﻿Two new species of Diphya Nicolet, 1849 (Araneae, Tetragnathidae) from Southwest China

﻿Abstract Two new species of tetragnathid spiders from Guizhou and Sichuang provinces of China are described: Diphyaguiyang J. Zhang & H. Yu, sp. nov. (♂♀) and Diphyaweimiani J. Zhang & H. Yu, sp. nov. (♀). Detailed descriptions, diagnoses, and photographs are provided for these two species, as well as a key and a distribution map for Chinese Diphya species. DNA barcodes (a partial fragment of the mitochondrial cytochrome oxidase subunit I gene, COI) of both new species were obtained for species delimitation, matching of different sexes, and future use in molecular studies.


Introduction
Diphya Nicolet, 1849 is a small spider genus with an unusual distribution, it is disjunctively distributed in South America, southern Africa, and East Asia Omelko et al. 2020;World Spider Catalog 2022). Diphya currently includes 18 described species, with seven species recorded from Asia, six of which are known from China (Omelko et al. 2020;World Spider Catalog 2022).
The genus has been revised both regionally and on a worldwide scale (Tanikawa 1995;Álvarez-Padilla and Hormiga 2011;Marusik 2017;Omelko et al. 2020). However, debate is ongoing on the genus's limit and subfamily placement (Álvarez-Padilla and Hormiga 2011;Marusik et al. 2017;Omelko et al. 2020). Marusik et al. (2017) have expressed doubts about the monophyly of the genus and thought that African, Asian, and South Neotropical species may in the future be considered to belong to separate genera. Despite of the dispute about the limits of this genus, most Diphya species have been well studied, especially several new species described in recent years. These species have been described in detail, alongside high-quality illustrations, to allow easy species recognition (Álvarez-Padilla and Hormiga 2011; Yu et al. 2014;Marusik 2017;Marusik and Omelko 2017a, b;Omelko et al. 2020).
While examining spiders recently collected from Guizhou and Sichuan provinces, southwestern China, we have found some Diphya specimens that belong to two undescribed species. With that, the total number of Diphya species in China reaches nine species, five known by both sexes. This makes China the country with the most Diphya species. The goal of this paper is to provide detailed descriptions, illustrations, and diagnosis of these two new species: D. guiyang J. Zhang & H. Yu, sp. nov. and D. weimiani J. Zhang & H. Yu, sp. nov. The DNA barcodes of these two species were obtained to confirm matching of the sexes (for D. guiyang sp. nov.) and future use in molecular studies. Additionally, an identification key and a distribution map for Chinese Diphya species are given.

Materials and methods
Specimens in this study were collected by beating vegetation. The type specimens are deposited in the Museum of Guizhou Education University, Guiyang, China (MGEU; Hao Yu curator). Specimens were preserved in 95% alcohol and examined using an Olympus SZX7 stereomicroscope. Left male palps were examined and illustrated after dissection. Epigynes were removed and cleared in a warm 10% potassium hydroxide (KOH) solution. The vulvae were imaged after being embedded in Arabic gum. Images were captured with a Canon EOS 70D digital camera (20.2 megapixels) mounted on an Olympus CX41 compound microscope and assembled using Helicon Focus v. 6.80 image-stacking software. All measurements were obtained using an Olympus SZX7 stereomicroscope and are given in millimetres. Eye diameters were measured at the widest part. The total body length does not include the chelicerae or spinnerets. Leg lengths are given as total length (femur, patella+tibia, metatarsus, tarsus). The terminology used in the text and figure legends follows Marusik (2017), Marusik et al. (2017), andOmelko et al. (2020).
The distribution map was generated with ArcGIS v. 10.5 (Environmental Systems Research Institute, Inc.). Due to lack of locality coordinates in previous publications, locality coordinates for all known species are derived from ArcGIS, except for D. qianica and D. tanasevitchi, which were copied from the original publications (see Zhu et al. 2003: 57;Zhang et al. 2003: 407).
To obtain the DNA barcodes, a partial fragment of the mitochondrial cytochrome oxidase subunit I (COI) gene was amplified and sequenced for four specimens, using the primers LCO1490 (5′-GGTCAACAAATCATCATAAA-GATATTGG-3′) and C1-N-2776 (5′-GGATAATCAGAATANCGNCGAGG-3′). For additional information on extraction, amplification, and sequencing procedures, see Wheeler et al. (2016). Sequences were trimmed to 651 bp. All sequences were analysed using BLAST and are deposited in GenBank. The accession numbers are provided in Table 1. Description. The genus is well described by Tanikawa (1995) and Álvarez-Padilla and Hormiga (2011).
Composition and distribution. For details see WSC (2022).
Comments. Although the debate on the limit of this genus remains open, a review of the genus Diphya is not within the scope of this work. Consequently, the present study follows WSC (2022) and Omelko et al. (2020) and temporarily places both new species in Diphya sensu lato for the lack of a better solution. Paracymbium complex, with at least 3 processes (or outgrowths); embolus short and stout, shorter than tegulum width, embolar tip C-shaped, laminar or blade-shaped ( Fig. 1A Epigynal atrium (or called fovea) distinct, lack of septum ( Fig. 5A, C, E) ...6 -Epigynal atrium indistinct, divided or covered by septum ( Fig. 3A, C, E) ...7 6

Key to Diphya species occurring in China
Epigynal atrium located at anterior part of epigynal plate; copulatory ducts short and simple, not longer than epigyne length, not convoluted . Septum T-shaped, with a wide head (anterior part of septum); septal base short, about 1/3 of septum length (Fig. 3A, C, E); abdomen dorsally with 5 pairs of irregularly shaped black marks (Fig. 4D) Yu, Zhang & Omelko, 2014 in having a similar complex paracymbium with several processes (other species have simple unbranched paracymbium and cymbial process) but can be distinguished from it by the different shape, locations, and number of paracymbial processes and by the different shape and degree of sclerotization of the embolus. In D. guiyang sp. nov., the paracymbium has 3 processes (vs 4), the intermediate process (Ip) is thumb-like and originates from the distal end of the paracymbium, close to tibia (Fig. 1A, B, D) (vs papilliform and located at the proximal margin of the paracymbium, well-separated from tibia); the embolar tip (Em T) is blade-shaped, hyaline, and with a relatively wide apex (Fig. 2B-E). (vs C-shaped, thick, heavily sclerotized, and with the apex relatively sharp; Yu et al. 2014: 31, figs 5, 10, 12;Marusik et al. 2017: 143, figs 13-15, 17). The female of D. guiyang sp. nov. also resembles that of D. wulingensis in having a similarly shaped vulva, but can be separated by having the septal base (Sb) relatively narrow (less than 1/3 of the epigynal plate width) (vs wide, about ½ of the plate width) (cf. Fig. 3A, C, E and Marusik et al. 2017: 143, figs 10, 11), and by the kidney-shaped posterior chamber of receptacle (Rp), which is distinctly larger than the anterior chamber (Ap) (vs both Ap and Rp nearly globular and Ap slightly larger than Rp) (cf. Fig. 3B, D and Yu et al. 2014: 31, figs 4, 9). In addition, the two species can be reliably separated by the abdominal pattern: dorsum of the abdomen centrally with a distinct symmetrical pattern in D. guiyang sp. nov. (Fig. 4A, D Etymology. The species name is derived from the type locality; noun in apposition. Description. Male. Holotype (Figs 3F, 4A-C): total length 4.18; carapace 2.04 long, 1.48 wide; abdomen 2.14 long, 1.46 wide. Carapace dark brown, slightly lighter between PER and cervical groove. Clypeus dark brown, distinctly higher than AME diameter. Eye sizes and interdistances: AME 0.07, ALE 0.14, PME 0.15, PLE 0.14, AME-AME 0.09, AME-ALE 0.08, PME-PME 0.16, PME-PLE 0.20, MOQL 0.66, MOQA 0.22, MOQP 0.48. Chelicerae light brown, with 3 promarginal and 4 retromarginal teeth. Sternum coloured the same as carapace, 0.76 long, 0.85 wide.    Abdomen dorsally dark with 5 pairs of spots (anterior pair circular, 2 nd pair comma-shaped and largest, posterior 3 pairs represented by 6 short transverse bands), surrounded by line consisting of small white spots. Lateral sides whitish. Ventrally with irregularly shaped black pattern.
Epigyne (Fig. 3A-E). Plate distinctly wider than long. Septum (Se) T-shaped, consisting of a transverse head (Sh), a narrow stem (Ss) and nose-shaped base (Sb); septal head wide, about 2/3 of the epigynal plate width; septal stem (Ss) slightly narrower than septal base, about twice longer that septal base length; septal base (Sb) shaped like a nose, nearly as wide as long. Copulatory openings indistinct, located in rebordered groove of lateral margins of septum. Copulatory ducts (Cd) diverging posteriorly, running along with lateral margin of septum. Receptacle subdivided in 2 chambers; anterior chamber (Ra) globular, relatively small, widely separated by ca 2.7 diameters; posterior chamber (Rp) kidney-shaped, distinctly larger than anterior chamber, 1.5 times longer than wide, separated by ca 1.3 widths. Fertilization ducts (Fd) acicular, membranous, located on posterior-interlateral surface of Rp.
Etymology. The specific name is a patronym after Mian Wei (Chengdu City, China), the collector of the type material.
Abdomen dorsally uniformly yellowish white, dorsum with two pairs of inconspicuous muscle depressions; laterally with lengthwise reticular pattern; ventrally white with no distinct pattern.

Male. Unknown.
Comments. According to WSC (2022), only two species of Diphya are known only from males: D. bicolor Vellard, 1926 from Brazil, andD. leroyorum Omelko, Marusik &Lyle, 2020 from South Africa. However, neither could be matched with D. weimiani sp. nov. due to the long distance between their type localities (China is tens of thousands of kilometres from Brazil and South Africa).