Nihonella gen. nov., a new troglophilic genus of dwarf spiders from Japan with a discussion on its phylogenetic position within the subfamily Erigoninae (Araneae, Linyphiidae)

A new monospecific genus belonging to the family Linyphiidae Blackwell, 1859, Nihonella gen. nov., is described using an integrative taxonomic approach based on the species N. chika gen. et sp. nov. The new genus is endemic to Western Honshu, Japan, and it shows distinctive genitalic and somatic characters of other genera of the subfamily Erigoninae Emerton, 1882. Nihonella gen. nov. is found only in the twilight and transition zones of caves in Okayama and Nara Prefectures. The phylogenetic position of Nihonella gen. nov. within the subfamily Erigoninae, and its relationship as a sister clade of the species of the group of Savignia Blackwell, 1833 (sensu Millidge 1977), is discussed on the basis of both, morphological and molecular evidence.

and species-rich (Tanasevitch 2020). At middle latitudes, these tiny spiders live in a wide range of habitats including forest leaf litter, empty spaces between rocks, and caves, where they build small sheet-webs. In Japan, 293 linyphiid species belonging to 109 genera are currently recorded (Tanikawa 2020). Although some Japanese species can colonize the entrances of caves or other semi-subterranean environments, the hypogean environment supports only a few species (Ono et al. 2009).
While collecting in caves in Central and Western Honshu, Japan, we found several specimens of spiders belonging to different families, including some Linyphiidae. A detailed morphological analysis of the collected material led to the discovery of an unknown linyphiid species belonging to the subfamily Erigoninae. The peculiar somatic characters, as well as the shape of the epigyne and male palp, excluded this species from the currently known genera. Thus, herein we describe this spider as a new species, and we establish a new genus on the basis of morphological and molecular evidence. To support this claim and to clarify the phylogenetic position of the new genus within the subfamily Erigoninae, we conducted a multi-locus phylogenetic analysis, the results of which are herein discussed.

Taxonomy
Specimens were collected inside caves in Okayama and Nara Prefectures (Western Honshu Island, Japan) and immediately preserved in 99% ethanol for morphological and molecular studies. Samples were studied at the Systematic Zoology Laboratory, Department of Biological Sciences, Tokyo Metropolitan University, Japan (TMU). A Nikon SMZ1270 stereo microscope and a Nikon Optiphot 2 microscope were used to observe habitus and genitalia of the specimens, and to take measurements. Photographs were taken using a Canon EOS60D digital camera mounted on the microscopes. Final images were merged with Helicon Focus 7 image stacking software. Additional scanning electron micrographs were taken using a Jeol JSM-6510LV scanning electron microscope at TMU. The male left palp was drawn and photographed. Epigyne was dissected using a sharp scalpel and cleared with lactic acid to show the inner structures. All measurements are reported in millimeters, leg measurements are given as total length (femur, patella, tibia, metatarsus, tarsus). The specified chaetotaxy (= tibial spine formula) refers to the number of dorsal spines on tibiae I-IV. All vouchers used in this study are preserved in the National Museum of Nature and Science, Tokyo, Japan (NMST).

Institutional abbreviations
NSMT = National Museum of Nature and Science, Tokyo, Japan TMU = Tokyo Metropolitan University, Japan

Molecular analysis
The complete genomic DNA was extracted from four legs of each sample using a Qiagen DNeasy Blood & Tissue Kit, following the standard protocol suggested by the manufacturer. Extraction and Polymerase Chain Reaction amplification (PCR) of the samples were performed at TMU. Genomic DNA from voucher specimens was stored in a freezer at -20°C at the same institute. Five gene fragments were amplified using standard primers: cytochrome c oxidase subunit I (COI), 16SrRNA (16S), 18SrRNA (18S), 28SrRNA (28S), and histone 3 (H3). PCR amplifications were performed using a SimpliAmp Thermal Cycler (Thermo Fisher Scientific, USA) with a final volume of 11µl. All primers and protocols used in this study follow Arnedo et al. (2009). In addition, the following primers were also used to amplify the first segment of 18S: 18S-1F (forward) TACCTGGTTGATCCTGCCAGTAG (Giribet et al. 1996) and 18S-SSU (reverse) GTGGTGCCCTTCCGTCAATT (Balczun et al. 2005). Purified PCR products were submitted and sequenced by Eurofins Genomics Company, Tokyo branch. Sequences of other linyphiid spiders used in the analysis were obtained from GenBank (https://www.ncbi.nlm.nih.gov/genbank/). In order to clarify the phylogenetic position of the new genus herein described, we reconstructed a simplified phylogenetic tree of Linyphiidae, including representative species of the five main subfamilies distributed in the Palearctic region: Stemonyphantinae (Wunderlich, 1986), Linyphiinae (Blackwell, 1859), Micronetinae (Hull, 1920), Ipainae Saaristo, 2007, and Erigoninae. A preliminary morphological study of our samples suggested that the new genus was closely related to the species of the Savignia group sensu Millidge, 1977. Thus, we focused the sampling especially on the Erigoninae subfamily, including distal Erigoninae, genera located in early branches, and species belonging to the Savignia group. Pimoa rupicola (Simon, 1884) from the family Pimoidae Simon, 1884 was preferentially selected as an outgroup to root the tree, due to the close relationship between Pimoidae and Linyphiidae (Wheeler et al. 2017;Fernandez et al. 2018). Fifty-eight taxa composed the final dataset. The complete list of sequences and related GenBank identification codes are reported in Table 1.
Final sequences were edited using Bioedit ver. 7.0.5 (Hall 1999) and aligned using the online version of MAFFT ver. 7.450 (Katoh et al.  In order to compare the genetic distance between the new genus and the main components of the Savignia group, partial fragments of the COI barcodes of nine species within the group were found on GenBank (see Table 1). The GenBank sequences were compared with the new genus sequences using an

Diagnosis
The new genus is distinguished from any other genera belonging to the distal Erigoninae clade by the following unique combination of somatic and genitalic characters: Femur I with 1 prolateral spine; Tibia I with 1 dorsal spine; tibial spine formula: 1.1.1.1; male palp with a well-developed distal suprategular apophysis and a hypertrophic ʻsecondaryʼ DSA (Figs 2A-B, 3A-C, 4A-E) (usually presented in Erigoninae as a simple tooth and protruding form a different side of the DSA); a well-developed and uniquely-shaped prolateral tibial apophysis, the same length as the cymbium and partially covering it (Figs 2A-C, 3A-B, D, 4E-F). The unusual chaetotaxy and unique shape of the epigyne, with anteriorly converging lateral walls and two distinct, flat, ovoid inflations of the copulatory ducts, also distinguishes the female of this genus from females of any other genera in distal Erigoninae.

Etymology
The generic name is a combination of the word ʻNihonʼ and the Latin suffix ʻ-ellaʼ. The former refers to the country of Japan where the genus is endemic; the latter is the feminine suffix of ʻ-ellusʼ commonly used in Latin to form the feminine diminutive of a noun. Name in apposition, feminine in gender.

Species included
Only the type species Nihonella chika gen. et. sp. nov.

Taxonomic remarks
The morphology of Nihonella gen. nov. suggests it may be closely related to the species of the Savignia group. However, the presence of numerous differences in genitalia shape and somatic characters does not allow us to include the new genus within this group of species. A close but distinct relationship with the Savignia group is also supported by the molecular analysis (see Fig. 5).
Nihonella gen. nov. male palps have a general morphology similar to those found in some genera of the Savignia group (e.g., Araeoncus Simon, 1884 or Diplocephalus Bertkau, 1883). They share a similar shape in the embolic division: a long, modified palpal tibia, and a well-developed DSA. However, the new genus shows a distinct hypertrophy of the SDSA, which is extremely long and clearly protruding outside the frontal part of the palpal bulb (Figs 2A Table 2), and by the shape of the epigyne and male palp (short SDSA, different shape of epigyne and internal ducts, see

Etymology
The specific name is derived from the Japanese word ʻchikaʼ (地下) meaning ʻunderground, subterraneanʼ and thus refers to the habitat of the species, but it is also the pronunciation of a feminine given name in the Japanese language. Name in apposition. ProsoMa. Carapace, chelicerae, labium, and sternum uniformly light brownish. Head distinctly raised, AME = 0.04, PME, ALE, PLA = 0.06. Anterior margin of cheliceral groove bearing 5 robust teeth.

Holotype
oPistHosoMa. Opisthosoma uniformly grayish, lacking any pattern, covered with numerous short hairs. Central area of ventral side of opisthosoma slightly lighter.  Fig. 4A-D. Palpal tibia bearing 1 trichobotrium and long prolateral tibial apophysis approximately as long as the cymbium and partially covering it along its median line. PTA triangle-shaped when observed dorsally, ending with triangular spiked structure. Cymbium ovoid when observed dorsally, covering whole bulb with exception of the tip of secondary branch of distal suprategular apophysis. Deep groove along middle line of cymbium, in which rests ventral part of PTA. Paracymbium stumpy and simple, lacking in apophyses. Tailpiece of radix slightly protruding, with well-developed, thin apophysis, hook-like and frontally-oriented. DSA well-developed, hook-like and ending with blunt tip. Secondary branch of distal suprategular apophysis hypertrophic, long and thin, strongly protruding frontally and ventrally, ending with sharp tip. Median membrane transparent and barely visible, protruding retrolaterally. Embolus hook-like, initial trait oriented frontally then ventro-posteriorly, stumpy, ending with blunt end near tip of DSA.
Female (based on three paratypes) Habitus. As shown in Fig. 1D-G.
ProsoMa and oPistHosoMa. Coloration and other details of carapace, chelicera, and opisthosoma as in male. Head only slightly raised.

Ecology and habitat
Although lacking extreme troglomorphic characters (e.g., eye loss), N. chika gen. et. sp. nov. shows troglophilic adaptations, such as body depigmentation. This species has only been found inside caves, several meters from the entrance, in the twilight and transition zones where the light is strongly reduced or absent. N. chika gen. et. sp. nov. builds small sheet-webs inside cracks or empty spaces between rocks on the cave floor.

Molecular analysis
The general structure of our Bayesian phylogeny (Fig. 5) shows strong similarity to phylogenies reported in previous studies on Linyphiidae (Miller & Hormiga 2004;Arnedo et al. 2009;Zhao & Li 2017). In our tree, the nodes of clades forming the main linyphiid subfamilies are strongly supported (PP = 1) and show monophyly. Some genera of Micronetinae appear mixed together with the basal Erigoninae; this is in line with previous research and confirms that these two taxa, as they are currently recognized, are not monophyletic. Within the distal Erigoninae, the node support is sometimes low (PP ≤ 0.85) and thus the phylogenetic positions of some genera are uncertain. Nevertheless, most of the main clades, including the Savignia group, are well-defined and with high node support (PP > 0.95). In particular, the genera Dicymbium Menge, 1868, Diplocephalus Bertkau, 1883, and Savignia Blackwall, 1833 form a wellsupported clade, as hypothesised by Frick et al. (2014). Our analysis highly supports (PP = 1) Nihonella gen. nov. as a distinct and well-defined genus within the subfamily Erigoninae, and in particular as part of the distal Erigoninae clade. Such results further suggest a close relationship between the new genus and the species forming the Savignia group sensu Millidge (1977).
The pairwise distance analysis (Table 2) shows a large genetic distance (13-15%) between Nihonella gen. nov. and the Savignia group. Genera within the Savignia group are also separated to each other by between 10% and 19% genetic distance.

Discussion
Both the molecular results and the genital morphology support a close relationship of Nihonella gen. nov. with the main genera forming the Savignia group. Thus, we reject the hypothesis of morphological similarities in palp and epigyne between Nihonella gen. nov. and the Savignia group as the result of evolutionary convergence. Nevertheless, despite this affinity, well-defined genetic differences and distinctive traits in somatic characters and genital morphology clearly show that the new genus belongs to an independent monophyletic clade, separate from the Savignia group. In particular, a tibial spine formula of 1.1.1.1, as shown in the new genus, is unusual and in contrast with the standard chaetotaxy of the species forming the Savignia group. According to our phylogenetic results, Nihonella gen. nov. can be considered a sister clade to the Savignia group to which it shares a common ancestor.
The Savignia group remains an unresolved taxon, in need of further study. Although the wide majority of its species seem to share the same common origin, recent phylogenetic studies suggest that this group, as was originally defined by Millidge in 1977, is probably not monophyletic (Frick et al. 2010). Thus, more comprehensive analyses are necessary to evaluate the correct composition and systematic position of the Savignia group within the distal Erigoninae clade. Due to its probable close relationship and potential common ancestry with the Savignia group, Nihonella gen. nov. might be a promising genus to shed further light on the phylogeny of this group of species.
As far as we know, Linyphiidae are not commonly found in caves within Japan or other Asian countries (Zhao & Li 2017). Some Japanese genera (e.g., Arcuphantes Chamberlin & Ivie, 1943) inhabit the entrance and inner areas of natural large cavities, but their species are not endemic to the subterranean environment. Such limited affiliation with the hypogean habitat is also highlighted by the retention of cuticle pigmentation in these spiders. Among other linyphiid genera, in Japan only Porrhomma Simon, 1884 and Micrargus Dahl, 1886 seem to contain species which are only found in caves. Among endemic linyphiid cave species, only P. ohkawai Saito, 1977 andP. rakanum Yaginuma &Saito, 1981 share marked troglobitic adaptations. No other obligate cave-dwelling linyphiid species were known in Japan until our research. With the present study, we add one more genus with clear troglophilic habits to the Japanese endemic fauna. Although Nihonella gen. nov. does not exhibit distinct troglobitic traits (e.g., eye loss), its depigmentation and preference for the inner traits of caves, as well as the lack of any known epigean record, suggest a strong affiliation with the subterranean environment.
Nihonella gen. nov. is currently monospecific, and has only been found in three localities. Nevertheless, the collection of specimens in caves in Okayama and Nara Prefectures, approximately 240 km away from each other, suggests that the new genus might be more widely distributed than currently known. Such lack of data is probably related to the scarcity of recent taxonomic studies on the Japanese cave spider fauna, with the last extensive surveys conducted in the 1970s. Further collections in Japanese caves, or other Asian karst areas, will probably lead to the discovery of new records of Nihonella chika gen. et. sp. nov. and, possibly, new congeners. The recent discovery of cave-adapted linyphiid genera in China (Zhao & Li 2017), and now Japan, further indicates that the study of the troglophilic linyphiid fauna is in general still largely unexplored in East Asia. Specific taxonomic studies on these small spiders in Asian caves may lead to new and interesting discoveries.