Description of a new species of Cybaeus (Araneae: Cybaeidae) Title from central Honshu, Japan Author(s) Matsuda, Kenji; Ihara, Yoh; Nakano, Takafumi Citation Species Diversity (2020), 25(2): 145-152 Issue Date 2020-08-07 URL http://hdl.handle.net/2433/253698 © 2020 The Japanese Society of Systematic Zoology; 許諾条 Right 件に基づいて掲載しています。 Type Journal Article Textversion publisher Kyoto University Species Diversity 25: 145–152 Published online 7 August 2020 DOI: 10.12782/specdiv.25.145 Description of a New Species of Cybaeus (Araneae: Cybaeidae) from Central Honshu, Japan Kenji Matsuda1, Yoh Ihara2, and Takafumi Nakano1,3 1 Department of Zoology, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan E-mail: [email protected] 2 Hiroshima Environment & Health Association, 9-1 Hirose-kita-machi, Naka-ku, Hiroshima 730-8631, Japan 3 Corresponding author (Received 9 March 2020; Accepted 27 April 2020) http://zoobank.org/8715C237-1C34-46E8-8877-8B877F083FA7 A new spider species, Cybaeus daimonji, from Kyoto, western-central Honshu, Japan is described based on both sexes. The shape of epigyne indicates that this new species is close to C. communis Yaginuma, 1972, C. kirigaminensis Komatsu, 1963, C. maculosus Yaginuma, 1972 and C. shinkaii (Komatsu, 1970), which are distributed in eastern to central Honshu. Nuclear internal transcribed spacer 1, 28S ribosomal RNA and histone H3 as well as mitochondrial cytochrome c oxidase subunit I, 12S ribosomal RNA and 16S ribosomal RNA sequences of the new species are provided for future phylogenetic studies. Key Words: Arachnida, RTA clade, epigeic, retreat, Mt. Daimonjiyama, molecular identification. dium-sized”), and body length greater than 10 mm (“large- Introduction sized”). Based on a combined molecular and morphological analysis, a phylogenetic backbone of Holarctic and Cali- Cybaeus L. Koch, 1868 is the type genus of Cybaeidae, a fornian clades for North American and European Cybaeus member of the “marronoid clade” of the RTA clade (Wheel- species has been established (Copley et al. 2009; Bennett et er et al. 2017) the members of which possess a retrolateral al. 2016, 2019); similar work has not yet been done for the tibial apophysis (RTA) on the male palp (Coddington and Japanese Cybaeus species. Levi 1991; Griswold et al. 1999, 2005). Cybaeus spiders are An unidentified “medium-sized” Cybaeus, collected from epigeic, inhabiting moist woodlands, and are widely distrib- the montane area of the eastern part of Kyoto City, central uted in the Holarctic region, i.e., Europe to Caucasus and Honshu Island, Japan, is described as a new species in this the southern Far East in the Palearctic and eastern and west- study. In addition, its DNA sequences of several nuclear and ern North America in the Nearctic (Bennett 2017; World mitochondrial genetic markers are provided to aid future Spider Catalog 2020). The genus now comprises 161 species phylogenetic studies to determine its phylogenetic position (World Spider Catalog 2020), and is most diverse in Far East within the Japanese Cybaeus fauna. Asia, especially on the Japanese Archipelago and in western North America (Copley et al. 2009). In Japan, 82 species of Cybaeus are known so far (World Spider Catalog 2020). In Materials and Methods addition, 14 species have been described from the Korean Peninsula (Seo 2016), and five species have been described Sampling and morphological examination. During the from the Russian Far East and Kuril Islands (Marusik and period October to November in 2019, Cybaeus spiders were Logunov 1991; Marusik and Kovblyuk 2011). These figures collected from 13 locations in montane regions in the north illustrate the significant diversity of the genus (over 100 spe- to eastern parts of Kyoto City, Honshu, Japan. When pos- cies) in Far East Asia. sible, geographical coordinates for the collection sites were To help delimit the diversity of this species-rich taxon, obtained using a Garmin eTrex® GPS unit. Specimens were informal species-groups have been recognized for the Japa- preserved in 70% ethanol; legs of some specimens were re- nese and North American Cybaeus species based on char- moved and preserved in 99% ethanol for DNA extraction. acters of the male palp and female genitalia (Ihara 2003b, Epigynes were dissected from several female specimens, 2007, 2008, 2009a, b; Ihara and Nojima 2004; Copley et al. and cleared with proteinase K (100 µg/ml) at 37°C for 2009; Bennett et al. 2016, 2019). In addition, the Japanese 8–10 hours, or at 55°C for 2–3 hours to observe the inter- species have been conveniently divided into three groups ac- nal structure. Morphological examination of the specimens cording to body length of mature spiders (Ihara 2004) as de- was conducted using a Leica M125C stereoscopic micro- fined by Roth (1993): body length less than 5.0 mm (“small- scope. Images of the specimens were captured with the aid sized”), body length ranging from 5.0 mm to 10 mm (“me- of a Leica MC170 HD digital camera mounted on the Leica © 2020 The Japanese Society of Systematic Zoology 146 Kenji Matsuda et al. M125C, and prepared using a 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 Kyoto Uni- versity (KUZ). Terminology of morphological characters follows Bennett (2005, 2017) and Bennett et al. (2016, 2019), nonetheless, a conductor is adopted for the structure on the bulb, which was defined as a proximal arm of the tegular apophysis in these studies. The chaetotaxy and arrangement of leg spines follow Komatsu (1968); abbreviations: p, prolateral; r, retro- lateral; and v, ventral. PCR and DNA sequencing. The methodology for ex- traction of genomic DNA from leg muscle was modified from Nakano (2012). Primer sets for the polymerase chain reactions (PCR) and cycles sequencing (CS) reactions used for nuclear histone H3 (H3), internal transcribed spacer 1 (ITS-1), and mitochondrial cytochrome c oxidase subunit I (COI) and 16S ribosomal RNA (16S) were shown in Nakano et al. (2017). Those for nuclear 28S ribosomal RNA (28S) were 28SO (Hedin and Maddison 2001) and 28SrCy500 (5′-GCC TGT TCA AGA CCC ATT GA-3′), and 28Sa and 28Sb (Whiting et al. 1997); and that for mitochondrial 12S Fig. 1. Cybaeus daimonji sp. nov. A, dorsal view of holotype male ribosomal RNA (12S) was 12Sai and 12Sbi (Simon et al. (KUZ Z2753); B, retreat of non-type male specimen (KUZ Z2782), 1990, 1994). The primer 28SrCy500 was newly designed at from Mt. Hieizan, Kyoto, Japan, without scale. Abbreviation: OP, the Primer3 website (Koressaar and Remm 2007; Untergas- opening of retreat. ser et al. 2012). All PCR reactions were performed using a GeneAmp also are “medium-sized” and have a similar posteromedi- PCR System 9700 (Applied Biosystems; ABI), or a GeneAt- ally located atrium and inverted V-shaped spermathecae las (ASTEC) using an Ex Taq Polymerase Kit (Takara Bio [figs 2-2-30-18–23 in Ihara (2009a)]. However, females of Inc.) The PCR mixtures were heated to 94°C for 6 min, fol- C. daimonji sp. nov. can be discriminated from those of the lowed by 35 cycles at 94°C (10 s), 40°C for COI and 16S or other four species by the copulatory ducts running toward 50°C for the other markers (20 s), and 72°C (42 s), with a the medial part of respective spermathecae, while in the final extension at 72°C for 6 min. The amplified DNA frag- other four congeners the copulatory ducts run directly to- ments were purified using polyethylene glycol (20% PEG ward the respective spermathecal heads [for C. communis, 6000) precipitation. based on an unpublished observation by Yoh Ihara; for C. All samples were sequenced in both directions. The CS kirigaminensis, pl. 4, fig. H in Komatsu (1963); for C. macu- reactions were performed using a BigDye Terminator v3.1 losus, fig. 38 in Yaginuma (1972); and for C. shinkaii, fig. 4 Cycle Sequencing Kit (ABI). Each CS reaction mixture was in Komatsu (1970)]. Males of C. daimonji sp. nov. possess incubated at 96°C for 2 min, followed by 40 cycles of 96°C a palpal tibia, which is shorter than the palpal patella, and (10 s), 50°C (5 s), and 60°C (42 s). The products were collect- thus can be clearly discriminated from those of C. communis ed by ethanol precipitation and sequenced on an ABI 3130xl [fig. 37 in Yaginuma (1972)], C. kirigaminensis [pl. 4, fig. D Genetic Analyzer. The obtained sequences were edited using in Komatsu (1963)] and C. shinkaii [fig. 2-2-30-17 in Ihara DNA BASER (Heracle Biosoft S.R.L.). The DNA sequences (2009a)] bearing a tibia that is longer than the patella [see obtained in this study were deposited with the International also figs 2-2-30-14–16 in Ihara (2009a)]. Nucleotide Sequence Database Collaboration through the Material examined. Holotype: KUZ Z2753 (Fig. 1A), DNA Data Bank of Japan. male, under rotten wood in Mt. Daimonjiyama, Sakyo- ku, Kyoto City, Kyoto Prefecture, Japan (35.027462°N, Taxonomy 135.801530°E), Kenji Matsuda (KM), 1 November 2019. Genus Cybaeus L. Koch, 1868 Paratypes (in total 13 specimens collected from the type Cybaeus daimonji sp. nov. locality by KM): 6 males, KUZ Z2754 (35.027378°N, [New Japanese name: Daimonji-namihagumo] 135.801555°E), KUZ Z2755 (35.027462°N, 135.801530°E), KUZ (Figs 1–5) Z2756–Z2757 (35.027280°N, 135.801641°E), KUZ Z2758 (35.026685°N, 135.801831°E), KUZ Z2759 (35.024971°N, Diagnosis. “Medium-sized” Japanese Cybaeus. Females of 135.802875°E), and 4 females, KUZ Z2761 (35.027302°N, C. daimonji sp.