First barcode-assisted annotated checklist of owlflies (Neuroptera, Myrmeleontidae, Ascalaphidae) of Georgia with the first record of genus Deleproctophylla Lefèbvre, 1842

The present study aims to provide an updated checklist of the owlfly subfamily As - calaphidae Lefèbvre, 1842, with the first records of the genus Deleproctophylla Lefèb-vre, 1842, from the country. The new records give an improved understanding of owlfly distribution within the country. The record of the genus Deleproctophylla in Georgia is based on a single female specimen of D. australis (Fabricius, 1787)


Introduction
Named after the custodian of the Hades' orchard, owlflies (Ascalaphidae) are the predaceous insects of the order Neuroptera, both in the imaginal and larval stages (Devetak 2007), consisting of less than 500 species worldwide, with the highest diversity in tropical and subtropical regions (Devetak and Janžekovič 2012).Based on phylogenomic results, a new classification for the antlions was proposed, which synonymizes Ascalaphidae with Myrmeleontidae and divides the family into four subfamilies (Ascalaphinae, Myrmeleontinae, Dendroleontinae, and Nemoleontinae) (Machado et al. 2019).This classification is based on a single publication, which is not unanimously supported (Badano et al. 2017;Michel et al. 2017;Jones 2019;Jones and Badano 2021), as it is believed that further studies are needed to confirm or reject this position, and that, for the time being, the Ascalaphidae should be retained as a family.
Most of the European species are univoltine or semivoltine; in some cases, like Bubopsis, the whole cycle lasts more than two years in nature, with the overwintering stage always represented by larvae (Badano et al. 2014).The Caucasiana 3: 5-18 (2024), DOI: 10.3897/caucasiana.3.e117039Japaridze et al.: First barcode-assisted annotated checklist of owlflies first data on owlflies in Georgia was published just after World War II (Shengelia 1947).Then, Zakharenko and Krivokhatsky (1993), list one species (Libelloides macaronius) from Georgia and Deleproctophylla australis (!) from the Caucasus (without specifying location and references).In general studies, such as the European Monograph of Neuropterida (Aspöck et al. 1980) and the Catalog of Neuropterida of the Western Palearctic (Aspöck et al. 2001), owlflies are generally reported from Georgia (L.macaronius) or even more broadly from the Caucasus (Deleoroctophylla variegata (Klug, 1834)).Until now, two genera consisting of three species were recorded from Georgia.Despite the recent studies on Ascalaphidae and Neuroptera in general in recent years (Duelli et al. 2015;Dobosz et al. 2017;Dobosz et al. 2018;Kerimova et al. 2023), D. australis (Fabricius, 1787) has always eluded collectors.Finally, we present the first record of this genus from Georgia, along with the specimens' photographs, collecting information, a distribution map, and the results of DNA barcoding for each studied species.

Sample collection and preparation
Most of the examined specimens were collected within the framework of the Caucasus Barcode of Life (CaBOL) project (https://ggbc.eu/) by entomological nets during the day, by hand, or in the light traps in the 2021-2023 period.Collected specimens were preserved in 96% ethanol, stored in a freezer under -22˚C at the scientific collections of Ilia State University, or kept dry in the personal collection of the first author.
The part of the species was determined in accordance with the key by Aspöck et al. (1980).Photographs of the live specimens were taken using a Canon EOS 5D Mark II camera equipped with a Canon EF 100mm f/2.8LMacro IS USM.Photographs of the preserved specimens were done using a Canon EOS 60D camera with a Canon EF 60 mm f/2.8 Macro USM lens.The digital images were then processed in Zerene Stacker version 1.04 image stacking software and Adobe Photoshop CS6.
In recent decades, the role and contribution of сitizen scientists in the field of entomology has been highly acknowledged.In entomology, their contributions to our comprehension of patterns and processes have been impressive for hundreds of years.Gathering information and collaborating across disciplines, volunteers have helped shape our understanding of this complex field (Gardiner and Roy 2021;Kittelberger et al. 2021), including discoveries of Neuroptera species new to science (Winterton et al. 2012) as well as defining their distributional patterns (Parenta et al. 2022).The present survey is not the first of its kind in Georgia, since citizen scientists have already contributed to local faunistic and distributional research (Iankoshvili and Tarkhnishvili 2021).Accordingly, it would have been unreasonable to neglect publicly available and valuable data from online platforms and databases such as iNaturalist and Georgian Biodiversity Database (GBD) (Tarkhnishvili et al. 2013), or specialized local groups on Facebook, as we attempted to compile the deposited data in the species list given below.The distribution map for studied species and sampling localities was created using QGIS v. 3.22.10.

DNA processing
Some of the collected specimens were submitted to the DNA barcoding pipeline at Ilia State Unviersity.DNA was extracted from whole samples using the Quick-DNA Magbead Plus Kit (Zymo Research).Partial sequences of cytochrome oxidase subunit I (COI) were amplified by polymerase chain reaction (PCR) using the primer pairs LCOI490-JJ and HCO2198-JJ (Astrin and Stüben 2008).Thermal conditions included denaturation at 95 °C for 1 min, followed by the first cycle set (15 cycles): 94 °C for 30 s, annealing at 55 °C for 1 min (-1 °C per cycle) and extension at 72 °C for 1:30 min.Second cycle (25 cycles set): 94 °C for 35 s, 45 °C for 1 min, 72 °C for 1:30 min, followed by 1 cycle at 72 °C for 3 min, and the final extension step at 72 °C for 5 min.PCR amplicons were visualized on 1% agarose gels using 1.7 μl of PCR product.The unpurified PCR products were sequenced in both directions at the Beijing Genomics Institute (Hong Kong, CN) using the amplification primers.Sequence analysis was performed using Geneious Prime 2022.1.1 (http://www.geneious.com).Extracted DNA was deposited in the scientific collections of Ilia State University, Tbilisi, Georgia, and aliquots will be deposited at LIB Biobank at Museum Koenig, Bonn, Germany, while the sequences have been submitted to Barcode of Life Data System (BOLD) databases.The newly obtained DNA barcodes of COI sequences were checked against the BOLD Systems database (http://www.boldsystems.org/index.php).Barcode Index Number (BIN) (Ratnasingham and Hebert 2013) for the sequenced taxa and their nearest neighbor in BOLD Systems (if they had a BIN) are also given.For the calculation of sequence differentiation, we used p-distance as performed in the BOLD Systems.

Results
In total, the data of 128 specimens was collected, comprising four species from three genera.We were able to obtain five quality barcodes (658 bp in length, with no stop codons, indels, or deletions) for all four species.Barcode information is given under each barcoded species listed below.The list is given in alphabetical order.An asterisk (*) is used to indicate species first recorded in Georgia.
Remarks.Bubopsis hamata is a species with a distribution ranging from northeastern Africa to West Asia (Hölzel 2004), and the Caucasus (Dobosz et al. 2017).From the neighboring countries, this species is known to occur in Armenia, Azerbaijan, Turkey, and Dagestan (Dobosz et al. 2017).From Georgia, this species is also reported by Kerimova et al. (2023).Bubopsis hamata is the only species of local owflies with a crepuscular lifestyle (Aspöck et al. 1980).For species distribution within the country, see Fig. 12. Remarks.Deleproctophylla australis exhibits considerable variability, especially the wing pattern in individual populations, which often lacks front-wing spots, making it challenging to identify the species using the key in Aspöck et al. (1980).Generally, D. australis is significantly larger than the similar species Deleproctophylla variegata.Also, specimens of D. variegata collected in Kyrgyzstan, from the collections of the Upper Silesian Museum in Bytom are smaller.Deleproctophylla australis has extensive brownish-red spots on both wings below the pterostigma.It happens that the spot on the forewing is missing, but the spot on the hindwing remains always brownish red.The spots on the hind wing of D. variegata are darker brownish black (visibly darker than in D. australis).Sometimes the wing is slightly smoky toward the base.There are also differences in the pattern of the pronotum and thorax.In the Upper Silesian Museum in Bytom collection, the two species show considerable, in the Figures 1. Bubopsis hamata (Klug, 1834); third instar larvae.case of the above features, differences.In D. variegata specimens from other regions, such as Kyrgyzstan or Afghanistan, the wing membrane is smokey and the specimens are a little darker than the others, but these are more just colour variations.In Georgia, both of the species are highly likely to occur.

*Genus
Prior to our work, there were no barcodes for the properly identified D. australis in BOLD Systems (or in GenBank).We assume that the very small p-distance between D. australis and D. variegata might be attributed to the potential misidentification of the specimen whose molecular data were mined from GenBank.Such cases are not uncommon and require an individual approach to address the issue, considering the possibility that the observed morphological differences could be indicative of a single species with variations across isolated populations.
Deleproctophylla australis is an element of the Mediterranean fauna that has previously never been reported in the Caucasus region.The nearest known report of the species lies in Edirne, Turkey (Popov 1977;Háva 2000;Canbulat 2007).Háva (2000), not knowing the publication by Alexi Popov, quotes the same specimens from the collection of the National Museum in Prague.In a European monograph of Neuropterida (Aspöck et al. 1980) and a Catalog of Neuropterida of the Western Palearctic (Aspöck et al. 2001) the authors incorrectly listed D. australis from Anatolia, which is a very broad geographical term, suggesting the occurrence of this species in almost the entire territory of Turkey, while it occurs in its European part near the border with Bulgaria.This species, most likely incorrectly, was recorded from the Caucasus and Middle Asia (Zakharenko and Krivokhatsky 1993).This information is not commented on in any publications by Zakharenko, Krivokhatsky, or other authors.For species distribution within the country, see Fig. 12.   (Kerimova et al. 2023).
Remarks.A highly variable species that requires revision.Of the numerous described subspecies and color varieties, there are currently three recognized taxa (Oswald 2023).An attempt to organize the taxonomy of L. macaronius of Crimea and related species from the Western Palearctic was made by Krivokhatsky et al. (2018).Unfortunately, in the taxonomy of this group, he adopted criteria that were inconsistent with the Code of Zoological Nomenclature, hence, despite many valid conclusions regarding population variability, they cannot be included in the taxonomy.In the Caucasus region, there is great variability within this species, which is also visible in the illustrations presented in this paper, e.g., Fig. 8 typical colored specimen, Fig. 9 white colored form (in Krivokhatsky (2018) [respectively] Libelloides macaronius kolyvanensis (Laxmann, 1770) m. typica and Libelloides macaronius kolyvanensis (Laxmann, 1770) m. alba).
Libelloides macaronius is a species with an expansive Ponto-Mediterranean distribution (Devetak 2007).It is the most widespread local species, reported from Georgia and all neighboring countries (Zakharenko and Krivokhatsky 1993;Aspöck et al. 2001).For species distribution within the country see Fig. 12. Barcoding.A single barcode was obtained from the specimen with CaBOL-ID 1010385 (BOLD: AEG2795) identical to the nearest neighbor in the BOLD Systems L. ustulatus (Eversmann, 1850) from Georgia, Abastumani (BOLD: AEG2795).

Discussion
Based on the distribution pattern (Fig. 12), it is evident that three species, specifically Bubopsis hamata, Deleproctophylla australis, and Libelloides macaronius, are primarily distributed within the arid habitats of the central and southeastern regions of Georgia.In contrast, the predominant population of L. ustulatus is concentrated along the Meskheti Range, extending to the vicinity of Tbilisi, at altitudes ranging from 700 to 2200 m a.s.l.There is a strong likelihood that the Caucasian owlfly (L.ustulatus) also inhabits the subalpine belt within the northern mountainous regions of Georgia.After analyzing the collected data, local owlflies can be classified into two conditional groups: late spring-late summer species, which include L. macaronius, B. hamata, and D. australis (flight period V-VIII), and late spring-early summer species represented solely by L. ustulatus (flight period V-VI).Typically, this species flies in May, but at high altitudes (> 2000 m a.s.l.) can be observed until the end of June.Our data corresponds to the species phenology given in Aspöck et al. (1980), except for L. ustulatus, which is treated as a subspecies of L. hispanicus.
To gain a more comprehensive understanding of the actual distribution and biological aspects of these sparsely studied and infrequently observed owlfly species (except for L. macaronius) within the local ecosystem, further extensive research is needed.It is also worth noting that there is no description of the larval stages available for B. hamatus and L. ustulatus.

Figures 12 .
Figures 12.The distributional map of Georgian owlflies (Ascalaphidae) discussed in the main text.