Two new species of the purse-web spider genus Atypus Latreille, 1804 from Hainan Island, China (Araneae, Atypidae)

Abstract Two species of the purse-web spider genus Atypus Latreille, 1804 collected from Hainan Island, China, are diagnosed and described as new to science based on genital morphology, A. baotingensis sp. n. (♂♀) and A. jianfengensis sp. n. (♀). The DNA barcodes of the two species are also provided for future use.


Introduction
The purse-web spider family Atypidae is an ancient branch of the infraorder Mygalomorphae. Atypidae is one of the burrowing mygalomorph families. However, unlike other burrowing mygalomorph spiders, atypids form a tough web with silk from the end of their burrows to the upper ground section, which is expanded and camouflaged as a trap for wandering arthropods (Fig. 1) (Jocque and Dippenaar-Schoeman 2006;Fourie et al. 2011). The family contains 52 species in three genera (Atypus Latreille, 1804, Calommata Lucus, 1837, and Sphodros Walckenaer, 1835 around the world. Atypus includes 32 species worldwide, of which, 13 are known from China (Zhang 1985;Schwendinger 1990;Zhu et al. 2006;Yin et al. 2012;Li and Lin 2016;World Spider Catalog 2018). Atypus can be distinguished from the other two genera as follows: male with marginal ridges in sternum; palp with short, straight, and spike-like embolus, as well as distally enlarged and straightforward conductor; female genitalia with bulbous or pyriform receptacula and with two lateral patches of pores on genital atrium (Fig. 2) (Schwendinger 1990;Zhu et al. 2006). Historically, Kraus and Baur (1974) gave detailed discussions on the taxonomic problems in Atypus species of Europe, and pointed out that the female genitalia are more reliable than the male bulb for atypid taxonomy. Schwendinger (1989) revised Atypus species in northern Thailand and also revised the whole genus in 1990 (Schwendinger 1990). Zhu et al. (2006) revised the genus Atypus of China. No new species has been described since the genus Atypus was revised a decade ago.
In this study, we diagnose and describe two new Atypus species collected from Hainan Island, China, using male and female genital morphology. To support our identifications in the future, here we provide COI barcode evidence. In addition, the female genitalia and male palp of the genus are illustrated for the first time with clear digital photographs.

Materials and methods
All specimens were excavated from their underground silk tubes by the roadside (Fig. 1A). They were collected alive and fixed in absolute alcohol, their right four legs were removed for molecular work, and the remains were preserved in 75% ethanol for morphological work. Male palp and female genitalia were dissected using a stereomicroscope SZM 45-B2 (Ningbo Sunny Instruments Co., Ltd.). After being cleared with Proteinase K by being incubated at 56° C for 3 hours, female genitalia were photographed with the Olympus BX51 compound microscope using a MicroPublisher 3.3 RTV camera. The others were photographed with a Leica M205C digital microscope. All measurements were given in millimetres. All the specimens were examined and deposited in the Centre for Behavioural Ecology  DNA barcodes were obtained for future use: a fragment of the mitochondrial gene cytochrome c oxidase subunit I (COI) was amplified and sequenced using the primer pairs: LCO1490 (5'-GGTCAACAAATCATAAAGATATTGG-3') (Folmer et al. 1994) and HCO2198 (5'-TAAACTTCAGGGTGACCAAAAAATCA-3') (Folmer et al. 1994). All molecular procedures on extraction, amplification and sequencing followed standard protocols (see Xu et al. 2015).
The genetic distance of the COI gene was calculated using MEGA version 6 (Tamura et al. 2013).
Palpal femur with furrow. Legs slender in red grey. Granular texture only on femur I present. Spines on all metatarsus; metatarsus IV with 17 dorsal spines. Leg formula: 1243.
Male palp (Fig. 2G-I): long conductor with a triangular folded part of its upper corner in retro-lateral view; embolus long, thin spike with a wide triangular space between embolus and conductor in lateral views.
Habitat. Purse webs were found attached to the soil slope along roadside (Fig. 1A-B). Remarks. We examined the holotype and two paratypes of A. ledongensis (Museum of Hebei University, Baoding, Hebei, China), and also successfully sequenced the COI barcode of the holotype specimen (LD-001), which is available on GenBank (GenBank accession number MH279560). The lowest pairwise distance between the holotype of A. ledongensis and the specimens of A. baotingensis sp. n. is 7.2% in mean Kimura 2-parameter distance (K2P) and 6.8% in p-distance. The previous study revealed that the interspecific COI barcode for North American tarantulas is at 5%   Etymology. The specific name refers to the type locality. Diagnosis. The female genitalia of the new species is similar to that of A. karschi Dönitz, 1887, but can be distinguished from the latter by the basal stalks of median pair of receptacula almost being as long as the diameter of their receptacula and much longer than the basal stalks of lateral pair (Fig. 4D-E); first pair of sigilla irregular shape (Fig. 4B).
Abdomen, oval and medium brown (Fig. 5B), with a yellow dark dorsal tergite on anterior half. ALS 0.61, PMS 1.12, four-segmented PLS with lengths as follows: basal 0.77, median 0.69, subapical 0.62, apical 0.42, total 2.5. Spines on all metatarsus; metatarsus IV with eleven dorsal spines. Leg formula: 1423. Vulva (Fig. 4D-E): Genital atrium very short, pore patches large and rounded, the median pair of receptacula attached to the atrium more basally; the basal stalks of median pair of receptacula almost being as long as the diameter of their receptacula and much longer than the basal stalks of lateral pair.
Habitat. Purse webs were found attached to the base of pine trees.  Remarks. Although A. jianfengensis sp. n. is collected from Ledong County, Hainan Island, it can be diagnosed from A. ledongensis found at the same area, Jianfeng mountains, by the latter having very short basal stalks of median pair of receptacula. In addition, it can be distinguished from A. baotingensis sp. n. by the latter having upper incrassate basal stalks of the median pair of receptacula. The intraspecific genetic distance for A. jianfengensis sp. n. based on both the mean Kimura 2-parameter distance (K2P) and p-distance is 1.4% and 1.0%, respectively. The molecular data also provide the evidence that A. jianfengensis sp. n. can be distinguished from A. baotingensis sp. n. as well as from A. ledongensis: the interspecific genetic distance based on K2P and p-distance for the two new species are 15.4% and 13.8%, respectively, and between A. jianfengensis sp. n. and A. ledongensis are 17.2% and 15.3%, respectively.