﻿Into the unknown: the first barcode-assisted checklist of Psocoptera (Insecta, Psocodea) of Georgia with a census on country species richness

﻿Abstract This checklist reports 47 species of Psocoptera from 15 families and three suborders from Georgia, of which 31 species are recorded for the first time, increasing the known fauna of the country by more than 65%. Of these, 37 species have been barcoded, representing 210 Barcode Identification Numbers (BINs). An additional 14 species are expected to occur in Georgia but remain undiscovered, meaning that only ≈ 77% of the fauna is currently documented. Barcodes, comments on distributions, and images of voucher specimens are given followed by a map of the sampling sites.


Introduction
Psocoptera, known as the booklice and barklice, is an order of hemimetabolous insects having approximately 6000 described species worldwide (Anonby 2019), often regarded as the most primitive hemipteroids alive today (Lyal 1985). Despite being called "lice", psocids are not parasites, instead they are free living, generally herbivoroues or detritivorous insects, feeding on organic debris and microflora (Lienhard 1998) and are classified into three suborders: Trogiomorpha (booklice), Troctomorpha (booklice), and Psocomorpha (barklice). While most of them (mainly species belonging to the suborder Psocomorpha) are bark-or leaf-dwellers found outdoors, there are domiciliary species exhibiting excellent powers of dispersal and readily spread by humans all over the world. For some species, this makes it difficult to reconstruct the original distribution or to distinguish between native and introduced once (Schneider 2010).
Although Psocoptera has been classified as an order for much of recent history, it was shown by Yoshizawa and Lienhard (2010) that Phthiraptera (the true lice) have evolved within the Troctomorpha suborder and, based on both morphological and molecular data, are probably sister group to Liposcelididae, or, various lines of Phthiraptera may have budded off independently in the infraorder Nanopsocetae within Troctomorpha.
Thus, to maintain monophyly, the former orders Psocoptera and Phthiraptera are now placed in the order Psocodea (Yoshizawa and Lienhard 2010). However, since psocids and true lice have quite different ecologies, and are studied by different methods, for practical reasons Psocoptera is still typically treated as a group in the traditional way, but referred to as Psocodea: 'Psocoptera' (Anonby 2019;Golub 2016Golub , 2019. This practical approach was also applied in the present work. The first attempt to assess the species richness of the psocid fauna of Georgia was made by Danka (1950), who documented seven families comprising eight species from Sokhumi Botanical Garden. In her later work (Danka 1955), ten species belonging to six families have been reported from Batumi and Sochi Botanical Gardens, six of which have been reported for the first time, thus raising the number of species known from Georgia to 13. Based on the data of subsequent articles (Danka 1957(Danka , 1968, the overall number of Georgian psocids has been raised to 16 local species from ten different families combined in two suborders (Table 1). It should be noted that all studies mentioned above were carried out in the western part of Georgia near the Black Sea coast, in the territories of Adjara and Abkhazia, and since the last review of Georgian Psocoptera (Danka 1968), there has been no survey conducted on this group, hence leaving the hidden psocid diversity of most of the country unrevealed for more than half a century.
In the present contribution, we provide the results of recent countrywide investigations and significantly improve the existing knowledge on the psocid diversity of Georgia.

Locations and methods
The main part of the studied material was collected within the framework of Caucasus Barcode of Life (CaBOL) project, being the most ambitious arthropod inventories ever performed in Georgia by the members of the GGBC (Georgian-German Biodiversity Center) and the  Fig. 1 and in the Suppl. material 1. Collected specimens were preserved in 96% ethanol, stored in a freezer at -22 °C at the scientific collections of Ilia State University. Unless otherwise stated, all material has been determined by the corresponding author, using a Zeiss Stemi Stereo Microscope with 8:1 Zoom and a Zeiss Apo 1.5× FWD 53 mm front lens attached and keys, provided by Lienhard (1998Lienhard ( , 2006 and Saville (2008). For the classification of psocids, we followed Johnson et al. (2022).
Photographs of preserved specimens were taken using a Canon EOS 60D camera with a Canon MP-E 65mm f/2.8 1-5× Macro Photo Lens mounted on a Novoflex Castel-L Focusing Rack. Digital images were prepared using Zerene Stacker image stacking software and Adobe Photoshop CS6.

DNA processing
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 pair LCOI490-JJ and HCO2198-JJ (Astrin and Stüben 2008). Thermal conditions included denaturation at 95 °C for 1 min, followed by 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 cycles set (25 cycles): 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 final extension step at 72 °C for 5 min. PCR amplicons were visualized on 1% agarose gels using 1.7 μl of PCR product. Sequencing of the unpurified PCR products in both directions was conducted at the Beijing Genomics Institute (Hong Kong, CN) by 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 out against the BOLD systems database (http://www.boldsystems.org/index.php). Barcode Index Number (BIN) (Ratnasingham and Hebert 2013) for the sequenced taxa and for 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 system.

Results
In total 2353 specimens were collected representing 45 species comprising 15 families (Fig. 2). New country records are marked with an asterisk (*) and detailed data on the collected material is provided in Suppl. material 1. From the collected material, 371 barklice specimens were submitted for barcoding pipeline and only 210 quality barcodes (658 bp length barcodes, with no stop codons, indels or deletions) representing 37 species were generated so far. Barcode information is given under each barcoded species listed below.

Annotated checklist of Georgian Psocoptera
Suborder Psocomorpha Badonnel, 1951 Family *Amphipsocidae Pearman, 1936 Note. Representatives of the family have not been previously known to occur in Georgia. One species has been recorded within the current study. Genetics. We obtained eleven barcodes from the specimens with CaBOL-IDs 1027555, 1027556, 1027557 (BOLD:AFC5937), 1027571, 1027572, 1012880, 1012881, 1012882, 1012883, 1012884 and 1012885 (BOLD:AFC5936) (maximum p-distance 1.52%) with the nearest neighbor in BOLD systems being K. quisquiliarum from Finland (BOLD:ACG2800, mean p-distance 7.2%). Examined specimens perfectly matched the description provided by Lienhard (1998). The large COI distance (over 5%) between the populations of K. quisquiliarum might indicate the existence of a cryptic species complex in western Palaearctic, and further study is needed to solve this puzzle.

Genus *Kolbia
Remarks. The species is characterized by micropterous females. Genetics. Eleven barcodes were obtained from the specimens with CaBOL-IDs 1012650, 1012720, 1012721, 1012722, 1012893, 1012895, 1012896, 1027603, 1027607, 1027611, 1027613 (BOLD:AEJ9302, maximum p-distance 1.56%) originating from two populations, with the nearest neighbor in BOLD systems being C. fuscopterus from Finland (BOLD:ACG3628, mean p-distance 5.43%). Examined specimens perfectly matched the description provided by Lienhard (1998) (e.g., wing venation and pattern). Given the large COI distance (over 5%) between the populations of C. fuscopterus might indicate the existence of a cryptic species complex in western Palaearctic, and further study is needed to solve this puzzle.

Genetics.
A single barcode was obtained from the specimen with CaBOL-ID 1027647 (BOLD:AEX9052) with the nearest neighbor in BOLD systems being V. burmeisteri from Germany (BOLD:AEX9052, p-distance 0.46%).
Remarks. The species is known to have asexual parthenogenetic populations in Europe, consisting of triploid females, but is believed to have sexual and asexual populations in North America as well (de Moya 2022).
Remarks. Aaroniella badonneli is the only psocid species originally described from Georgia (Danka 1950). It was named after a French entomologist Andre Badonnel, who specialized in Psocoptera. No males of the species are known to be found.

Family Psocidae Roesler, 1940
Note. Nine species are recorded from Georgia.
Remarks. Cryptogenic species of unknown origin (Schneider 2010). Mainly found in buildings with food storages in Central Europe (Schneider 2010; Svensson and Hall 2010) and the south, especially in the Mediterranean, where it is found in more natural places, like dried grass or leaf litter (Lienhard 1998). Lepinotus reticulatus is parthenogenetic and so there are only females, although a few times (non-functional) males have been reported (Lienhard 1998).

Discussion
Danka laid a profound basis with her research on the Psocoptera fauna of Georgia, even though it was limited to only a few locations almost exclusively in Batumi and Sokhumi Gardens located at the Black Sea coast. The material collected and examined in our study mostly also originates from the western part of Georgia (see Suppl. material 1 and Fig. 1) and had more of a bycatch character than a purposeful collection of Psocoptera, but still resulted in 31 species, three families (Amphipcosidae, Elipsocidae, Psyllipsocidae) and one suborder (Trogiomorpha) that have never been previously reported from the country. Of 31 species reported for the first time from Georgia, 24 have been known to occur in the adjacent countries, 37 from the European part of the post-Soviet space and six (Embidopsocus enderleini, Dorypteryx domestica, Cerobasis guestfalica, Liposcelis rufa, Ectopsocus meridionalis and Trichadenotecnum alexanderae) have never been reported from the territories mentioned above ( Table 2). Lack of specialists and targeted studies from other parts of the country on this particular group of minuscule animals has left many more species undiscovered/undescribed. After analyzing the fauna of neighboring countries of Georgia, as well as countries of the Western Palaearctic European part of the post-Soviet space (Table 2) there are at least 14 species still expected to be found in Georgia. New taxa could also be discovered in unexplored areas such as for instance caves (Lienhard 2021) and other underground habitat, thus the psocid fauna of Georgia still remains understudied. This study, originally conceived as a small addition to the fauna of Georgian psocopterans, eventually turned into a large-scale exciting journey through the study of a new group of arthropods for the corresponding author. In the end, we hope that our study inspires the readership to spend more time in nature observing and unveiling the hidden gems, reminding them that science begins with subsiding the mind's curiosity.