Five new species of the long-legged sac spider genus Cheiracanthium C.L. Koch, 1839 (Araneae: Cheiracanthiidae) from China

. Five species of the cheiracanthiid spider genus Cheiracanthium C.L. Koch, 1839 collected from China are diagnosed and described as new to science based on morphological characters: Cheiracanthium arcilongum sp. nov. (♂♀) from Yunnan, C. circulum sp. nov. (♂♀) from Yunnan, C. digitatum sp. nov. (♂♀) from Anhui, C. jiuquan sp. nov. (♂♀) from Guangxi and C. xinjiangense sp. nov. (♂♀) from Xinjiang. In addition, the photos of the habitus, copulatory organs and distribution map are provided for all species. However, DNA barcodes information is only provided for four species.


Introduction
The genus Cheiracanthium C.L. Koch, 1839, with 214 valid species mainly from the Old World, is the largest genus of the family Cheiracanthiidae (World Spider Catalog 2022).Members of Cheiracanthium are slim and pale-colored, with long and slender legs, and often build silk nests in the folds of foliage, thus also earning these spiders the common name long-legged sac spiders (Lotz 2007;Deelemen-Reinhold 2001).
Although several major taxonomic studies of Cheiracanthium have been conducted (Bonaldo & Brescovit 1992;Ramírez et al. 1997;Song et al. 1999;Deelemen-Reinhold 2001;Bayer 2014;Ramírez 2014;Zhang et al. 2020;Esyunin & Zamani 2020), the global diversity of this genus still remains to be discovered.Currently, 43 species of Cheiracanthium have been recorded from China (mostly from southern regions), but the diversity of it is still poorly understood.For example, 14 species were described from a single female (9) or male (5) (Li & Lin 2016;Li & Zhang 2019;World Spider Catalog 2022).Affi rming correct sex matching is one of the challenges for taxonomic studies on Cheiracanthium.Some species are mismatched or conspecifi c male and females have been described as separate species (Bonaldo & Brescovit 1992;Dankittipakul & Beccaloni 2012;Marusik et al. 2020).Therefore, the application of the molecular tools such as DNA barcoding is valuable for correct sex matching.Hebert et al. (2003) proposed the use of cytochrome c oxidase subunit I (COI) sequences on the mitochondrial gene in DNA barcoding techniques, which enable species identifi cation in most animal phyla.DNA barcoding of spiders has been commonly used in the identifi cation of species or delimitation of similar species, association of separate sexes and specimens collected at different localities (Zhang & Li 2014;He et al. 2016;Coddington et al. 2016;Tyagi et al. 2019;Zhang et al. 2020;Wang et al. 2021).Lo et al. (2021) applied DNA barcoding information to correct sex matching of Cheiracanthium insigne O. Pickard-Cambridge, 1874, the results showing that the signifi cance of integrated morphological and the standard barcode sequence can be used as an effective tool for the identifi cation of species and correctly matching sexes in species of Cheiracanthium.
In the current study, fi ve new species of Cheiracanthium are recognized.The intraspecifi c and interspecifi c genetic distances based on the DNA barcode gene, cytochrome c oxidase subunit I (COI) are studied to match male and female of new species except C. xinjiangense sp.nov., which was failed to extract genomic DNA.

Material and methods
All measurements are given in millimeters.Leg measurements are shown as: total length (femur, patella, tibia, metatarsus, tarsus).Eye diameters as AME, ALE, PME, PLE and interdistances as AME-AME, AME-ALE, PME-PME, PME-PLE.The leg spination pattern follow the model of Lotz (2015) and maintains the femur, tibia, metatarsus order, and each segment are divided equally into basal, middle and distal parts.First, the prolateral spines are counted, then the retrolateral ones, the dorsals and, lastly, the ventral spines.Epigynes were removed and cleared in a pancreatin solution and then transferred to 75% ethanol for images captured.Photographs were taken using the Leica M205A.All specimens examined were deposited in Museum of Hebei University, Baoding, China (MHBU).
We extracted total genomic DNA from spider legs using the QIAGEN DNeasy Blood & Tissue Kit (Qiagen Inc., Valencia, CA) following the manufacturer's protocols.We used the primer pair LCO1490/ HCO2198 (Folmer et al. 1994) to amplify COI sequences under the following PCR reaction protocol: initial denaturation at 94°C for 5 min; 35 cycles of denaturation at 94°C for 30 s, annealing at 45°C for 40 s, and elongation at 72°C for 1 min; and fi nal extension at 72°C for 7 min.The 25 μl PCR reactions included 12.5 μl of 2×Taq MasterMix (KangWei Biotech, Beijing, China), 0.8 μl of each forward and reverse 10 μM primer, 4 μl of genomic DNA, and 6.9 μl of double-distilled H2O.The PCR products were visualized by agarose gel electrophoresis (1% agarose).All PCR products were purifi ed and sequenced at GENEWIZ Inc. (Suzhou, China).

DNA barcodes
All sequences were deposited in GenBank.The accession numbers of the generated DNA barcodes are provided in Table 1.The K2P genetic distance of intraspecifi c and interspecifi c nucleotide divergences of four Cheiracanthium are shown in Table 2.
The male (ZYL130) and female (ZYL122) were collected at different localities, but they have a relatively lower genetic distance (1.2%), which was much lower than the interspecifi c genetic distance within other species (7.4%-11.9%)(Table 2) in our dataset.Consequently, we consider the male (ZYL130) and female (ZYL122) conspecifi c.
The intraspecifi c genetic distance ranged from 0 to 1.2%, and the interspecifi c genetic distance ranged from 7.4% to 11.9%.All maximum intraspecifi c distances were much lower than minimum interspecifi c distances for all species.The results of Kimura two-parameter genetic distances confi rm the correct matching of male and female in our dataset.

Diagnosis
The new species can be easily distinguished from all the other species of Cheiracanthium by the long (ca equal to tibia+patella length), curved and fi liform cymbial spur with distal end tapering, the long median apophysis with a broad base and hook-shaped apex (Fig. 1C-F), the large (ca 2.5 × as long as wide), spindle shaped spermathecae, and the wide copulatory ducts (almost as wide as spermathecae) (Fig. 2C-D).

Variation
The total length in male specimens examined ranges from 6.04 to 7.26 (n = 3), in female specimens from 5.01 to 9.07 (n = 6).

Etymology
The specifi c epithet is an adjective from the Latin 'circulus', referring to the circular tegulum in ventral view.3A-B).Prosoma with distinct radial striae, cephalic region yellow, thoracic region yellowish brown.All eyes with black rings, AER slightly recurved in frontal view; PER slightly wider than AER, almost straight in dorsal view.Chelicerae yellowish brown, with 3 promarginal and 4 retromarginal teeth.Sternum yellowish brown.Labium and endites colored as sternum.Legs pale yellow, without distinct markings.Leg spines: I missing II 1p1r-1p1r-0, .Opisthosoma elongate-oval, dorsally grey, with scattered white small patches and numerous black hairs, narrow longitudinal band in middle, enclosed by light spots, two pairs of muscular impressions; venter greyish, with sparse white spots.Spinneret coniform, ALS larger and closed each other; PMS smallest; and PLS with two segments, length of distal segment nearly length of base segment.PALP (Fig. 4C-E).Tibia long, ca ⅔ of cymbium 's length; RTA short, ca ¼ of tibia's length, with wider base and hook-shaped tip, and pointed dorsally from ventral view; DTA slightly shorter than RTA, thin and rodlike; cymbium almost 2 × as long as wide; tip of cymbium long, ca ⅓ of cymbium's length; cymbium retrolateral margin with shallow dent, cymbial furrow short and shallow; cymbial spur slightly longer than RTA; tegulum circular; median apophysis long, ca ½ of tegulum's length, with wider base and hook-shaped apex in ventral view; embolus originating on retrolateral fl ank, at about 3 o'clock position, extending clockwise along tegular margin, tip covered by conductor; conductor membranous, with broad apex; sperm duct indistinct.BODY (Fig. 3C-D).Prosoma reddish brown, thoracic region darker than other parts except the eye area.Chelicerae with 3 promarginal and 5 retromarginal teeth.Sternum, labium and endites colored as carapace.Legs pale yellow.Leg spines: I 0-0-0, 0-0-0, 1v-0-2v; II 1p1r-1p-0, .Opisthosoma greyish brown, with scattered white spots and two pairs of muscular impressions.EPIGYNE (Fig. 4A-B).Atrium heavily sclerotized, located at posterior portion of epigynal plate, with arch-shaped anterior margin and crescent-shaped lateral margins; two copulatory openings located at postero-lateral margins of atrium; part of dark copulatory duct and spermathecae visible through tegument of epigynal plate in ventral view; spermathecae dumbbell-shaped, connected with simple copulatory duct, copulatory ducts ascend, then fold backwards and lastly descend to spermathecae.
Cheiracanthium digitatum sp.nov.urn:lsid:zoobank.org:act:2AD7B3F8-7752-4952-B7D3-E1EEA35DC51C Figs 5-6, 11, Table 1 Diagnosis The male of this new species (Fig. 6C-E) can be distinguished from other species of Cheiracanthium by having a short, robust cymbial spur, a needle-shaped median apophysis, and an infl ated tegulum with indistinct markings.Among the congeners, the male of this new species resembles (3) the long RTA, extending beyond the tegular base in retrolateral view (vs.extending only to the tegular base in the other two species).The female (Fig. 6A-B) is similar to C. solidum Zhang, Zhu & Hu, 1993(Zhang et al. 1993: fi gs 5-6) in having a broad epigynal atrium and spherical spermathecae, but can be distinguished by: (1) the copulatory ducts curved for two circles, while curved only one circle in C. solidum; (2) the anterior margin of atrium with an inward median process, while with an arch-shaped margin in C. solidum.

Variation
The total length in male specimens examined ranges from 6.96 to 8.28 (n = 3).

Etymology
The specifi c epithet is derived from the Chinese pinyin 'jiǔ quān', which means 'nine loops', and refers to the coiled copulatory ducts, forming nine entwined coils; noun in apposition.

Variation
The total length in male specimens examined ranges from 4.01 to 7.74 (n = 5), in female specimens from 4.56 to 7.02 (n = 3).

Diagnosis
The male of this new species (Fig. 10C-E

Etymology
The species epithet is an adjective derived from the type locality.

Discussion
The genus Cheiracanthium is the most diverse among cheiracanthiids, with 214 extant species and represent 59% of the total number of species of Cheiracanthiidae (World Spider Catalog 2022).Due to cryptic habitat and almost half of the species are known from a single sex or juveniles, the identifi cation of species in Cheiracanthium is often challenging (Zhang et al. 2020;Lo et al. 2021).All interspecifi c divergences were well above the largest intraspecifi c values (minimum interspecifi c 7.4% vs 1.2%).These results mirror those found in the closely related study of Cheiracanthium from Taiwan, whose mean intraspecifi c and interspecifi c COI genetic distances range are 0.33-1.95%and 3.32-14.67%,respectively (Lo et al. 2021).Therefore, the combination of DNA barcodes with locality information may ease the identifi cation of species of Cheiracanthium.

Fig. 11 .
Fig. 11.Distribution records of the fi ve new species of Cheiracanthium in China.

Table 2 .
Intraspecifi c and interspecifi c nucleotide divergences for four species of Cheiracanthium, using Kimura two-parameter model.