Zoosyst. Evol. 100 (1) 2024, 279-289 | DOI 10.3897/zse.100.115633 eee Ee BERLIN Taxonomic study of four closely-related species of the Pholcus yichengicus species group (Araneae, Pholcidae) from China’s Qinling Mountains: An integrated morphological and molecular approach Lan Yang", Qiaogiao He!, Zhiyuan Yao? 1 College of Life Science, Shenyang Normal University, Shenyang 110034, Liaoning, China https://zoobank. org/45A 2695 2-6A37-4C42-99D6-DBS5E6ADIBCC4 Corresponding author: Qiaoqiao He (heqq@synu.edu.cn); Zhiyuan Yao (yaozy@synu.edu.cn) Academic editor: Danilo Harms # Received 24 November 2023 # Accepted 21 February 2024 Published 14 March 2024 Abstract Four morphologically similar species of the Pholcus yichengicus species group, occurring in geographic proximity of China’s Qin- ling Mountains, were recognised, based on morphology and four methods of molecular species delimitation. They comprise two new species, namely Pholcus ankang sp. nov. and P. baoji sp. nov. and two previously described species: P. ovatus Yao & Li, 2012 and P. taibaiensis Wang & Zhu, 1992. Their DNA barcodes were obtained to estimate p-distances and K2P distances. In addition, an identification key for the four closely-related species is presented. Key Words Biodiversity, daddy-long-legs spider, identification key, molecular species delimitation, new species Introduction The family Pholcidae C.L. Koch, 1850 is a highly di- verse group of spiders, with 97 genera and 1,937 species (World Spider Catalog 2023), classified under five sub- families: Arteminae Simon, 1893, Modisiminae Simon, 1893, Ninetinae Simon, 1890, Pholcinae C.L. Koch, 1850 and Smeringopinae Simon, 1893 (Huber 2011a; Dimitrov et al. 2013; Huber et al. 2018). Pholcus Walckenaer, 1805 is the most diverse genus within Pholcinae, with 389 de- scribed species belonging to 21 species groups distribut- ed mainly in the Palaearctic, Oriental, Afrotropical and Australasian biogeographic realms (Huber 2011b; Huber et al. 2018; World Spider Catalog 2023). Bestriding the Palaearctic and Oriental Regions, China harbours a high diversity of Pholcus spiders. Recently, a large number of new species of Pholcus have been re- ported from northern China, based on morphological and molecular data. For instance, the extensive 2020 expedi- tion into the Changbai Mountains, at the border between north-eastern China and North Korea, brings the species count of Pholcus in the Changbai Mountains to 27 spe- cies, including 13 new species (Lu et al. 2021; Yao et al. 2021; Zhao et al. 2023a). The systematic investigation in the Yanshan-Taihang Mountains in northern China in 2021 recorded 36 Pholcus species, of which 14 species were new to science (Lu et al. 2022a, b). In 2022, Pholcus spiders were collected for the first time during an expedi- tion to the Luliang Mountains in Shanxi Province, north- ern China. The study identified one known species and eight new species (Zhao et al. 2023b). So far, 169 species, 43% of the genus, have been recorded in China (World Spider Catalog 2023). China’s Qinling Mountains is generally regarded as a geographical dividing line between northern China and southern China. It straddles the Provinces of Shanxi, Shaanxi and Henan. To date, 14 species of Pholcus have been found to be recorded in the Qinling Moun- tains (Zhang and Zhu 2009; Yao and Li 2012; Dong et al. 2016; World Spider Catalog 2023). This paper identifies four morphologically similar species from the Qinling Mountains (Shaanxi part, Fig. 1), based on Copyright Yang, L. et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 280 morphological and molecular evidence, including two new species to be described and all belong to the Phol- cus yichengicus species group. Materials and methods Specimens were examined and measured with a Leica M205 C stereomicroscope. Left male pedipalps were photographed. Epigynes were photographed before dis- section. Vulvae were illustrated after treating them in a 10% warm solution of potassium hydroxide (KOH) to dissolve soft tissues. Images were captured with a Canon EOS 750D wide zoom digital camera (24.2 megapixels) mounted on the stereomicroscope mentioned above and assembled using Helicon Focus v. 3.10.3 image stacking software (Khmelik et al. 2005). All measurements are giv- en in millimetres (mm). Leg measurements are shown as: total length (femur, patella, tibia, metatarsus, tarsus). Leg segments were measured on their dorsal side. The dis- tribution map was generated with ArcGIS v. 10.2 (ESRI Inc.). The specimens studied are preserved in 75% etha- nol and deposited in the College of Life Science, Shen- yang Normal University (SYNU) in Liaoning, China. Terminology and taxonomic descriptions follow Hu- ber (2011b) and Yao et al. (2015, 2021). The following abbreviations are used in the descriptions: ALE = anterior lateral eye, AME = anterior median eye, PME = posteri- or median eye, L/d = length/diameter ratio; used 1n the il- lustrations: a = appendix, b = bulb, da = distal apophysis, e = embolus, fa = frontal apophysis, pa = proximo-lateral apophysis, pp = pore plate, pr = procursus, u = uncus. The mitochondrial gene fragment encoding COI and two nuclear gene fragments encoding H3 and wnt were obtained for 19 samples (Table 1). Primers are listed in Table 2. Two species Pholcus paralinzhou and Yang, L. et al.: Taxonomic study of four closely related Pho/cus spiders P. taishan were selected as outgroups. DNA sequences were checked and edited with BioEdit 7.2.2 (Hall 1999). P-distances and K2P distances from COI were computed using MEGA 5 (Tamura et al. 2011). Phylogenetic trees were constructed using the Maximum Likelihood (ML) method for molecular species delimitation, using both COI and a combined dataset. ML analyses were conduct- ed using RAxML 8.2.9 under a GIRCAT model for all partitions, with 500 rapid bootstrap replicates followed Table 1. Voucher specimen information. Species Voucher GenBank accession number Collection code Col H3 wnt locality P.ankang | W265 PP082941 PP349964 PP349983 China, Sp. Nov. W266 PP082942 PP349965 PP349984 Shaanxi, W267 PP082943 PP349966 PP349985 AMKang W268 PP082944 PP349967 PP349986 W269 PP082945 PP349968 PP349987 P. baoji W270 PP082946 PP349969 PP349988 China, Sp. Nov. W271 PP082947 PP349970 PP349989 Shaanxi, W272 PP082948 PP349971 PP349990 ~—~P2 W273 PP082949 PP349972 PP349991 W274 PP082950 PP349973 PP349992 P. ovatus W220 PP082951 PP349955 PP349974 China, W221 PP082952 PP349956 PP349975 Shaanxi, W222 PP082953 PP349957 PP349976 ~—*Van W223 PP082954 PP349958 PP349977 P. W224 PP082955 PP349959 PP349978 China, taibaiensis W225 PP082956 PP349960 PP349979 Shaanxi, W226 PP082957 PP349961 PP349980 —~PA0! W227 PP082958 PP349962 PP349981 W228 PP082959 PP349963 PP349982 P. yO46 MW721825 ON375203 ON375294 China, paralinzhou Henan, Jiaozuo P. taishan y133 MW721826 ON375204 ON375293 China, Shandong, Taian CHINA Shaanxi Figure 1. Distribution records of four closely-related species of Pholcus from the Qinling Mountains, China. 1. Pholcus ankang sp. nov.; 2. P. baoji sp. nov.; 3. P. ovatus; 4. P. taibaiensis. zse.pensoft.net Zoosyst. Evol. 100 (1) 2024, 279-289 Table 2. Primers used for amplification and sequencing. Gene Primer F/R Sequence 5’-3’ Reference COl LCO1490 F GGTCAACAAATCATAAAGATATTGG — Folmer et al. (1994) C1-N-2776 R GGATAATCAGAATANCGNCGAGG Vink et al. (2005) H3 H3af F ATGGCTCGTACCAAGCAGACVGC Colgan et al. H3ar R ATATCCTTRGGCATRATRGTGAC (1998) wnt Spwefl F GYAAATGCCAYGGWATGICMGG _ Blackledge Spwerl R ACTTGRCAACACCARTGAAAWG et al. (2009) Wnt2¢ F CAGTGRAATGTRCARTTG Wnt2r R CNGTTCAAACTTGYTGGATG by a thorough Maximum Likelihood tree search (Stamat- akis 2014). The sequences are deposited in GenBank. For additional information on extraction, amplification and sequencing procedures, see Yao et al. (2016). We applied four methods for molecular species delim- itation. The Automatic Barcode Gap Discovery (ABGD) analyses were conducted using both Jukes—Cantor and Kimura 2-P distance matrices with options: Pmin = 0.001, Pmax = 0.1, Steps = 10, X = 1.0, Nb bins = 20 (Puillandre et al. 2012). The Bayesian implementation of the Poisson Tree Processes (bPTP) analysis was run for 100,000 generations, with a thinning of 100 and burn- in of 0.25 (Zhang et al. 2013). The Generalised Mixed Yule Coalescent (GMYC) analysis was performed un- der the single threshold model using the R 4.2.2 pack- age SPLITS (R Development Core Team 2023). The phylogenetic tree was converted to an ultrametric format for GMYC analysis using BEAST 1.8.2 (Drummond et al. 2012). Bayesian Phylogenetics and Phylogeography (BPP) requires data from multiple genes and pre-defined candidate species. We used BPP to test the results be- tween the analyses mentioned above. Like Yang (2015), we conducted four different sets of analyses with dif- ferent values of a and £: G@(2, 1000) and Grx(2, 2000), GO@2, 100) and Gx(2, 200), GA(2, 100) and Gr(2, 2000), G@(2, 1000) and Gr(2, 200). The analyses were per- formed using the following settings: species delimitation = 1, algorithm = 0, finetune = 5. The reversible-jump MCMC analyses were run for 100,000 generations and sampled every two generations, with 25,000 samples be- ing discarded as burn-in. Identification key 281 Results We obtained a concatenated alignment of 1767 bp (COI, 1184 bp; H3, 293 bp; wnt, 290 bp). Separate phylogenet- ic analyses of the individual gene COI and concatenated data found compatible topologies. Fig. 2 presents the phy- logenetic tree from the concatenated data. The tree clear- ly divided the samples into four deeply divergent clades. We defined the four major clades as four candidate spe- cies, because the ABGD, GMYC and bPTP analyses un- ambiguously support their status as separate species and the results were fairly consistent with morphology. We used BPP to validate the four species. The BPP analyses found speciation probabilities of one for all of the nodes tested using all four prior combinations. Furthermore, the smallest p-distance and K2P distance amongst the species is 0.068 and 0.072, respectively (between P. ovatus and P. taibaiensis) (Table 3). Of the four species, two are new and descriptions are provided below. Table 3. The average uncorrected p-distances (below diagonal) and K2P distances (above diagonal) amongst the species and the maximum p-distances (on diagonal) within each species. P. ankang’ P.baoji =P. ovatus P. taibaiensis Sp. nov. Sp. nov. P. ankang sp. nov. 0.001 0.099 0.102 0.119 P. baoji sp. nov. 0.092 0 0.096 0.102 P. ovatus 0.094 0.090 0.001 0.072 P. taibaiensis 0.109 0.095 0.068 0.001 Taxonomy Family Pholcidae C.L. Koch, 1850 Subfamily Pholcinae C.L. Koch, 1850 Genus Pholcus Walckenaer, 1805 Type species. Aranea phalangioides Fuesslin, 1775 Pholcus yichengicus species group Diagnosis and description. See Huber (2011b). Note that males and females must be present for this key to work. 1 Sclerotised prolatero-subdistal apophysis of procursus prolatero-proximally strongly widened (figs 134C, 137A in Yao and Li (2012)); raised prolatero-subdistal membranous edge of procursus laterally strongly curved (figs 134D, 137B in Yao and Li (2012)); appendix with slender median branch (branch length/appendix length ratio: 0.5); vulval anterior arch postero-medially strongly protruding (figs 135B, 137D in Yao and Li (2012)).... 0. ce cece ce eeeeeeeeeeeeeeees P. ovatus - Sclerotised prolatero-subdistal apophysis of procursus not widened prolatero-proximally (e.g. fig. 169C in Yao and LI (2012)); raised prolatero-subdistal membranous edge of procursus laterally angular or rectangular (e.g. fig. 169D In Yao and Li (2012) and Fig. 5D); appendix with slender median branch (branch length/appendix length ratio: 1 or 0.2) or angular branch; vulval anterior arch not protruding postero-medially (e.g. Fig. 4B) 0.0... ccc cece eee seeee eee eeseeeeeeaeeeeeeneeees 2 zse.pensoft.net 282 Yang, L. et al.: Taxonomic study of four closely related Pho/cus spiders 2 Raised prolatero-subdistal membranous edge of procursus laterally rectangular (Fig. 5D); appendix with slender medi- an branch (branch length/appendix length ratio: 1, arrow 2 in Fig. 6C); epigynal plate nearly triangular and posteriorly straight (Fig. 6A); vulval pore plates relatively close to each other (Fig. 6B) ..............::ceceeeeeeeeeeeeeeeeeeeeeees P, baoji sp. nov. - Raised prolatero-subdistal membranous edge of procursus laterally angular; epigynal plate posteriorly strongly curved (e-ooF ig. 4A); vulval -pore-platés: widely separated (e:8. Fig: AB is. sic. .canekuee ten eens decried guile oldie cnet calden sapilnac oslo a vn eidiwieedidiens 3 3 Sclerotised prolatero-subdistal apophysis of procursus not protruding latero-distally (arrow 4 in Fig. 3D); appendix with angularcmedian-Dranen=- Carrow 2aINurig A Cy tas oct ecko Mads, berks BET coco 0) pha agdes 21,2 tes ee needy ddesoe BONY ocho’ ch. P, ankang sp. nov. - Sclerotised prolatero-subdistal apophysis of procursus latero-distally protruding (fig. 169D in Yao and Li (2012)); ap- pendix with slender median branch (branch length/appendix length ratio: 0.2) ..........cecceeeceeeeeeeeeeeeeeeeeee tees P, taibaiensis W271 56 M272 = w274 | P. baoji sp. nov. 9WV270 W273 W269 81 77 a8 W266 we7_ | P. ankang sp. nov. s0NV268 W265 W225 100 75 W226 w227 | P. tailbaiensis 95 45 W228 W224 64 W222 W220 a P. ovatus bg221 W223 133 100 x y046 Figure 2. The results of species delimitation conducted by the ABGD, GMYC, bPTP and BPP analyses; different colours of the bars represent the different species (Phylogenetic tree was inferred from ML analysis, bootstrap values are provided at the nodes). Pholcus ankang sp. nov. https://zoobank.org/D586922C-E2C7-4C52-BE9F-A9A A9F38BD06 Figs 3, 4 Type material. Holotype @ (SYNU-Ar00398) and Paratypes 24 (SYNU-Ar00399-400) 292 (SY- NU-Ar00401-02), China, Shaanxi, Ankang, Shiquan County, Chengguan Town, Guiguling Scenic Spot (33°12.32'N, 108°20.15'E, 1585 m elev.), 24 July 2022, Z. Yao, L. Yang & L. Zhang leg. Etymology. The specific name refers to the type local- ity and is a noun in apposition. Diagnosis. The species resembles P. baoji sp. nov. with similar male chelicerae and uncus (Fig. 4C, D), but it can be distinguished by raised prolatero-subdistal mem- branous edge of procursus laterally angular (arrow 3 in Fig. 3D; laterally rectangular in P. baoji sp. nov., arrow 3 in Fig. 5D), by appendix with angular median branch (arrow 2 in Fig. 4C; slender median branch, same length as appendix in P. baoji sp. nov., arrow 2 in Fig. 6C), by epigynal plate posteriorly strongly curved (Fig. 4A; zse.pensoft.net posteriorly straight and epigynal plate nearly triangular in P. baoji sp. nov., Fig. 6A) and by vulval pore plates widely separated (Fig. 4B; relatively close to each other in P. baoji sp. nov., Fig. 6B). Description. Male (holotype): Total length 5.17 (5.26 with clypeus), carapace 1.53 long, 1.62 wide, opisthoso- ma 3.64 long, 1.58 wide. Leg I: 39.46 (10.13, 0.66, 9.94, 16.54, 2.19), leg IT: 26.96 (7.50, 0.63, 6.79, 10.57, 1.47), leg HI: 18.30 (5.38, 0.60, 4.45, 6.79, 1.08), leg IV: 24.35 (7.24, 0.61, 6.03, 9.29, 1.18); tibia I L/d: 70. Eye inter- distances and diameters: PME-PME 0.25, PME 0.19, PME-ALE 0.06, AME-AME 0.06, AME 0.15. Sternum width/length: 1.12/0.94. Habitus as in Fig. 4E, F. Cara- pace yellowish, with brown radiating marks and marginal brown bands; ocular area yellowish, with anterior brown marks; clypeus brown; sternum yellowish, with brown marks. Legs yellowish, but dark brown on patellae and whitish on distal parts of femora and tibiae, with dark- er rings on subdistal parts of femora and proximal and subdistal parts of tibiae. Opisthosoma yellowish, with dorsal and lateral spots. Chelicerae (Fig. 4D) with pair of Zoosyst. Evol. 100 (1) 2024, 279-289 283 C, D. Distal part of procursus (C. Prolateral view, arrow | indicates distal membranous process, arrow 2 indicates sclerotised prolatero-subdistal apo- physis; D. Dorsal view, arrows 1, 2 indicate dorsal spines, arrow 3 indicates angular part of raised prolatero-subdistal membranous edge, arrow 4 indicates latero-distal part of sclerotised prolatero-subdistal apophysis). Abbreviations: a = appendix, b = bulb, e = embolus, pr = procursus, u = uncus. Scale bars: 0.20 mm (A, B); 0.10 mm (C, D). zse.pensoft.net 284 Yang, L. et al.: Taxonomic study of four closely related Pho/cus spiders Figure 4. Pholcus ankang sp. nov., holotype male (C—F) and paratype female (A, B, G, H) A. Epigyne, ventral view; B. Vulva, dorsal view; C. Bulbal apophyses, prolateral view, arrow | indicates latero-median protrusion, arrow 2 indicates angular median branch; D. Chelicerae, frontal view; E-H. Habitus (E, G. Dorsal view; F. Lateral view; H. Ventral view). Abbreviations: a = appen- dix, b = bulb, da = distal apophysis, e = embolus, fa = frontal apophysis, pa = proximo-lateral apophysis, pp = pore plate, u = uncus. Scale bars: 0.20 mm (A—D); 1.00 mm (E-—H). zse.pensoft.net Zoosyst. Evol. 100 (1) 2024, 279-289 proximo-lateral apophyses, pair of distal apophyses with two teeth each and pair of frontal apophyses. Pedipalp as in Fig. 3A, B; trochanter with long (longer than wide), retrolaterally strongly bulged ventral apophysis; femur with small retrolatero-proximal apophysis and distinct ventral protuberance; tibia with prolatero-ventral pro- jection; procursus simple proximally, but complex dis- tally, with raised prolatero-subdistal membranous edge bearing distal membranous process (arrow | 1n Fig. 3C), sclerotised prolatero-subdistal apophysis (arrow 2 in Fig. 3C) and two strong dorsal spines (arrows | and 2 in Fig. 3D); uncus latero-medially protruding (arrow 1 in Fig. 4C), with proximal apophysis and distal scaly edge; ap- pendix curved, with angular median branch (arrow 2 in Fig. 4C); embolus weakly sclerotised, with some trans- parent distal projections (Fig. 4C). Retrolateral trichobo- thrium of tibia I at 7% proximally; legs with short ver- tical setae on tibiae, metatarsi and tarsi; tarsus I with 38 distinct pseudosegments. Female (paratype, SYNU-Ar00401): Similar to male, habitus as in Fig. 4G, H. Total length 4.60 (4.75 with cly- peus), carapace 1.31 long, 1.44 wide, opisthosoma 3.29 long, 1.22 wide; tibia I: 7.18; tibia I L/d: 51. Eye inter- distances and diameters: PME-PME 0.23, PME 0.16, PME-ALE 0.05, AME-AME 0.05, AME 0.13. Sternum width/length: 0.90/0.76. Epigyne (Fig. 4A) posteriorly strongly curved, laterally and medially strongly sclero- tised, with column-shaped knob. Vulva (Fig. 4B) with lat- erally strongly curved, sclerotised anterior arch and pair of nearly elliptic pore plates. Variation. Tibia I in two paratype males (SY- NU-Ar00399-400): 9.04, 9.36. Tibia I in another para- type female (SYNU-Ar00402): 7.12. Natural history. The species was found on the under- side of an overhang on rocky cliffs. Distribution. China (Shaanxi, type locality; Fig. 1). Pholcus baoji sp. nov. https://zoobank. org/EE28 1 8D8-A3D6-4E90-BE29-9 1278647C486 Figs 5, 6 Type material. Holotype ¢ (SYNU-Ar00403) and Para- types 13 (SYNU-Ar00404) 22 (SYNU-Ar00405-06), China, Shaanxi, Baoji, Long County, Xinjichuan Town, Longmendong Scenic Spot (35°2.33'N, 106°40.22'E, 1489 m elev.), 29 July 2022, Z. Yao, L. Yang & L. Zhang leg. Etymology. The specific name refers to the type local- ity and is a noun in apposition. Diagnosis. The species resembles P. ankang sp. nov. with similar male chelicerae and uncus (Fig. 6C, D), but it can be distinguished by raised prolatero-subdistal mem- branous edge of procursus laterally rectangular (arrow 3 in Fig. 5D; laterally angular in P. ankang sp. nov., arrow 3 in Fig. 3D), by appendix with slender median branch (same length as appendix, arrow 2 1n Fig. 6C; angular me- dian branch in P. ankang sp. nov., arrow 2 in Fig. 4C), by 285 epigynal plate nearly triangular and posteriorly straight (Fig. 6A; posteriorly strongly curved in P. ankang sp. nov., Fig. 4A) and by vulval pore plates relatively close to each other (Fig. 6B; widely separated in P. ankang sp. nov., Fig. 4B). Description. Male (holotype): Total length 4.96 (5.05 with clypeus), carapace 1.48 long, 1.78 wide, opisthoso- ma 3.48 long, 1.52 wide. Leg I: 42.86 (10.77, 0.75, 11.15, 17.56, 2.63), leg II: 28.13 (7.82, 0.70, 6.98, 10.96, 1.67), leg III missing, leg IV: 25.71 (7.69, 0.65, 6.28, 9.62, 1.47); tibia I L/d: 70. Eye interdistances and diameters: PME- PME 0.26, PME 0.16, PME-ALE 0.06, AME-AME 0.05, AME 0.12. Sternum width/length: 1.08/0.93. Habitus as in Fig. 6E, F. Carapace yellowish, with brown radiating marks and marginal brown bands; ocular area yellow- ish, with anterior brown marks; clypeus brown; sternum yellowish, with brown marks. Legs yellowish, but dark brown on patellae and whitish on distal parts of femora and tibiae, with darker rings on subdistal parts of femo- ra and proximal and subdistal parts of tibiae. Opisthoso- ma yellowish, with dorsal and lateral spots. Chelicerae (Fig. 6D) with pair of proximo-lateral apophyses, pair of distal apophyses with two teeth each and pair of frontal apophyses. Pedipalp as in Fig. 5A, B; trochanter with long (longer than wide), retrolaterally strongly bulged ventral apophysis; femur with small retrolatero-proximal apoph- ysis and distinct ventral protuberance; tibia with prolat- ero-ventral projection; procursus simple proximally, but complex distally, with raised prolatero-subdistal mem- branous edge bearing distal membranous process (arrow 1 in Fig. 5C), sclerotised prolatero-subdistal apophysis (arrow 2 in Fig. SC) and two strong dorsal spines (arrows 1 and 2 in Fig. 5D); uncus latero-medially protruding (arrow | in Fig. 6C), with proximal apophysis and distal scaly edge; appendix curved, with slender median branch (same length as appendix, arrow 2 in Fig. 6C); embolus weakly sclerotised, with some transparent distal projec- tions (Fig. 6C). Retrolateral trichobothrium of tibia I at 8% proximally; legs with short vertical setae on tibiae, metatarsi and tarsi; tarsus I with 35 distinct pseudoseg- ments. Female (paratype, SYNU-Ar00405): Similar to male, habitus as in Fig. 6G, H. Total length 4.75 (4.80 with cly- peus), carapace 1.35 long, 1.62 wide, opisthosoma 3.40 long, 1.70 wide; tibia I: 7.10; tibia I L/d: 51. Eye interdis- tances and diameters: PME-PME 0.22, PME 0.15, PME- ALE 0.06, AME-AME 0.05, AME 0.11. Sternum width/ length: 1.01/0.79. Epigyne (Fig. 6A) nearly triangular, laterally and medially strongly sclerotised, with col- umn-shaped knob. Vulva (Fig. 6B) with laterally strongly curved, posteriorly sclerotised anterior arch and pair of nearly elliptic pore plates. Variation. Tibia I in paratype male (SYNU-Ar00404): 10.44. Tibia I in another paratype female (SY- NU-Ar00406): 7.18. Natural history. The species was found on the under- side of an overhang on rocky cliffs. Distribution. China (Shaanxi, type locality; Fig. 1). zse.pensoft.net 286 Yang, L. et al.: Taxonomic study of four closely related Pho/cus spiders - = — os =~ - C ee “a ta D Figure 5. Pholcus baoji sp. nov., holotype male A, B. Pedipalp (A. Prolateral view; B. Retrolateral view); C, D. Distal part of pro- cursus (C. Prolateral view, arrow | indicates distal membranous process, arrow 2 indicates sclerotised prolatero-subdistal apophysis; D. Dorsal view, arrows 1, 2 indicate dorsal spines, arrow 3 indicates rectangular part of raised prolatero-subdistal membranous edge, arrow 4 indicates latero-distal part of sclerotised prolatero-subdistal apophysis). Abbreviations: a = appendix, b = bulb, e = embolus, pr = procursus, u = uncus. Scale bars: 0.20 mm (A, B); 0.10 mm (C, D). zse.pensoft.net Zoosyst. Evol. 100 (1) 2024, 279-289 287 Figure 6. Pholcus baoji sp. nov., holotype male (C—F) and paratype female (A, B, G, H) A. Epigyne, ventral view; B. Vulva, dorsal view; C. Bulbal apophyses, prolateral view, arrow 1 indicates latero-median protrusion, arrow 2 indicates slender median branch; D. Chelicerae, frontal view; E—H. Habitus (EK, G. Dorsal view; F. Lateral view; H. Ventral view). Abbreviations: a = appendix, b = bulb, da = distal apophysis, e = embolus, fa = frontal apophysis, pa = proximo-lateral apophysis, pp = pore plate, u = uncus. Scale bars: 0.20 mm (A—D); 1.00 mm (E-H). zse.pensoft.net 288 Pholcus ovatus Yao & Li, 2012 Pholcus ovatus Yao and Li (2012: 28), figs 134A—D, 135A-E, 136A-—D, 137A-D. Material examined. 1 (SYNU-Ar00120F) 19 (SY- NU-Ar00121F), China, Shaanxi, X1’an, Zhouzhi Coun- ty, Banfangzi Town (type locality), roadside of G108 (33°48.02'N, 107°59.08'E, 1165 m elev.), 31 July 2022, Z. Yao, L. Yang & L. Zhang leg. Diagnosis. The species resembles P. taibaiensis Wang & Zhu, 1992 (Yao and Li 2012: 34, figs 169A—D, 170A-— C) with similar male chelicerae and epigyne (figs 135A, 136B, C, 137C in Yao and Li (2012)), but it can be dis- tinguished by sclerotised prolatero-subdistal apophysis of procursus prolatero-proximally strongly widened (figs 134C, 137A in Yao and Li (2012); not widened in P. tai- baiensis, fig. 169C in Yao and Li (2012)), by raised pro- latero-subdistal membranous edge of procursus laterally strongly curved (figs 134D, 137B in Yao and Li (2012); laterally angular in P. taibaiensis, fig. 169D in Yao and Li 2012), by appendix median branch length/appendix length ratio: 0.5 (figs 134A, 136A in Yao and L1 (2012); 0.2 in P. taibaiensis, fig. 169A in Yao and Li (2012)) and by vulval anterior arch postero-medially strongly pro- truding (figs 135B, 137D in Yao and Li (2012); not pro- truding in P. taibaiensis, fig. 170B in Yao and Li (2012)). Natural history. The species was found on the under- side of an overhang on rock cliffs. Distribution. China (Shaanxi, Fig. 1). Pholcus taibaiensis Wang & Zhu, 1992 Pholcus taibaiensis Wang and Zhu (1992: 20), figs 1-6. Song, Zhu and Chen (1999: 63), fig. 25I-K. Zhang and Zhu (2009: 90), fig. S2A-I. Huber (2011b: 451), figs 2097-2099, 2124, 2125, 2178-2183, 2185 and 2199. Yao and Li (2012: 34), figs 169A—D, 170A-C. Material examined. 34 (SYNU-Ar00130F—Ar00132F) 32° (SYNU-Ar00133F—Ar00135F), China, Shaanxi, Baoji, Mei County, Yingtou Town, near Haopingsi Tem- ple (type locality) (34°5.32'N, 107°42.33'E, 1101 m elev.), 30 July 2022, Z. Yao, L. Yang & L. Zhang leg. Diagnosis. The species resembles P. ovatus Yao & Li, 2012 (Yao and Li 2012: 28, figs 134A—D, 135A—-E, 136A—D, 137A—D) with similar male chelicerae and epigyne (fig. 170A in Yao and Li (2012)), but it can be distinguished by sclerotised prolatero-subdistal apophy- sis of procursus not widened prolatero-proximally (fig. 169C in Yao and Li (2012); strongly widened in P. ova- tus, figs 134C, 137A in Yao and Li (2012)), by raised prolatero-subdistal membranous edge of procursus lat- erally angular (fig. 169D in Yao and Li (2012); laterally strongly curved in P. ovatus, figs 134D, 137B in Yao and Li (2012)), by appendix median branch length/appendix length ratio: 0.2 (fig. 169A in Yao and Li (2012); 0.5 in P. ovatus, figs 134A, 136A in Yao and Li (2012)) and by zse.pensoft.net Yang, L. et al.: Taxonomic study of four closely related Pho/cus spiders vulval anterior arch not protruding postero-medially (fig. 170B in Yao and Li (2012); strongly protruding in P. ova- tus, figs 135B, 137D in Yao and L1 (2012)). Natural history. The species was found on the under- side of an overhang on rocky cliffs. Distribution. China (Shaanxi, Fig. 1). Acknowledgements The manuscript benefits greatly from comments by Dani- lo Harms, Bernhard Huber, Yanfeng Tong and an anon- ymous reviewer. Joseph KH Koh checked the English. This study was supported by the Science & Technology Fundamental Resources Investigation Program of China (2023FY 100200) and the National Natural Science Foun- dation of China (NSFC-32170461, 31872193). References Blackledge TA, Scharff N, Coddington JA, Sziits T, Wenzel JW, Ha- yashi CY, Agnarsson I (2009) Reconstructing web evolution and spi- der diversification in the molecular era. Proceedings of the Nation- al Academy of Sciences of the United States of America 106(13): 5229-5234. https://doi.org/10.1073/pnas.0901377106 Colgan DJ, McLauchlan A, Wilson GDF, Livingston SP, Edgecombe GD, Macaranas J, Cassis G, Gray MR (1998) Histone H3 and U2 sn- RNA DNA sequences and arthropod molecular evolution. Australian Journal of Zoology 6(5): 419-437. https://doi.org/10.1071/Z098048 Dimitrov D, Astrin JJ, Huber BA (2013) Pholcid spider molecular systematic revisited, with new insights into the biogeography and the evolution of the group. Cladistics 29(2): 132-146. https://do1. org/10.1111/j.1096-0031.2012.00419.x Dong T, Zheng G, Yao Z, Li S (2016) Thirteen new species of the spider genus Pholcus Walckenaer, 1805 (Araneae: Pholcidae) from China. Zootaxa 4170(1): 1-40. https://doi.org/10.11646/zootaxa.4170.1.1 Drummond AJ, Suchard MA, Xie D, Rambaut A (2012) Bayesian Phy- logenetics with BEAUti and the BEAST 1.7. Molecular Biology and Evolution 29(8): 1969-1973. https://doi.org/10.1093/molbev/ mss0O75 Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994) DNA prim- ers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology 3(5): 294-299. Hall TA (1999) BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41: 95-98. Huber BA (201 1a) Phylogeny and classification of Pholcidae (Araneae): An update. The Journal of Arachnology 39(2): 211-222. https://doi. org/10.1636/CA10-57.1 Huber BA (2011b) Revision and cladistic analysis of Pholcus and close- ly related taxa (Araneae, Pholcidae). Bonner Zoologische Monogra- phien 58: 1-509. Huber BA, Eberle J, Dimitrov D (2018) The phylogeny of pholcid spiders: A critical evaluation of relationships suggested by molec- ular data (Araneae, Pholcidae). ZooKeys 789: 51-101. https://doi. org/10.3897/zookeys.789.22781 Zoosyst. Evol. 100 (1) 2024, 279-289 Khmelik VV, Kozub D, Glazunov A (2005) Helicon Focus 3.10.3. https://www.heliconsoft.com/heliconsoft-products/helicon-focus/ [Accessed 1 November 2023] Lu Y, Yang F, He Q (2021) Pholcus maxian sp. nov., the fifth endem- ic spider species of Pholcus phungiformes species-group (Araneae: Pholcidae) at the border between Jilin, China and North Korea. Biodiversity Data Journal 9: e72464 [1-7]. https://doi.org/10.3897/ BDJ.9.e72464 Lu Y, Chu C, Zhang X, Li S, Yao Z (2022a) Europe vs. China: Pholcus (Araneae, Pholcidae) from Yanshan-Taihang Mountains confirms uneven distribution of spiders in Eurasia. Zoological Research 43(4): 532-534 [& Suppl. 1-78]. https://doi.org/10.24272/j. issn.2095-8137.2022.103 Lu Y, Yao Z, He Q (2022b) A new species of Pholcus yichengicus spe- cies-group (Araneae, Pholcidae) from Hebei Province, China. Bio- diversity Data Journal 10: e81800 [1-7]. https://doi.org/10.3897/ BDJ.10.e81800 Puillandre N, Lambert A, Brouillet S, Achaz G (2012) ABGD, Auto- matic barcode gap discovery for primary species delimitation. Mo- lecular Ecology 21(8): 1864-1877. https://doi.org/10.1111/).1365- 294X.2011.05239.x R Development Core Team (2023) R Foundation for Statistical Com- puting. http://www.R-project.org/ [Accessed on 1 November 2023] Song D, Zhu M, Chen J (1999) The spiders of China. Hebei Science and Technology Publishing House Shijiazhuang, 640 pp. Stamatakis A (2014) RAxML Version 8: A tool for phylogenetic analy- sis and post-analysis of large phylogenies. Bioinformatics (Oxford, England) 30(9): 1312-1313. https://doi.org/10.1093/bioinformatics/ btu033 Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGAS: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony meth- ods. Molecular Biology and Evolution 28(10): 2731-2739. https:// doi.org/10.1093/molbev/msr121 Vink CJ, Thomas SM, Paquin P, Hayashi CY, Hedin M (2005) The effects of preservatives and temperatures on arachnid DNA. Invertebrate Systematics 19(2): 99-104. https://doi.org/10.1071/ 1S04039 289 Wang X, Zhu M (1992) One new species of the genus Pholcus from China (Araneae: Pholcidae). Acta Arachnologica Sinica 1(1): 20-22. World Spider Catalog (2023) World Spider Catalog, Version 24.5. Nat- ural History Museum Bern. http://wsc.nmbe.ch [Accessed 12 No- vember 2023] Yang Z (2015) The BPP program for species tree estimation and spe- cies delimitation. Current Zoology 61(5): 854-865. https://doi. org/10.1093/czoolo/61.5.854 Yao Z, Li S (2012) New species of the spider genus Pholcus (Araneae: Pholcidae) from China. Zootaxa 3289(1): 1-271. https://doi. org/10.11646/zootaxa.3289.1.1 Yao Z, Pham DS, Li S (2015) Pholcid spiders (Araneae: Pholcidae) from northern Vietnam, with descriptions of nineteen new species. Zootaxa 3909(1): 1-82. https://doi.org/10.11646/zootaxa.3909. 1.1 Yao Z, Dong T, Zheng G, Fu J, Li S (2016) High endemism at cave entrances: a case study of spiders of the genus Uthina. Scientific Reports 6: 35757 [1-9 & Suppl. 1-52]. https://doi.org/10.1038/ srep35757 Yao Z, Wang X, Li S (2021) Tip of the iceberg: species diversity of Pholcus spiders (Araneae, Pholcidae) in the Changbai Mountains, Northeast China. Zoological Research 42(3): 267-271 [& Suppl. 1-60]. https://doi.org/10.24272/}.issn.2095-8137.2021.037 Zhang F, Zhu M (2009) A review of the genus Pholcus (Araneae: Pholci- dae) from China. Zootaxa 2037(1): 1-114. https://doi.org/10.11646/ zootaxa.2037.1.1 Zhang J, Kapli P, Pavlidis P, Stamatakis A (2013) A general species delimitation method with applications to phylogenetic placements. Bioinformatics (Oxford, England) 29(22): 2869-2876. https://doi. org/10.1093/bioinformatics/btt499 Zhao F, Jiang T, Yang L, He Q, Zheng G, Yao Z (2023a) Pholcid spiders of the Pholcus phungiformes species group (Araneae, Pholcidae) from Liaoning Province, China: An overview, with description of a new species. ZooKeys 1156: 1-14. https://doi.org/10.3897/zook- eys.1156.98331 Zhao F, Yang L, Zou Q, AliA, Li S, Yao Z (2023b) Diversity of Pholcus spiders (Araneae: Pholcidae) in China’s Liliang Mountains: an in- tegrated morphological and molecular approach. Insects 14(4): 364 [1-34]. https://doi.org/10.3390/insects 14040364 zse.pensoft.net