Arrup akiyoshiensis 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.

Arrupinae, Chilopoda, Kagekiyo-ana, limestone, taxonomy, 18S rRNA gene

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 and Geophilus 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 and Minelli 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.

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, National Museum of Nature and Science, Tokyo (NMST).

Figure 1. 

A map of Kagekiyo-ana cave. Contour lines are shown with altitudes every 50 m.

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 MgCl₂, 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).

The two adult female specimens examined were morphologically almost identical (except for the body size), and were therefore concluded to be conspecific. Male characteristics are unknown at present.

Arrup akiyoshiensis sp. n. exhibits unique characteristics which are not observed in other valid named congeners, i.e., the entire areolation of the crypeal pleurite, elongation of distal part of female gonopod, and tiny tubercle on forcipular article II. It is most similar to A. holstii (Pocock, 1895) and A. ishiianus Uliana, Bonato & Minelli, 2007 (Fig. 10) known from Japan, but can be easily distinguished from them by clearly developed, outwardly pointed tooth of the forcipular segment I (sharp and short tooth in A. holstii; well developed, rounded tooth in A. ishiianus), and triangular basal tooth of forcipular tarsungulum (sharp basal tooth in A. holstii; rounded or slightly pointed basal tooth in A. ishiianus). It is also easily distinguished from other similar congeners (Uliana et al. 2007) by a combination of the characteristics shown in Table 7. In addition, the 18S rRNA gene sequences of A. akiyoshiensis sp. n. and A. ishiianus differed by four bp out of 1821 bp (sequence of three individuals of A. akiyoshiensis sp. n. are all identical). Therefore, the undetermined species is herein concluded to be new species, named A. akiyoshiensis.

Arrup akiyoshiensis sp. n. and A. holstii can be found in the same area, Akiyoshi-dai. However, it is unclear whether the both species occur in the same cave or not, because the cave where A. holstii was found is not clearly mentioned (Kuramoto 1980). At least, A. holstii was not collected in our survey of Kagekiyo-ana, where A. akiyoshiensis sp. n. was collected. Many endemic species with small geographic ranges may occur in isolated caves (Barr Jr and Holsinger 1985). For further understanding of endemism of Akiyoshi-dai, a thorough inventory of cave invertebrates is needed.

Arrup akiyoshiensis sp. n. has no troglomorphic traits such as exceptionally long antennae, legs, and claws (Stoev et al. 2015). In the collection site (shown in Fig. 1), four troglobionts species Nesticus akiyoshiensis akiyoshiensis (Uyemura, 1941) (Araneae); Coecobrya akiyoshiana (Yosii, 1956) (Collembola); Trechiama pluto kanekiyo Ueno, 1958 (Coleoptera); an undescribed species of Campodeidae (Diplura), and one troglophiles species: Epanerchodus etoi etoi Miyosi, 1955 (Polydesmida) can be found. Thereuopoda clunifera (Wood, 1862) (Scutigeromorpha) and species of Rhaphidophorid (Orthoptera), both of which have strongly indicated epigeal ecology, cannot be found (T Murakami pers. obs.). Considering the above facts, the habitat of A. akiyoshiensis sp. n. seems to be confined to cave environments. Further surveys are needed to consider adaptations of its ecology for cave environment.

We are grateful to Dr Manabu Hori (Yamaguchi University) for helpful support. We also thank Mr Tatsumi Suguro (Keio Yochisha Elementary School), Ms Mari Ishida (Akiyoshi-dai Museum of Natural History), Mr Keisuke Kawano (The Firefly Museum of Toyota Town), Dr Kiyoshi Ishii (Professer Emeritus, Dokkyo University), and Dr Makiko Hasegawa (Showa University) for much support and advice. A part of this study was supported by Nippon Life Insurance Foundation (Leader: Takeshi Yamasaki, FY2017–FY2018), Asahi Glass Foundation (Leader: Katsuyuki Eguchi; FY2017–FY2020) and JSPS KAKENHI Grant Numbers 15H02858 for Satoshi Shimano.