A new Diplura species from Georgia caves, Plusiocampa (Plusiocampa) imereti (Diplura, Campodeidae), with morphological and molecular data

1 Colecciones Entomológicas Torres-Sala, Servei de Patrimoni Històric, Ajuntament de València, Passeig de la Petxina, 15, 46008 València, Spain. 1 Departament de Didàctica de les Ciències Experimentals i Socials, Facultat de Magisteri, Universitat de València, Avda. Tarongers 4, 46022 València, Spain. 2 Institut Cavanilles de Biodiversitat i Biologia Evolutiva, Universitat de Valencia, C/ Catedrático José Beltrán 2, 46980 Paterna, Spain. 2 Department of Life Sciences, The Natural History Museum, Cromwell Road, London SW7 5BD, UK. 3 Departament de Botànica i Geologia, Facultat de Ciències Biològiques, Universitat de València, C/ Dr. Moliner 50, 46100 Burjassot, València, Spain. 3 Division of Invertebrate Zoology, American Museum of Natural History, Central Park West at 79th Street, New York, NY 10024-5192, USA. 4 Universidad de Alcalá, Research Team on Soil Biology and Subterranean Ecosystems, Department of Life Sciences, Faculty of Science, Campus Universitario Crta. A-2 Km. 33.6, E-28805, Alcalá de Henares, Madrid, Spain. 5 Laboratori d’Investigació d’Entomologia, Departament de Zoologia, Universitat de València, C/ Dr. Moliner 50, 46100 Burjassot, València, Spain. 6, 7 Institute of Zoology, Ilia State University, Giorgi Tsereteli 3, 0162, Tbilisi, Georgia.


Introduction
is a relatively well-known genus of the subfamily Plusiocampinae, part of the most diverse family of Diplura (i.e., Campodeidae Lubbock, 1873;Sendra et al. 2021a). Seventyfi ve Plusiocampa species have been described so far, mostly cave-dwellers distributed throughout the Mediterranean and Black Sea regions but some inhabiting soil ecosystems (Sendra et al. 2021a(Sendra et al. , 2021b (Fig. 1). Four species of Plusiocampa are already known from caves around the Black Sea, at the western side in the well-known Movile Cave in Dobroudja (Condé 1993(Condé , 1996; caves from the Crimean Peninsula in the north (Silvestri 1949;Sendra et al. 2020a), and a cave near the Abkhazia coast in the east (Sendra et al. 2020a). Recently collected specimens of Plusiocampa, found in three caves in the Imereti Plateau (Georgia), have allowed us to describe one more species using morphological and, for the fi rst time, molecular data.
Campodeoidea DNA sequences are poorly represented in current databases (only 14.6% of the dipluran sequences; NCBI accession date 29/04/2021), and data are highly skewed towards a handful of taxa. Only 15 Campodeoidea identifi ed to species level are represented in Genbank, and only 5 of those include a DNA barcode (Remycampa herbanica Sendra & Oromí, 2020, Campodea fragilis Meinert, 1865, C. lubbocki Silvestri, 1912, C. tillyardi Silvestri, 1931and Lepidocampa weberi Oudemans, 1890. Previous studies have focused on sequence variation in nuclear ribosomal genes (18S rDNA and 28S rDNA), but these genes provide little resolution for evolutionary relationships within Campodeidae (Luan et al. 2005;Sendra et al. 2020b). The present study contributes the fi rst CO1 sequences for Plusiocampinae taxa and the fi rst molecular data for cave-dwelling species of Plusiocampa.

Material and methods
Diplurans were sampled by hand using an aspirator in the dark zones of the Datvis (Bear), Melouri, and Shvilobisa caves in 2018 and 2019. Specimens were transferred to vials containing 70% ethanol.

Morphological study
In the laboratory, specimens were washed with distilled water, mounted on a slide with Marc André II solution, and examined under a phase-contrast optical microscope (Leica DMLS). Illustrations were made with a drawing tube and measurements were taken with an ocular micrometre. To determine body length, specimens were mounted in toto and measured from the base of the distal macrochaetae on the frontal process to the supra-anal abdominal valve. Four specimens were coated with palladium-gold for SEM photography (Hitachi S-4900) and sensilla measurements. The morphological descriptions and abbreviations follow Condé (1956). The term ʻgouge sensillaʼ is used for the concavo-convex shaped sensilla on the antennae, following Bareth & Condé (1981).

Institutional abbreviations
Coll AS = private collection of Alberto Sendra, València, Spain IZISU = Institute of Zoology at Ilia State University, Georgia MZB (MCNB) = Museu de Ciències Naturals de Barcelona, Spain

Molecular analysis
Total genomic DNA was isolated from ethanol-preserved tissues using commercial extraction kits (NucleoSpin kit, Macherey-Nagel™). Therefore, we decided to amplify the most variable CO1 gene fragment with the universal primers LCO1490 and HCO2198 (Folmer 1994), commonly used for DNA barcoding. The thermal profi le of the polymerase chain reaction (PCR) used was 94°C for 15 min for polymerase activation (HotStart), followed by 38 cycles of 94°C for 30 s, 50°C for 30 s, 72°C for 30 s, and a fi nal extension at 72°C for 20 min. Amplifi ed PCR products were cleaned with Exo-SAP enzyme prior to direct product sequencing in an ABI Prism 3770 (Macrogen, Spain).
Chromatograms were checked using BioEdit ver. 7.2.5 (Hall 1999), and all sequences were translated into amino acids to detect possible insertions and/or stop codons to rule out the presence of pseudogenes, and a sequence alignment was performed using the MAFFT program with default parameters. To improve reliability, conserved (ungapped) blocks of sequence were extracted from each alignment using the Gblocks server under default settings (Castresana 2000). The CO1 gene has been suggested as an informative molecular marker at several taxonomic scales, particularly at the species level. Therefore, K2P genetic distances were obtained for the CO1 dataset using MEGA ver. 7 (Kumar et al. 2016) to compare with estimates in other taxa. The best-fi tting substitution model was tested using MrAIC ver. 1.4.6 (Nylander 2004) and selected with a correction for small sample sizes according to the Akaike information criterion (AICc). The maximum-likelihood (ML) phylogenetic tree construction method was applied as implemented in Phyml ver. 3.0 (Guindon et al. 2010).

Etymology
The specifi c epithet refers to the Imereti region, the location of the Shvilobisa Cave, treated as a noun in apposition.
SECONDARY SEX CHARACTERS. Male urosternite I (Fig. 8) with slightly enlarged subcylindrical appendages, each bearing up to 21 glandular a 1 setae. Female appendages slightly thinner, with up to 11 glandular a 1 setae. Spermatozoid fascicles 40 μm in diameter without apparently spiral fi lament.

Molecular analysis
The nucleotide substitution model selected was GTR+G+I (BIC = 6998.6), with the proportion of invariant sites (I = 0.46) and estimated alpha parameter for the gamma distribution (α = 1.39), indicating a signifi cant heterogeneity in the DNA substitution among sites. The Campodeidae sequences formed a well-supported clade, clearly distinct from that of Japygidae (Fig. 13)

Habitat
Plusiocampa (Plusiocampa) imereti Sendra & Barjadze sp. nov. inhabits the deep zone (over 50 m from the entrance) of three caves. The Shvilobisa Cave, the type locality, is a 1000 m long, tunnellike, easily accessible sub-horizontal cave with a small subterranean water stream (Tatashidze et al. 2009b). The others two nearby caves are about 55 km away from the Shvilobisa Cave; the Melouri Cave is 5300 meters long and has the status of natural monument (Tatashidze et al. 2009b), whereas the Datvis Cave is a poorly known cavern (K. Tsikarishvili, pers. comm.). The distance between the Datvis and Melouri caves is ca 3.5 km. The Melouri Cave -easily accessible -has dried halls and a permanent subterranean water stream near its end. This cave has gigantic stalagmites and fallen stones. The Datvis Cave is a horizontal, dry, and easily accessible cave with several halls, which are rich in different speleothems like the Shvilobisa Cave (Tatashidze et al. 2009b).
These phyletic relationships based on morphological evidence have been supported for the fi rst time in Campodeidae by molecular evidence. Thus, the molecular results confi rm Plusiocampinae to be a monophyletic group, as previously proposed in Condé (1956), Paclt (1957), and Sendra et al. (2020a). Furthermore, the CO1 tree suggests that Plusiocampa s. str., with medial posterior macrosetae on the mesonotum and metanotum, is probably monophyletic, including P. (P.) humicola, P. (P.) aff. elongata Ionescu, 1955 and P. (P.) imereti Sendra & Barjadze sp. nov. These phyletic affi nities confi rm those pointed out in several contributions (Condé 1956;Sendra et al. 2019Sendra et al. , 2020a) based on morphological features and suggest that further analyses using both nuclear and mitochondrial genes should be completed in order to clarify the evolutionary relationships within Plusiocampinae.

Biogeographical notes
Plusiocampa is the most diverse genus of Plusiocampinae, with 74 species including the new one, spread around the Euro-Mediterranean Basin and the Black Sea. Furthermore, Plusiocampa and Plusiocampinae are also totally absent north of 50º N latitude, which roughly marks the southern limit of the ice during the Last Glacial Maximum from Belgium to Crimea (Sendra et al. 2020a;Sendra et al. 2021b). Most species of Plusiocampinae inhabit cave ecosystems, with only nine dwelling in moist, soil habitats.
All the localities of species of Plusiocampa in the Black Sea region are found in karst caves located at low altitudes, not far away from the coastline (Fig. 1). None of them exceed the ice-covered areas (glacier or permafrost) during the Last Glacial Maximum (Fig. 1) Sendra & Reboleria 2012). The southern area of the Black Sea lacks records of any caveadapted species of Plusiocampa or troglobitic diplurans in spite of occasional biospeleological visits, which also occurs in the caves of other regions such as the Pontic Mountains (Sendra et al. 2006(Sendra et al. , 2010.