from Germany.