A new species of the genus Arrup from a limestone cave in Akiyoshi-dai, Western Japan (Chilopoda, Geophilomorpha, Mecistocephalidae)

Abstract Arrupakiyoshiensis Tsukamoto & Shimano, sp. n. is described from a limestone cave, Kagekiyo-ana, in Akiyoshi-dai, one of the largest karst regions in Japan, Yamaguchi prefecture. It is distinguishable from 14 valid named congeners by some unique characteristics including entire areolation on the cephalic pleurite, elongation of distal part of female gonopod, and a tubercle on forcipular segment II. In addition, the 18S rRNA gene sequences of A.akiyoshiensis Tsukamoto & Shimano, sp. n. and A.ishiianus, one of the most morphologically similar species, differed by four bp out of 1821 bp. The fact that only troglobionts and troglophilic species are found in the collection site suggests that this new species might be a cave-dweller.


Introduction
Centipedes are for the most part soil-dwellers, and common in various habitats such as forests, grasslands, coastal areas and so on (Lewis 1981). Although the most of soil-dwelling animal taxa have troglobionts species, few troglobiotic centipedes have so far been recorded (Chagas-Jr and Bichuette 2018). Especially, despite high adaptation for subterranean habitats, only two troglobionts species are hitherto known in Geophilomorpha: Geophilus persephones Foddai &Minelli, 1999 andGeophilus hadesi Stoev, Akkari, Komerički, Edgecombe &Bonato, 2015. Both of them have unusual traits, which are common among troglobiotic arthropods (exceptionally elongated antennae, legs, and claws) (Foddai andMinelli 1999, Stoev et al. 2015). In Japan, several centipede species can be found in both the inside and outside cave, and Shinohara (1966) referred two species were considered to be troglobiotic centipedes; Brachygeophilus polyporus Takakuwa, 1942 (Geophilomorpha) and Monotarsobius minor Takakuwa, 1942 (Lithobiomorpha). Commonly, the troglobiotic fauna has a high proportion of endemic species in each cave or cave group (Gibert and Deharveng 2002;Christman et al. 2005). Many endemic species with small geographic ranges may occur in isolated caves (Barr Jr and Holsinger 1985); therefore, the inventory of the troglobiotic fauna is important to clarify the formulation of endemism.
Akiyoshi-dai, where is a one of the largest karst regions in Japan, has a spread of 16 km in northeast direction and 6 km northwest direction, with more than 400 limestone caves (Fujii 2009). Thirteen invertebrate species are endemic to the area (Kuramoto 1980). For Chilopoda, 13 species are recorded from Akiyoshi-dai, but all of them are not endemic species (Kuramoto 1980). In the course of our recent survey of cave invertebrates in Kagekiyo-ana cave of Akiyoshi-dai, six individuals belonging to the genus Arrup Chamberlin, 1912 were collected, and later confirmed as a new species based on our careful morphological examination and comparison with 14 valid named congeners (Uliana et al. 2007, Bonato et al. 2016) by using cephalic capsule, mandible, maxillae, the number of coxal pore and genital segments. We herein describe this species as A. akiyoshiensis sp. n.

Materials and methods
Two adult female specimens and four juvenile specimens of A. akiyoshiensis sp. n. were collected by hand under rocks inside Kagekiyo-ana cave (a limestone cave; 34°17.50'N, 131°20.00'E), in Akiyoshi-dai (a karst region), Mitou-cho, Mine-shi, Yamaguchi Prefecture, Japan. The exact position of the collection site is shown in Fig. 1. This was 130 m below the surface, 500 m from the northern entrance, and 900 m from the southern entrance of the cave. In addition, one specimen of A. ishiianus Uliana, Bonato & Minelli, 2007 from Imperial Palace, Tokyo was used for comparing morphology and 18S rRNA gene sequence with A. akiyoshiensis sp. n. Each specimen examined in the present study is specified by its specimen identification number, in the form "TS-YYYYMMDD-XX"; where TS is an abbreviation of the first author, Tsukamoto Sho; YYYYMMDD designates the date on which the specimen was collected; and XX is the identification number given to each specimen collected on that date (e.g., TS-20180330-01). All specimens are deposited at the Collection of Myriapoda, Department of Zoology, National Museum of Nature and Science, Tokyo (NMST).
Specimens were observed and drawn in lactic acid on temporary cavity slides using a Nikon Eclipse E600 microscope, and were then mounted with Hoyer's medium (gum arabic, chloral hydrate and glycerol). Some characters were photographed by using Panasonic LUMIX DMC-GX8 and Canon EOS Kiss X9, and focus stack images were produced from a series of pictures at different focal planes by Helicon Focus Pro version 6.6.1 on a desktop PC. Note that the external shape might be slightly distorted when immersed in lactic acid because of expansion of internal tissue. Besides, specimens were measured with their each part mounted with Hoyer's medium in order to avoid distortion of the external shape. The morphological terminology used below is mainly based on Bonato et al. (2010).
Genomic DNA was extracted from part of the appendage using a DNeasy Blood & Tissue Kit (Qiagen), with modifications from Johnson et al. (2004). An appendage of each specimen was incubated at 55 °C for 48 h to lyse the tissue. Before each lysis mixture was pipetted into a spin column, the exoskeleton was retrieved and preserved in 100% Ethanol. Table 1 lists all primers used in this study. Partial sequences of 18S rRNA gene were amplified by polymerase chain reactions (PCR) using the primer sets, 18S-F1 and 18S-R9 (Yamaguchi and Endo 2003). The PCR amplification was performed in a Thermal Cycler Dice (Takara) in a 10 μl volume containing 0.5 μl of template solution, 2 mM MgCl 2 , 2.5 mM each dNTP, 10 pmol each primer, and 0.25 U Ex Taq polymerase Hot Start version (Takara) in 1× buffer provided by the manufacturer. Amplification conditions were 95 °C for 2 min; 35 cycles of 95 °C for 30 sec, 50 °C for 30 sec, and 72 °C for 2 min; and 72 °C for 7 min.
Amplification products were purified with the ExoSAP-IT kit (Thermo Fisher Scientific). All nucleotide sequences were determined by direct sequencing using a BigDye Terminator Cycle Sequencing Kit ver. 3.1 with an ABI 3500XL automated sequencer (Thermo Fisher Scientific). The amplification primers and internal primers were used in sequencing 18S rRNA gene. Nucleotide sequences were assembled and edited with MEGA7 (Kumar et al. 2016). Sequences have been deposited in DDBJ/ EMBL/GenBank database under accession numbers LC460298-LC460301 (Table 2). Etymology. The species name is derived from the name of Akiyoshi-dai Karst region, which includes the type locality.
Diagnosis. Arrup akiyoshiensis sp. n. can be distinguished from the all named congeners by a combination of the following morphological characteristics: frontal line curved; seven pectinate lamellae in mandible; comma-shaped distal lobe of coxal projection in first maxillae; a tiny tubercle on outer-distal corner of each article of the telopodite; distal article of the telopodite of the second maxillae without claw; the welldeveloped tooth of forcipular article I; the triangular basal tooth in tarsungulum; the poison calyx overreaching forcipular article I; 31-35 pores on lateral and ventral sides on coxopleura.
First maxillae (Figs 4E, 8D) undivided, without mid-longitudinal suture in coxosternite, convergent forward; anterior corners not projecting; ventral surface areolate, except for anterior and lateral margins; setae absent. Coxal projection well developed, with six spines on each internal margin and 4-5 setae at the each middle position. Basal part of medial projection round, with distal lobe; distal lobe clavate as commashaped. Basal part 1.7 times as long as distal lobe.
Second maxillae (Figs 4E, 8D) undivided, without mid-longitudinal suture in coxosternite; 5 + 5 setae arranged along the anterior margin, 3 + 3 setae on lateral side. Isthmus areolate. Anterior and posterior margins concave. Lateral margins       parallel. Telopodites triarticulated, reaching the telopodite of first maxillae. Claw of the telopodite absent. A tiny tubercle present on outer-distal corner of each article. Article I 2.8 times as long as wide; article II 1.6 times as long as wide; article III 3.2 times as long as wide. Forcipular segment (Figs 5A-D, 7B, C, 9A, B) with setae both on dorsal and ventral surface; setae arranged almost symmetrically. Coxosternite with distinct 1 + 1 projections in anterior margin. Chitinous lines absent. Forcipular tergite trapeziform. When telopodites closed, tarsungulum reaching anterior margin of cephalic plate. Article I 1.9 times as long as wide, with a well-developed pointed tooth at the distal internal corner. Article II 0.40 times as long as wide, with a tubercle at the internal margin (arrows in Fig. 5A, B). Article III 0.37 times as long as wide, with a tubercle at the internal margin. Tarsungulum with a triangular basal denticle. Claw of tarsungulum with numerous tiny sensilla. Calyx of poison gland overreaching article I. Duct opening of poison gland on dorsal tip of tarsungulum (triangle in Fig. 5C).
Leg-bearing segments (excepting last leg-bearing segment) (Fig. 6A-D) without pore field on sternites. Median longitudinal sulcus present on sternites I-XVII. Forty-one leg-bearing segments in both the holotype and paratype. All legs weakly areolate. First pair of legs much shorter than the others. All leg claws with anterior and posterior accessory spines; posterior one with a subsidiary spine at its bottom (arrows in Fig. 6C, D).
Last leg-bearing segment (Figs 6E-H, 9C, D) with numerous setae both on tergite and sternite; setae arranged almost symmetrically. Sternite as long as wide, sub-triangular, with posterior margin round. Tergite sub-pentagonal. Coxopleura with 31-35 pores on lateral and ventral sides. Telopodite having six articles, but without claw.
Remarks. Arrup akiyoshiensis sp. n. is morphologically similar to several other congeners, especially A. holstii (Pocock, 1895) and A. ishiianus Uliana, Bonato & Minelli, 2007 (Fig. 10), but can be easily distinguished from them by a combination of the characteristics shown in Table 7.