Description and molecular characterization of Chrysopilus kafkai sp. nov. (Diptera: Rhagionidae) from Serra da Bodoquena (Mato Grosso do Sul, Brazil)

Rhagionidae (Brachycera, Tabanomorpha) is a widespread family of the order Diptera, containing ca. 720 species in 22 genera worldwide distributed. Chrysopilus Marquart is one of the most speciose genus of Rhagionidae. Here we describe Chrysopilus kafkai sp. nov. Cegolin & Santos, the first record of a rhagionid species from Midwest Brazilian region, and includes an identification key and a distribution map to the Brazilian species of Chrysopilus. The species description is complemented by a molecular characterization of the 28S rRNA sequence. Key-Words. 28S; Morphology; Neotropical; SISBIOTA; Taxonomy.

Rhagionidae lacks any clear synapomorphies. The family may be identified by the absence of a scale on the postspiracular sclerite (Kerr, 2010) and by the relative position of fork R₄-R₅ with respect to cell dm and the curvatures of R₅ and R₄ (Grimaldi & Cumming, 1999). Although molecular-based analyses, such as Wiegmann et al. (2000), suggests the family may be paraphyletic with respect to the other families of Tabanomorpha, Kerr (2010)'s extensive analysis, including both morphological and molecular analyses, depicts Rhagionidae as monophyletic.
Only four of the 22 extant genera of Rhagionidae are found in the Neotropical region: Chrysopilus Macquart 1826, Rhagio Fabricius 1805, Atherimorpha White 1914and Sierramyia (Lindner 1924 Kerr 2010 (Santos, 2008;Kerr, 2010;González et al., 2020). Chrysopilus, diagnosed mainly by the presence of a fusion between the sternite IX and the gonocoxites, and along with features such as wing veins R₄₊₅ fork almost in a right angle, is the most diversified and widespread rhagionid genus (Santos & Amorim, 2007).
Although 18 Chrysopilus species have been described in Brazil (Santos, 2008), none of them are known for the Midwest Brazilian region. Made up by the Federal District and three states (Goiás, Mato Grosso and Mato Grosso do Sul), this region holds one of the most diverse savannahs of the world, the Cerrado, and the largest tropical wetland, the Pantanal, along with some dry forest areas (IBGE, 2012). Despite its high number of endemic species (Junk et al., 2006), few collecting expeditions have been conducted in the area or used specimens collected in the two biomes. As a result, there is still an important lack of knowledge on the dipterofauna in the region.
In the present paper, we describe a new species of Chrysopilus collected during expeditions at ISSN On-Line: 1807-0205 ISSN Printed: 0031-1049 ISNI: 0000-0004-0384-1825 the Serra da Bodoquena (Mato Grosso do Sul, Brazil), a national park located in the Cerrado. These expeditions were organized by the SISBIOTA-Diptera project (Lamas et al., 2014). The new species described here is the first record of the family Rhagionidae in the Brazilian Midwest Brazilian region. The description includes both morphological and molecular data.

Taxonomy
The morphological analysis was based on direct observation of dry material, and dissections of male and female terminalia, preserved in vials with glycerin. Illustrations were made using a Nikon Eclipse E200 optical microscope with camera lucida and then vectorized in Adobe Illustrator CS 2018. Photographs were made with a Carl Zeiss SteREO Discovery.V12 Stereo Microscope attached to a Canon EOS Rebel T2i digital camera. All deep focus images were stacked in Adobe Photoshop CS 2018. Scanning electron microscope (SEM) photographs were made using a FEI Quanta 250 scanning electron microscope. The distribution map was made in QGIS 3.6.1, based on distribution registers presented on Santos & Amorim (2007) and Santos (2008).
All the material of the new species is now pinned and deposited in the collection of the Museu de Zoologia da Universidade de São Paulo (MZUSP).

Molecular sequencing
We used the DNA extraction method proposed by Santos et al. (2018). The specimens were clarified with Proteinase K preserving its external morphology. After clarification, the specimen was stored in ethanol 70%. The DNasy extraction kit purchased from Qiagen was applied following manufacturer's instructions, and the solution dissolved in a final volume of 30 μL (see the website protocols.io: http://doi.org/10.17504/protocols. io.kkqcuvw for the protocol used in here).
Concentration and quality of extracted DNA were determined by spectrophotometric evaluation of 1 μL of sample, employing BioDrop μLITE1. An aliquot of 2 μL of each sample was analyzed by 1% agarose gel electrophoresis to verify DNA quality and quantity. Afterwards, 100 ƞg of each DNA sampled was submitted to PCR under conditions 72°C 7 min/40 cycles of 94°C 30 sec, 50°C 30 sec, 72°C 30 sec/72°C 5 min, with the GoTaq DNA polymerase from Promega, to amplify a 464 bp fragment corresponding to D7 fragment of the 28S rRNA encoding gene from Diptera (Tautz et al., 1988). The primers are shown in Table 1. PCR products were analyzed by agarose gel electrophoresis stained with GelRed. The amplified products were purified with ThermoScientific PCR purification kit and submitted to Sanger's sequencing using Big Dye Terminator version 3.1 (ABI foster City, CA), according to manufacturer's instructions. The sequences were analyzed through the Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990).
The new sequences were deposited in GenBank (MK039530). Preservation: Holotype and paratypes dry and well preserved, with a few imperfections; additional material in 70% alcohol, with some missing parts, but complete enough to be identified as the new species.

Chrysopilus
Diagnosis: Gonostylus crest dark brown, strongly curved distally with thick long setae (Fig. 4), yellowish brown anteriorly. Another diagnose characteristic is the wing non-hyaline with only the middle of r₃ and r₄ cells lighter than the borders (Fig. 3).
Male (Figs. 1-4): length: body ca. 6.5 mm, wing ca. 7.3 mm. General color dark brown (Fig. 1a). Head in shades of brown (Fig. 1b). Eyes holoptic, slightly grayish brown with the borders in blackish brown, ommatidium facets round; ocellar tubercle dark brown, ocellus light brown. Gena brown, lighter than the eyes. Bare and strongly curved clypeus. Antenna uniformly light brown with short thin setae, 1.6 times smaller than head; scape flattened, pedicel two times longer than scape, first flagellomere tapering toward apex, clearly narrower than pedicel and approximately as wide as scape.
Maxillary palpus light brown, distally angled with thick medium-sized setae. Proboscis yellowish brown. Occiput grayish brown, with long-sized setae. Thorax dark brown (Fig. 1a), wider than long. Meron almost two times wider than long, katepisternum almost as wide as high (Fig. 2). Coxa dark brown, femur, tibia, and tarsus light yellow (Fig. 1a). Wing membrane brownish along most veins (Fig. 3); pterostigma, approximately six times smaller than the wing length; R₁ and R₂₊₃ running parallel for the most part, only last third of R₂₊₃ converging towards R₁; R₄ barely curved; CuA and CuP joining far from wing margin, CuA+CuP as long as r-m crossvein; discal cell approximately 4.2x longer than wide, bm cell five times longer than wide, br cell six times longer than wide, cua cell six times longer than wide, c cell placed before pterostigma; alula broad. Halter light brown (Fig. 1a).
Etymology: Based on its general dark color, the species is named after the fiction author Franz Kafka, whose literature mostly deals with the darkness and absurdity of human behavior.

Comments: Chrysopilus kafkai sp. nov. resembles
Chysopilus plaumanni Santos & Amorim in the general aspect of the male terminalia. However, C. kafkai sp. nov. has a distinctive crest occupying almost half of the gonostylus, while the crest in C. plaumanni is restricted to the distal region of the gonostylus (Fig. 9). Our molecular characterization of 28S rRNA of Chrysopilus kafkai sp. nov. expands the geographical representation of molecular data for the genus, now with sequences from species distributed in three biogeographical regions (Nearctic, Neotropical, and Australian).

Key to the Brazilian species of Chrysopilus
This key is adapted from Santos & Amorim (2007) and includes Chrysopilus kafkai sp. nov.

CONCLUSION
Until the description of Chrysopilus kafkai sp. nov., just over a dozen of species of Rhagionidae were known to Brazil, concentrated in the Amazon basin and the Brazilian Atlantic Forest (Santos & Amorim, 2007;Santos, 2008). Despite containing a unique and diversified biome -the Cerrado -the biodiversity of the Midwest Brazilian region is still poorly known (Junk et al., 2006). The lack of knowledge on the species of Diptera in the states of Mato Grosso, Mato Grosso do Sul and Goiás is significant (Lamas et al., 2014). Any new species description in a poorly known area such as the Midwest Brazilian region is important to diminish the knowledge gap about biological diversity in the Neotropics. As seen in the distribution map, there is a remarkable absence of species of Chrysopilus described for the Midwest Brazilian region, probably due to lack of published studies, since the region is a hotspot for a number of insect species (Junk et al., 2006;Lamas et al., 2014). Furthermore, the fact that C. kafkai sp. nov. is the first record of such a widespread family as Rhagionidae in the second largest region in Brazil shows the need for increasing taxonomic efforts for documenting the Brazilian dipterofauna. There may be a vast diversity of species of Diptera, including rhagionids and other Tabanomorpha, waiting for formal description.