Four new species of the trapdoor spider genus Conothele Thorell, 1878 (Araneae, Halonoproctidae) from China

Abstract Herein four species of the trapdoor spider genus Conothele Thorell, 1878 collected from China are described as new to science based on the female genital morphology: C.baishasp. n. (Hainan Province), C.baotingsp. n. (Hainan Province), C.linzhisp. n. (Tibet), and C.jinggangshansp. n. (Jiangxi Province). For two Hainan species, C.baishasp. n. and C.baotingsp. n., between which it is difficult to distinguish solely based on female genital morphology, additional diagnoses derived from species-specific nucleotide substitution information and genetic distances using the mitochondrial gene, cytochrome c oxidase subunit I are provided.


Introduction
Conothele Thorell, 1878 is a genus of trapdoor spiders belonging to the family Halonoproctidae Pocock, 1901 (Opisthothelae: Mygalomorphae) that was recently elevated from the family Ctenizidae based on molecular-based evidence (Godwin et al. 2018).
Like many poor-dispersal, ground-dwelling trapdoor spiders (although some species of Ummidia Thorell, 1875 disperse by ballooning (Coyle 1985;Fisher et al. 2014)), Conothele spiders construct underground burrows which are lined with silk and opened to the surface with a trapdoor. The trapdoor is usually covered with a layer of soil, leaf litter, and/or moss, which blend well in the surrounding environment, making them difficult to locate in nature (Fig. 1;Bond and Coyle 1995;Xu et al. 2017a;Yang and Xu 2018).
The two ummidiin genera Conothele and Ummidia share some common morphological and behavioral characters, thus they were considered as undistinguishable (Main 1985;Decae 2010). One of the most obvious shared features by two genera is the presence of a saddle depression on tibia III (Gertsch 1979;Coyle 1981;Ortiz 2007), leading some authors to consider both genera as synonyms (Decae 2010). However, these two genera are completely separated geographically, with Conothele being distributed in the Orient and Australasian regions, and Ummidia being found in the New World and Mediterranean regions (Xu et al. 2017a;Godwin et al. 2018;Yang and Xu 2018). In addition, they are reciprocally monophyletic, and currently considered as valid genera based on phylogenetic analyses (Godwin et al. 2018).
In this study, we diagnosed and described four new Conothele species collected in China based on female morphology as we were unable to obtain adult males (Fig. 2). As in other halonoproctid studies (Xu et al. 2017a;Yang and Xu 2018), both male and female morphology should be described for a new species; however, often it is impractical or impossible to collect adult males by direct searching or by excavating burrows. The standard DNA alignment of the mitochondrial cytochrome c oxidase subunit (COI), which provides the species-specific nucleotide substitution information in the animal barcoding gene region, has been widely used to diagnose species (Brower 2010;Cook et al. 2010;Planas and Ribera 2015;Xu et al. 2015Xu et al. , 2017b. Therefore, for the two new species from Hainan Province (Conothele baisha sp. n. and C. baoting sp. n.) that show similar morphology and considerable intraspecific variations in female genitalia, we provided additional evidence of species-specific nucleotide substitutions and genetic distances based on COI to support our identifications and for future verification of males.

Materials and methods
All specimens were collected from Tibet, Hainan, Jiangxi Provinces, China (Fig. 2). The right four legs of adult females were removed and stored in 100% ethanol at -80 °C for the molecular work. The rest of each specimen was stored as a voucher in 75-80% ethanol for morphological examination. All the voucher specimens were examined under an Olympus SZX16 stereomicroscope, and they were photographed using a Leica M205C digital microscope. Genitalia were cleaned by Protease K digest for 3 hrs at 56 °C. All the voucher specimens were deposited at the CBEE (Centre for Behavioural Ecology and Evolution), School of Life Sciences, Hubei University, Wuhan, China. All measurements were carried out under a Leica M205C digital microscope and given in millimeters. Standard measurements were made following Decae (2010). Measurements of legs and palps are given in the following order: Leg total length (femur + patella + tibia + metatarsus + tarsus), palp total length (femur + patella + tibia + tarsus).

ALE
anterior lateral eye; AME anterior median eye; PLE posterior lateral eye; PME posterior median eye; MOA median ocular area;
We extracted the total genomic DNA using the universal genomic DNA extraction kit (CWBIO) from one or two right legs per specimen depending on the size of the legs. The 25 μL PCR reaction included 12.5 μl 2 × TaqMaster Mix (TIANGEN), 9.5 μl double-distilled H 2 O (ddH 2 O), 1 μl genomic DNA and 1 μl of each forward and reverse primer (10 μM). The primer pairs of COI were LCO1490 (5'-GGT-CAACAAATCATAAAGATATTGG-3') and HCO2198 (5'-TAAACTTCAGG GTGACCAAA AAATCA-3') (Folmer et al. 1994). The PCR reaction protocol: initial denaturation at 94 °C for 5 min; 35 cycles of denaturation at 94 °C for 30 s, annealing at 40 °C for 45s and elongation at 72 °C for 1 min, and final extension at 72 °C for 10 min. The PCR products were visualized by agarose gel electrophoresis (1% agarose). All PCR products were purified and sequenced at the TSINGKE Biological Technology (Wuhan China) or Sunny Biological (Shanghai China). The species-specific nucleotide substitutions in the standard DNA barcode alignment and genetic distances were identified using MEGA v6 (Tamura et al. 2013;Xu et al. 2017b).
Diagnosis. The genus Conothele can be distinguished from all other Halonoproctidae genera other than Ummidia by the presence of a saddle depression on tibia III (Coyle 1981;Ortiz 2007;Decae 2010). Conothele differs from Ummidia by their burrowing habits. The former constructs a short, parallel to the surface of ground, superficial burrow, whereas the latter digs a several centimeters long burrow in the soil (Haupt 2006). Moreover, the geographical ranges of Ummidia and Conothele are completely separated, with Conothele being distributed in the Orient and Australasian regions, and with Ummidia being distributed in the New World and Mediterranean regions (Xu et al. 2017a;Godwin et al. 2018;Yang and Xu 2018;World Spider Catalog 2019).

Conothele linzhi sp. n.
http://zoobank.org/93117D0B-1A52-4CC3-9E67-044B44BB7DAF Diagnosis. Females of C. linzhi sp. n. can be distinguished from those of the other Conothele species by an obviously large irregularly duct-like sigillum in the sternum center (Fig. 3C); by the terminal lobes of spermathecae hemisphere-shaped; by the distal part of stalks Z-shaped and tilted slightly anteriorly .
Male. Unknown. Etymology. The species epithet, a noun in apposition, refers to the type locality. Distribution. Tibet (Linzhi City).  (Fig. 4C); by the distal part of stalks which are outwardly and then inwardly bend, somewhat semi-circle-like (Fig. 4G).
Legs brown, light brown ventrally, with long and short brown sparse setae. Basal part of tibia III with saddle-like depression dorsally (Fig. 4F). Palp with a single tar- sal claw and with two denticles on the claw. Legs each with three tarsal claws, paired claws with one denticle. Opisthosoma ellipsoid, black, scattered with slender short black setae. Spinnerets brown (Fig. 4D), PMS short and one-segmented, 0.64 long, PMS-PMS 0.19; PLS divided into three sections, 1.38 long. Genitalia with a pair of spermathecae, each stalk slender, long, distally sclerotized and folded, which is first bent outwards and then inwards, semi-circle-like; with bowl-shaped lobes (Fig. 4G).

Conothele baisha
Legs brown, with long and short brown sparse setae. Basal part of tibia III with saddle-like depression dorsally (Fig. 5F). Palp with a single tarsal claw, with two denticles on the claw. Legs each with three tarsal claws, paired claws with one denticle. Opisthosoma ellipsoid and black, scattered with many slender, short black setae. Spinnerets brown (Fig. 5D), PMS short and one-segmented, 0.48 long, PMS-PMS 0.08; PLS divided into three sections, 0.78 long. Genitalia with a pair of spermathecae, terminating with face-to-face bowl-shaped lobes; stalks sclerotized distally, each stalk sturdy, short, simple and direct, without the trench between the distal part of the stalks and the lobes (Fig. 5G). Variation. The female genitalia show considerable intraspecific variations: the spermathecae stalks of the holotype (Fig. 5G) and some paratypes are unbent E), or slightly curved (Fig. 6D), or the stalk on the left is tilted to the right by ca. 30°, and the right stalk is curled distally (Fig. 6F). The spermathecae of all samples are face to face, except for one (Fig. 6F).