A new species of Cybaeus L. Koch, 1868 (Araneae, Cybaeidae) with simple genitalia from central Japan is the sister species of C. melanoparvus Kobayashi, 2006 with elongated genitalia

Spiders of the genus Cybaeus L. Koch, 1868 exhibit two major centers of diversity: Western North America and Japan. Several Japanese Cybaeus possess an elongated embolus in the male palp and elongated tubular spermathecae in the female genitalia. Here we describe Cybaeus koikei sp. nov. from central Honshu, Japan, which has an unelongated embolus and bulbous spermathecae. Phylogenetic analyses using nuclear and mitochondrial gene markers clearly support the monophyly of C. koikei sp. nov. and Cybaeus melanoparvus Kobayashi, 2006, a species with elongated genitalia. Both species share a similar habitus and a cluster of robust setae on the lateral surface of the male palpal patella. The latter is considered a synapomorphy for C. koikei sp. nov. and C. melanoparvus. A supplementary description of the spermathecae of C. melanoparvus is also provided.

The extraordinary species-richness of Japanese Cybaeus may be caused by ecological attributes, such as poor dispersal abilities. Cybaeus spiders are not known to disperse via ballooning (Copley et al. 2009) and this may lead to range restriction in many species. Closely related species of Cybaeus generally share a similar body size and genital morphology but their ranges are parapatric and do not overlap (see fig. 1 in Ihara 2009b). Accordingly, these species may be classified as species groups (= 'superspecies' in Ihara 2008) but the monophyly of each group is yet to be confirmed by molecular phylogenetic analyses.
The morphological diversity of genitalia in Cybaeus is also noteworthy (Ihara 2007(Ihara , 2008. A previous study divided the genitalia of Japanese Cybaeus into two types based on features of the embolus and spermathecae ('types 1 and 2' by Ihara et al. 2021). In 'type 1' the embolus is not elongated and each spermathecae consists of three distinct bulbous parts: the spermathecal head, the stalk, and the base. In 'type 2' the embolus is elongated and the spermathecal head and stalk form an elongated tubular duct but the spermathecal base remains bulbous. The spermathecal head (= 'secondary spermatheca' in Ramírez 2014) of Cybaeus can be distinguished by the presence of primary pores on its surface (Bennett 1992(Bennett , 2006. In Cybaeus, additionally, Bennett's gland, which is a round-shaped patch of developed gland pores on the spermathecae, is typically located at the connection between the spermathecal base (= 'primary spermatheca' in Ramírez 2014) and the stalk (Bennett 1992;Ramírez 2014). Therefore, the homology of spermathecal structures can be determined by the relative position of primary pores and Bennett's gland. The 'type 2' spermathecal head can also be recognized by examining the primary pore positions (Ihara et al. 2021). The number of species that bear the 'type 2' genitalia is small and eight, or possibly nine, species have been recognized to date (Kobayashi 2006;Ihara 2009a;Ihara et al. 2021): six species inhabiting the Ryukyu Islands, plus C. melanoparvus Kobayashi, 2006in central Honshu, C. ishikawai (Kishida, 1940) (in Komatsu 1940 on Shikoku Island, and possibly C. monticola Kobayashi, 2006 in central Honshu. Since 'type 2' genitalia are exclusive to some Japanese species, it is considered to be apomorphic at the species level although this condition may have evolved several times independently (Ihara et al. 2021) and the closest relatives of each 'type 2' species have not yet been fully established.
The Cybaeus fauna in central Honshu, especially around Lake Biwa, was well documented by Kobayashi (2006). However, an unidentified 'small-sized' Cybaeus was collected recently from montane habitats on the western side of Lake Biwa. This unidentified species shows external similarity with C. melanoparvus but possessed bulbous 'type 1' spermathecae. Here we describe it as a new species, Cybaeus koikei sp. nov., and analyze its phylogenetic position with special focus on the relationship between C. koikei sp. nov. and C. melanoparvus.

Samples and morphological observation
Cybaeus spiders were collected from central Honshu ( Fig. 1) between 2007 and 2020. All specimens were collected either by hand or using a pooter. Where possible, geographical coordinates of the collection sites were obtained using a portable GPS unit (Garmin eTrex, Garmin Ltd., Olathe, KS). Specimens were preserved in 70% ethanol but legs of some specimens were removed and preserved in 99% ethanol for DNA extraction. Epigynes were dissected from female specimens and then cleared to observe their internal structure following the method described by Matsuda et al. (2020). Examination of the specimens was conducted using a stereoscopic microscope (M125C, Leica Microsystems, Wetzlar, Germany). Images of the specimens were captured with the aid of a Leica MC170 HD digital camera mounted on the Leica M125C, and analyzed using Leica Application Suite (LAS) v. 4.12 software. Measurements were taken to the nearest 0.01 mm using LAS. Specimens examined in this study have been deposited in the Zoological Collection of  Kobayashi (2006); star, the type locality of C. melanoparvus (Kobayashi 2006). The symbols and numbers correspond to those in Fig. 3. Shoreline data were based on Wessel and Smith (1996).
Kyoto University (KUZ), and at the National Museum of Nature and Science (NSMT), Tsukuba, Japan.

Molecular phylogenetic analyses
Three nuclear and three mitochondrial markers for phylogenetic analyses were selected following previous studies (Matsuda et al. 2020;Ihara et al. 2021): (1) 28S rRNA, (2) internal transcribed spacer 1 (ITS-1), (3) histone H3, (4) cytochrome c oxidase subunit I (COI), (5) 12S rRNA, and (6) 16S rRNA. However, 12S and 16S sequences could not be obtained from C. koikei sp. nov. and C. melanoparvus. Methods for genomic DNA extraction and cycle sequencing (CS) reactions were as described by Matsuda et al. (2020); primer sets and conditions for the polymerase chain reactions (PCR) and CS reactions used in this study are also listed in Suppl. material 1: Table S1. All PCRs were performed using a GeneAmp PCR System 9700 (Thermo Fisher Scientific, Waltham, MA, USA) using an Ex Taq Polymerase Kit (Takara Bio Inc., Kusatsu, Japan). In total, 20 sequences were newly obtained in this study and deposited with the International Nucleotide Sequence Databases (INSD) through the DNA Databank of Japan.
Phylogenetic relationships of the Cybaeus spiders were inferred based on the dataset comprising 28S, ITS-1, H3, COI, 12S and 16S sequences. In addition to the newly obtained 8 sequences of C. koikei sp. nov. and C. melanoparvus, 85 sequences of 17 species (see dataset in Ihara et al. 2021) were also included in our dataset (Table 1). Cybaeus striatipes Bösenberg & Strand, 1906 was treated as the outgroup a priori because this species seems to be a close relative of Eurasian-continental Cybaeus species (see Ihara 2009a). The alignments of H3 and COI were trivial and no indels were observed. The ITS-1, 12S, and 16S sequences were aligned using MAFFT L-INS-i v. 7.475 (Katoh and Standley 2013) and the 28S sequences were aligned using MAFFT G-INS-i. The fragments of 28S, ITS-1, H3, COI, 12S, and 16S were 792, 769, 328, 763, 335, and 441 bp, respectively. The concatenated sequences thus yielded 3428 bp of aligned positions.
Phylogenetic trees were reconstructed using maximum likelihood (ML) and Bayesian inference (BI). The bestfit partition scheme and models for both analyses were identified based on the corrected Akaike information criterion (AICc) using PartitionFinder v. 2.1.1 (Lanfear et al. 2017) and the 'greedy' algorithm (Lanfear et al. 2012). The selected partition scheme and models are shown in Suppl. material 2: Table S2. The ML phylogenetic tree was calculated using IQ-TREE v. 1.6.12 (Nguyen et al. 2015) with non-parametric bootstrapping (BS) conducted with 1000 replicates. BI tree and Bayesian posterior probabilities (PP) were estimated using MrBayes v. 3.2.7a (Ronquist et al. 2012) with 15 million generations and tree sampling every 100 generations. The parameter estimates and convergence were checked using Tracer v. 1.7.1 (Rambaut et al. 2018), and the first 30001 trees were discarded based on the results.
To confirm whether males and females of the present specimens belong to C. koikei sp. nov., and also to recognize their geographic structures, genetic relationships among 12 samples of C. koikei sp. nov. and two individuals of C. melanoparvus were inferred using relatively fast-evolving nuclear ITS-1 sequences. A neighbor network analysis was conducted using Neighbor-Net (Bryant and Moulton 2004) implemented in SplitsTree v. 4.16.2 (Huson and Bryant 2006) based on uncorrected p-distances. The ITS-1 sequences were aligned using MAFFT L-INS-i, and the length was 681 bp of aligned positions.

Molecular analyses
The obtained BI (mean ln L = -10901.95; Fig. 2) and ML (ln L = -10848.99; not shown) trees had almost identical topologies, and were almost identical to those of the previous analyses (Ihara et al. 2021). The monophyly of C. koikei sp. nov. and C. melanoparvus was strongly supported (BS = 100%, PP = 1.0) but the sister-group of this clade remains unclear since the monophyly of a lineage including C. koikei sp. nov., C. melanoparvus, and C. daimonji Matsuda, Ihara & Nakano, 2020 was not fully supported (BS = 63%, PP = 0.70).
The SplitsTree network ( Fig. 3) using ITS-1 sequences showed all males and females forming a cluster, which was clearly separated from that of C. melanoparvus, confirming that all specimens belong to C. koikei sp. nov. Although specimens of C. koikei sp. nov. collected from its type locality and adjacent locations formed a cluster, no further geographic structure was detected among the present specimens of C. koikei sp. nov. Interestingly, one sample of C. koikei (KUZ Z3761) and two samples of C. melanoparvus were geographically close to each other, but they showed the greatest genetic divergence.

Family Cybaeidae Banks, 1892
Genus Cybaeus L. Koch, 1868 Type species. Amaurobius tetricus C.L. Koch, 1839. Table 1. Samples of the Cybaeus species used for the molecular analyses. The information on the voucher is accompanied by the collection locality and the INSD accession numbers. Sequences marked with an asterisk (*) were obtained for the first time in this study. Samples marked with two asterisks (**) were only used for the neighbor network analysis.  6B) whereas C. melanoparvus have a well-developed palpal bulb (Fig. 8B). Females of C. koikei are clearly distinguishable by their relatively simple spermathecae (Fig. 6E); C. melanoparvus has highly complex spermathecae (Fig. 8F).     Carapace (Fig. 5A). Head narrow, ca. 0.59× as wide as thoracic region; thoracic region almost as high as head. AER straight in frontal view; PER almost straight in dorsal view; AME smallest, < 1/2 diameter of other eyes; ocular area ca. 2.1× wider than long. Clypeus shorter than median ocular area.
Color (Fig. 5A, B). Carapace: head brown, with reticulate dark brown markings; thoracic region yellowish brown, with radiating dark brown bands. Chelicerae, maxillary lobe and labium yellowish brown. Sternum beige. Legs beige to yellowish brown, darker distally, with pale to dark brown annulations. Abdomen: dorsally dark olive-brown with pale brown chevron-like markings; ventrally pale brown.
Carapace (Fig. 5C). Head ca. 0.66× as wide as thoracic region; thoracic region slightly higher than head. AER almost straight in frontal view; PER slightly recurved in dorsal view; AME smallest, < 1/2 diameter of other eyes; ocular area ca. 2.1× wider than long. Clypeus shorter than median ocular area.
Mouthparts. Chelicerae slightly geniculate, teeth and denticles of fang furrow not observable and covered by long setae; basally with lateral condyle. Labium wider than long.
Genitalia (Figs 6D, E, 7). Posterior margin of epigynal plate weakly 'M'-shaped. Atrium located posteromedially on epigyne and with 2 distinct copulatory openings.  CPs separated on both sides of atrium; CD long running anteromedially. Each of SH and SS slightly bulbous; SH small, with a few primary pores anteromedially; SS running posteromedially; SB large, globular, directed anterolaterally; Bennett's gland located anteriorly at connection between SS and SB.
Colour (Fig. 5C, D). Carapace: head brown, with reticulate dark brown markings; thoracic region yellowish brown, with radiating dark brown bands. Chelicerae, maxillary lobe and labium dark yellowish-brown. Sternum light yellowish-brown. Legs pale brown to yellowish brown, darker distally, with medium to dark brown annulations. Abdomen: dorsally dark olive-brown with pale brown chevron-like markings; ventrally light yellowish-brown to brown.
Etymology. The specific name is dedicated to Mr Naoki Koike who assembled the large collection of Japanese Cybaeus spiders that is now kept at Kyoto University, including the specimens of this new species.
Remarks. This species constructs a 'V-shaped' retreat ( Fig. 4B), which is the most common type of retreat among Japanese Cybaeus (Ihara 2006(Ihara , 2009bIhara et al. 2021). Because all specimens collected in 2019 and 2020 were found under stones, it is possible that this species prefers to construct its retreat on the underside of stones rather than in decaying substrates.
According to the nuclear ITS-1 sequences obtained from the holotype male (KUZ Z3744) and the paratype female (KUZ Z3743), the males and females examined in this study clearly belong to the same species newly described here. Their ITS-1 sequences through the overlapping aligned positions (451 bp) are completely identical to each other. Two species, C. daimonji and C. kiiensis Kobayashi, 2006 whose spermathecae are grouped into 'type 1', occur in sympatry with C. koikei sp. nov. through montane habitats on the western side of Lake Biwa. However, they are clearly distinguishable by body size: the body length of C. koikei sp. nov. reaches ca. 4 mm whereas that of C. daimonji and C. kiiensis reaches ca. 6 mm and ca. 2.5 mm, respectively (Kobayashi 2006;Matsuda et al. 2020). Additionally, males of C. koikei sp. nov. are conclusively distinguishable from those of C. daimonji and C. kiiensis by the lack of PA on its palp. Males of both C. daimonji and C. kiiensis possess a PA on their pedipalp (Kobayashi 2006;Matsuda et al. 2020). Kobayashi, 2006 Figures 8, 9 Cybaeus melanoparvus Kobayashi, 2006: 41-42, figs 53-57;Ihara 2009a: 167, figs 2-2-30-228-230. Material examined. Japan • 1 ♂, 1 ♀; Gifu Prefecture, Ibi-gun, Ibigawa Town, Sakauchihirose;35°36.77'N, 136°25.16'E;27 Oct. 2012 (Figs 8F, 9). CD, SH, and SS continuous tubular, and similar in diameter from CP through to SB; this duct starting from CP running on ventral side anteriorly, then bending posteromedially and coiling once toward anterior tip; this duct then turning at anterior tip, coiling 2.5 times in the opposite direction, undulating twice on ventral side, and then a half coil to SB; PP located on dorsal side, 1 coil before turning point at anterior tip; SB globular, extending anterolaterally, and expanded at connection with SS; BG well-developed, located dorsally at connection between SS and SB; each FD not running from basal part of SB, but extending from small atrium located beneath SB.

Supplementary description of female spermathecae
Remarks. The present specimens were unquestionably identified as C. melanoparvus by features of the male palp and female genitalia; that is, the remarkably developed conductor and complicated tubular spermathecae.
The present study could successfully document the positions of PP and BG in the spermathecae of this species for the first time. Because PPs were located at the dorsal surface of the duct near its anterior tip, this tubular structure, which was referred to as an 'anterior duct' by Kobayashi (2006), must comprise CD, and also SH and SS.

Discussion
Cybaeus koikei sp. nov. shares generally similar external features with C. melanoparvus; that is 'small-sized' body length and blackish color, as well as a cluster of robust setae on the lateral surface of the male palpal patella (see also Kobayashi 2006). Although the new species does not possess 'type 2' female spermathecae, the morphological similarities between the two species suggest that they are closely related to each other, forming a species-group. Our molecular phylogenies support the monophyly of C. koikei sp. nov. and C. melanoparvus and thus, they corroborate their close relationship. The present findings of remarkably different genitalia between the 'type 1' C. koikei sp. nov. and 'type 2' C. melanoparvus represent an exception because members of the other species groups have been identified mainly based on similarities in their genitalia (Ihara 2009b).
Given the close sister relationship between C. koikei sp. nov. and C. melanoparvus, their morphological similarities, especially the cluster of robust setae on the palpal patella, are probably synapomorphies between the two species. The positions of primary pores and Bennett's glands in their spermathecae are noticeable as well. Although the tubular spermathecae of C. melanoparvus are complex, features of its primary pores and spermathecal base are essentially concordant with those of C. koikei sp. nov.; the primary pores of both species are located mid-anteriorly and their globular spermathecal bases are directed antero-laterally. These similarities imply that the spatial positions of the spermathecal head and base in C. melanoparvus have not been drastically changed from their most recent common ancestor.
The geographical range of C. koikei sp. nov. and C. melanoparvus is also noteworthy. The new species inhabits montane forests along the western side of Lake Biwa and C. melanoparvus is known only from montane regions at the eastern side of the lake (Fig. 1). These data suggest a parapatric distribution for both species and a distributional boundary somewhere at the northern side of the lake.
Our previous study (Ihara et al. 2021) and the results shown herein also confirm the monophyly of the 'type 1' species C. kompiraensis (Komatsu, 1968) and 'type 2' species C. ishikawai (Komatsu 1968;Ihara 2009a); however both species were deeply divergent based on the phylogenies and the closest relative of the 'type 2' C. ishikawai probably remains unknown. Close relatives of six 'type 2' species endemic to the Ryukyu Islands also remain unresolved (Ihara et al. 2021). Another possible 'type 2' species Cybaeus monticola might be an eastern neighbor of C. melanoparvus that inhabits montane regions in central Honshu. Although the phylogenetic position of C. monticola remains uninvestigated, the morphological features of C. monticola, including a palpal patella with an apophysis and posteriorly directed spermathecal base (Kobayashi 2006), suggest that this species may not be closely related to C. koikei sp. nov. and C. melanoparvus. Nevertheless, further investigations into the underlying phylogeny and comparative morphology of Japanese Cybaeus are essential to elucidate their evolutionary history. Figure 9. Cybaeus melanoparvus Kobayashi, schematic drawing of epigyne and spermathecae, based on female specimen (KUZ Z3765). A. Ventral view; CD, copulatory duct; CP, copulatory pore; FD, fertilization duct; PME, posterior margin of epigynal plate; SB, spermathecal base; SH, spermathecal head; B. Dorsal view; BG, Bennett's gland; FD, fertilization duct; PP, primary pore; SB, spermathecal base; SS, spermathecal stalk; SS, spermathecal stalk. Duct from CP to before PP colored in red; duct from PP to before SB colored in blue; SB colored in green. Scale bar: 50 µm.