The Lonchaeidae (Diptera) of the GBOL project, with the description of a new Priscoearomyia species

The investigation of 331 specimens of the acalypterate dipteran family Lonchaeidae within the GBOL-project resulted in a list of 29 species from which one is new to science, Priscoearomyia bausenbergensis sp. nov. , and four species represent new records for Germany. For all voucher specimens detailed metadata are provided including validated DNA barcodes. These barcodes build a sound reference basis for future molecular identification of lonchaeid flies and will also allow the inclusion of female specimens in biodiversity studies, when morphological characters for separating these females are not available.


Introduction
Lonchaeidae is a small family of acalypterate flies, which is distributed worldwide.In Europe, approximately 100 species are known to occur with about 60 in Germany (see Maca 1999, Werner 1995, MacGowan 2004, MacGowan 2015, MacGowan et al. 2007, MacGowan and Rotheray 2008, Reimann and Rulik 2014, Reimann and Stuke 2016, MacGowan and Reimann 2021).The adults are small usually shiny black, sometimes somewhat dulled or slightly green/blue metallic flies.The females always have a strongly sclerotized retractable aculeus.The male genitalia vary in shape and structure in and between the genera.There is a wide range of larval habitats, e.g., under the bark of dead wood, in conifer cones, in rotting plant material, in fruits, in bulbs or in galls in the stems of plants.For more details on the biology see Morge (1963) and MacGowan and Rotheray (2008).In many cases, especially in small dipterans, it has been difficult to proper identify female specimens.Often there is a group of closely related species, where females look very similar and the morphological assignment to the respective males is very difficult or even impossible.Barcoding provides a tool for solving this problem.In Lonchaeidae the barcode sequences of the different species analyzed so far are very distinct and females can be identified in comparing their barcode with those of the males.Afterwards it is possible to search for additional morphological characters, which can aid in identification keys.Furthermore, barcoding can be used to identify torn or incomplete specimens which are always present in Malaise trap samples, especially when the emptying cycles are longer than one week.
Since 2011 the German Barcode of Life project (GBOL) acts as a national barcoding campaign and builds up a voucher-based barcode reference library (Geiger et al. 2016).So far roughly half of the German animal diversity is documented, while the megadiverse groups of Diptera or Hymenoptera are highly underrepresented.Both are present with more than 10.000 species in Germany.Therefore, the recent phase -GBOL III: Dark Taxa -is concentrating especially on micro flies and wasps.But even other small dipteran taxa need some serious attention and will reveal more diversity as known before.
In the present study we combine results with reverse identification approaches out of the Global Malaise trap Program (GMP).

Material and methods
The majority of the material examined was caught with three Malaise traps in Rhineland-Palatine in the administrative district Ahrweiler.The remaining samples are from different localities in other federal states of Germany and France (Fig. 1).For a detailed list of localities see Suppl.material 2.
All specimens except the non-GBOL material are stored in purified ethanol at -20 °C.For morphological identification genitalia were dissected, if needed and treated with KOH-solution (5% in aq.) at 70 °C for 30-60 min., then neutralized with acetic acid (5% in aq.) and rinsed in distilled water.The genitalia are stored in small silicon tubes with the respective specimen.From the non GBOL material one specimen is pinned and the genitalia are mounted on a glass slide; the others are stored in denaturated ethanol (70% in aq).
After morphological identification most of the specimens were barcoded following the established GBOL procedure: Tissue was subsampled from each specimen and transferred into 96 well plates for subsequent DNA extraction.For specimens larger than 2 mm 1-3 legs were used for lysis.For very small specimens (≤ 2 mm) the whole body was used non-destructively for lysis (i.e., subsequent voucher recovery).Genomic DNA was extracted using the BioSprint96 magnetic bead extractor and respective kits by Qiagen (Hilden, Germany).Polymerase chain reaction (PCR) was carried out in a total reaction volume of 20 μl, including 2 μl of undiluted DNA template, 0.8 μl of each primer (10 pmol/μl), 2 μl of 'Q-Solution' and 10 μl of 'Multiplex PCR Master Mix', containing hot start Taq DNA polymerase and buffers.The latter components are available in the Multiplex PCR kit by Qiagen (Hilden, Germany).
A minor fraction of specimens was processed within the Global Malaise trap Program (GMP) and the CCDB standard barcoding procedures were applied (https:// ccdb.ca/resources/).
Sequences were semi-automatically edited, assembled using the MUSCLE alignment approach (Edgar 2004) and checked for the occurrence of stop-codons or hints of nuclear mitochondrial DNA segments (NUMTs) in GENEIOUS version 7.1.9(http://www.geneious.com;Kearse et al. 2012).All metadata of specimens treated here were deposited in GBIF (https://biocase.zfmk.de/ipt/resource?r=gbolonch);further details like voucher information such as locality data, habitat, collector, identifier, taxonomic classifications, DNA barcode sequences, primer pairs, the sequence data and trace files were deposited in BOLD (https://doi.org/10.5883/DS-GBO-LONCH)and subsequently also transferred to Gen-Bank (accession numbers: OP831582-OP831877 and KT781862-KT781863).A neighbour-joining tree was obtained using the Tamura-Nei model without outgroup (Tamura and Nei 1993).Morphological identifications were checked on taxonomic consistency via the TaxCI script (Rulik et al. 2017).Inconsistent specimens were either morphologically re-identified (males) or assigned by topology within the genetic distance tree (NJ) and BLASTing against reliable barcode compliant entries of BOLD (females).Additionally, the following species delineation methods: Assemble Species by Automatic Partitioning (ASAP; Puillandre et al. 2021) and the BOLD built-in RESL algorithm for Barcode Index Numbers (BINs; Ratnasingham and Hebert 2013) were applied.
Distance statistics were calculated using DiStats (Astrin et al. 2016).For the alignment only barcode sequences with full length of 658 base pairs (n = 274) were used.
The Description.Male: Head.Eye with sparse very short hairs.Frons and face completely greyish brown dusted.9 to 10 frontal setulae in a row along the eye margins, 16 scattered interfrontal setulae.Orbital seta strong and long (0.35 mm), as long as outer vertical seta.Inner vertical seta slightly longer than outer vertical seta.Orbital plate dusted, with a shining central streak, bare apart from orbital seta.Ocellar seta strong, as long as orbital seta.Few scattered ocellar setulae, 0.25 times as long as ocellar seta.Lunula bare.Four anterior genal setulae in a single row.Postpedicel short, oval, not reaching mouth edge, 1.5 times as long as wide, distinct orange spot on the basal medial surface, which occupies approximately one third of the inner surface.Arista two times as long as postpedicel, short pubescent.
Thorax.Thoracic dorsum black, dusted greyish brown, covered in setulae one third length of orbital seta.Scutellum completely dusted greyish brown, no obvious contrast to thoracic dorsum, bare apart from the 4 marginal setae.Lateral sclerites completely dusted.Anepisternum with anterodorsal seta absent, three strong posterior setae in a dorsoventral row, approximately 12-16 scattered setulae on disc.Katepisternum with two strong posterior seta and one to two weaker anterior setae in an irregular row close to the dorsal margin, central part bare, approximately 4-7 setulae ventrally.One seta on proepimeron and one on proepisternum.Calypter yellow-white with light brown fringe.Wings yellowish in anterior half, turning slightly brownish towards wing tip, posterior half whitish yellow.Veins yellowish basally turning brownish towards wing tip.All legs entirely black.
Abdomen.Tergite 5 2.2 times the length of tergite 4, funnel-shaped in dorsal view, strongly narrowing in posterior half, with a closely spaced group of 6 stiff setulae on each side before the apex.A small triangular membraneous area on central anterior margin (Fig. 3C).In lateral view anteriorly bulged, abruptly narrowing in posterior half, beak-shaped (Fig. 3D).
Male terminalia.Epandrium (ep) short, belt like, three times as high as long (Figs 2A, 3A), with a terminal fringe of setulae on each side, gradually increasing in length from ventral to dorsal.Ventral lobes (ve) not sclerotized, only present as a membranous strip not exceeding the width of the epandrium, covered in small setulae.Fused cerci (ce) not sclerotized, hardly visible, with a row of setae.Surstyli (su) extending ventromedially from the shell of the epandrium as two lightly sclerotized processes covered in very short thick-based setulae (Fig. 3B).No prensisetae visible.Hypoproct (hy) a cap-shaped plate,   3B), both lobes with a fringe of hairs increasing in length towards the middle.Phallus Z-shaped with a massive basiphallus (bp) attached to a cross shaped plate with a heavy sclerotized rod extending from this plate anteriorly.Distiphallus (dp) long and straight, less sclerotized (Fig. 2C).Parameres (pa) belt like, not well sclerotized, strongly curved before ending close to base of phallus, not fused in midline (Fig. 2C, D).Hypandrial apodeme (ha) strongly sclerotized.Measurements: Body length 3.74 mm.Wing length 3.60 mm.
Female: Head.As in male.Postpedicel oval, reaching mouth edge, 1.6 times as long as wide.Distinct orange spot at the basal inner surface.Arista 1.75 times as long as postpedicel, short pubescent.
Thorax.Thoracic dorsum sub-shining black, completely dusted greyish brown, anteriorly covered in setulae one third length of orbital seta, posterior to the suture some setulae reaching almost half the length of orbital seta.Scutellum completely dusted greyish brown, dusting stronger than on thoracic dorsum, with 4 marginal setae (only bases visible in paratype).Lateral sclerites completely dusted.Anepisternum with no anterodorsal seta and three strong posterior setae in a dorsoventral row.With a few scattered setulae on disc.Katepisternum with two strong posterior setae and two weaker (half the length of the posterior) in an irregular row close to the dorsal margin, central part bare, 7 setulae ventrally.One seta on proepimeron and one on proepisternum.Calypter yellow-white with light brown fringe.Wings yellowish in anterior half, turning slightly brownish towards wing tip, posterior half yellowish white.Veins yellowish basally turning brownish towards wing tip.Only hind legs present in female paratype, entirely black.
Abdomen.Tergites completely brownish dusted.Sternites completely dusted.Aculeus black with yellow tip.Apical segment as in Fig. 2E.In dorsal view slightly constricted in the middle.In side view a long ventro-apical seta with three very small setulae anterior to these on each side and a tiny setula close to the tip.Dorsal surface with a pair of long setae at postion 0.6 of the total length measured from the base of the segment.Apical dorsal setae missing in paratype female specimen.
Etymology.This species is named after the collecting site of the male holotype.Biology.Unknown.Distribution.Only known from Europe (Germany, France).

Differential diagnosis.
With the plate-like hypoproct and epandrium without well sclerotized ventral lobes, this species belongs to a group with P. greciana (McAlpine, 1983) and P. hermoensis MacGowan & Freidberg, 2008.It differs from both mentioned species by the shape of the phallus, which is more or less U-shaped in P. greciana and P. hermoensis (see MacGowan 2014a, figs 22-31), but Z-shaped in P. bausenbergensis.Furthermore, the shape of the hypoproct is quite different.In P. greciana the hypoproct is a dome-shaped plate with two very short apical lobes.In P. hermoensis these lobes are broader and longer and have numerous finger-like processes on the outer margin (MacGowan 2014a).In P. bausenbergensis the apical lobes are very broad with smooth outer margins and enclosing a narrow oval incision.The extremely narrowed and elongated tip of the abdomen separates the new species from all other known species of Priscoearomyia, except P. greciana, which shows this character to a lesser extent.The small triangular membraneous area on the anterior margin of tergite 5 is not present in P. greciana and clearly seperates P. bausenbergensis from the latter species.The large distinct orange spot at the base of postpedicel separates the described species from all others, except P. withersi Mac-Gowan, 2014, which can have a small spot present.The new species can be included in the key to the Priscoearomyia species by MacGowan (2014a) as follows: 1 Epandrium without an obvious ventral lobe, hypoproct wider than long, in form of a Remarks.This specimen could not be assigned to L. spicata with confidence and holotype comparison was not possible due to unavailability.Therefore, we could only assign the specimen to L. cf.spicata.
Remarks.There was no respective male in our sampling.Therefore, this female could not be assigned to species level by barcoding.Remarks.There was no respective male in our sampling.Therefore, this female could not be assigned to species level by barcoding.Remarks.There was no respective male in our sampling.Therefore, this female could not be assigned to species level by barcoding.

Genetic analysis
Our sampling includes 331 specimens of which 68 are males and 263 are females.Barcoding resulted in COI-sequences of 298 specimens, which allowed us to identify most of the female specimens which could not be identified by morphology.All specimens and the according BOLD and Gen-Bank accession numbers for successfully sequenced material are listed in Suppl.material 2.
Average COI sequence length for the 298 sequences was 653 bp of 658 bp full barcode length, including twenty-four shorter sequences composed from 561 to 648 residues, respectively.
Among nucleotides, there was a compositional bias towards AT: 66.6% especially at third codon positions (average 90.1%) which is close to levels previously reported for other Diptera groups (e.g., Bernasconi et al. 2000;Cywinska et al. 2010;Rivera and Currie 2009).In detail, overall base composition was: A 28.5, C 16.7, G 16.7, T 38.1%.
Altogether, 37,401 pairwise distances were computed for our dataset: of these, 6,560 were intraspecific distances.
Our combined analysis resulted in a list of 29 species from which one, Priscoearomyia bausenbergensis is new to science.Furthermore, it includes eight well separated female only molecular clusters (see Suppl.material 1).Four species are new to the list of German Lonchaeidae.Included in the list are also those clusters of female specimens which could not be identified to species level.If possible, a closer relation to known species is given.One specimen could only be assigned with cf.due to taxonomic issues.

Discussion
In our sampling there were many more females than males.For most genera in the Lonchaeidae aerial swarming behavior of the males is known (McAlpine and Munroe 1968).This might be a more general habit in this taxon as already mentioned in MacGowan and Rothery (2008) and would partly explain why fewer males are obtained by Malaise trap sampling at ground level.The females are actively searching for substrates suitable for egg laying and may therefore be more often encountered by this method.As already mentioned, it was possible to identify most of the female specimens by barcode comparison or group females based on clustering.This is especially important in those species groups where the females are extremely similar and a morphological identification is impossible at the moment, but as far as barcodes of the corresponding males become available this may change immediately.Otherwise, we would not have been able to identify all the female specimens of the genus Priscoearomyia and the female specimens of e.g. the fraxina group of species within the genus Lonchaea (L.fraxina and L. iona in our sampling).Additionally, the assignment of the female paratype of P. bausenbergensis with certainty was only possible based on the barcoding results.The finding of a new species in our sampling is not that surprising.As already mentioned by MacGowan and Reimann (2021), there were several new species descriptions in the genus Priscoearomyia in the last decades.At the moment the species can only be separated by means of the male genitalia.This indicates that there may be many more distinct species in collections, which are not yet recognized.Another reason, of course, is the lack of taxonomic expertise in Lonchaeidae and in a broader sense in many other acalypterate dipteran taxa.Here barcoding of trap samples or other material can aid in finding new species.If there are clusters of specimens which cannot be assigned to known species, these clusters are worthy of deeper morphological investigation.But, at first, there is the need of barcode references for as many species as possible.Our study provides a sound reference basis for future molecular identification of lonchaeid specimens.This can significantly influence the data accessible for biogeographic studies, if all data of female records can be included.

Figure 1 .
Figure 1.Map with the sample locations for the data presented here.Map created at GPSVisualizer.com.Map data from Open-StreetMap.orgunder Open Data Commons Open Database License (opendatacommons.org).

Figure 2 .
Figure 2. Drawings of male (A-D) and female (E) genitalia of Priscoearomyia bausenbergensis sp.nov.: A. Lateral view of epandrium; B. Posterior view of epandrium and associated structures; C. Lateral view of phallus and parameres; D. Ventral view of phallus and parameres; E. Lateral (above) and dorsal (below) view of female ovipositor.
two broad triangular apical lobes, almost touching in midline, leaving a narrow oval incision (Figs 2B,

Figure 3 .
Figure 3. Abdomen and genital capsule of male holotype of Priscoearomyia bausenbergensis sp.nov.: A. Lateral view of male genital; B. Apicoventral view of male genital; C. Dorsal view of tip of abdomen; D. Lateral view of tip of abdomen.

Figure 4 .
Figure 4. Barcode gab analysis.Relationship between maximum intraspecific and nearest neighbor p-distances.Points above the diagonal line indicate species with a barcode gap.Molecular species delimitation by ASAP and BINs suggests 38 putative species, which fits well with our morphological findings (Fig. 5 and Suppl.material 1).