New species of tube web spiders of the genus Ariadna from South Australia (Araneae, Segestriidae)

Two new species in the tube-web spider genus Ariadna Audouin, 1826 (Segestriidae Simon, 1893) are described from South Australia based on morphological features of both males and females. Ariadna clavata sp. n. and Ariadna tangara sp. n. are widespread and sympatric on eastern Kangaroo Island, where they are found beneath bark, in borer holes in dead wood, and in short burrows under rocks. They have also been found in south-eastern mainland South Australia and bring the total number of described Australian Ariadna to 13 species. We showcase intraspecific variation in both species based on a significant number of specimens, including substantial size variation in females and variations in patterns of leg spination. For male Ariadna, we also establish the previously unknown functions of apophyses and spines on the metatarsi and tibiae on the first legs, which are used during mating to clasp the female. Key Words


Introduction
Tube web spiders (Segestriidae Simon, 1893) are medium-sized, six-eyed spiders with a distinctive body form; unlike most other spiders the pair of third legs when resting is directed forward, not rearward (e.g. Simon 1893a). Segestriids have simple genitalia and the male pedipalp consists of a simple bulb and embolus whilst the female genitalia lack a sclerotised epigynum.
World-wide four segestriid genera have been described, two of which have been recorded in Australia; Ariadna Audouin 1826, represented by eleven Australian The taxonomy of Australian Ariadna is at an early stage, with important diagnostic features showing little consensus between the descriptions. Only a single species, A. kiwirrkurra, has been described based on modern taxonomic methods (Baehr and Whyte 2016). Many of the historic descriptions overlook diagnostically important features, for example most descriptions of Australian Ariadna spp. do not include a description of the preening comb which is an important diagnostic feature. The morphological identification of Australian Ariadna based upon the original descriptions is therefore often difficult and current species delimitations may be unreliable. This is confounded by the fact that most descriptions are based upon females, which can display considerable intraspecific variation (Beatty 1970). Additionally, due to the lack of external genitalia, it is not always easy to assess the maturity of a female, resulting in descriptions based upon immature specimens. This is the case, for example, for the type specimen of the Australian segestriid Gippsicola raleighi Hogg, 1900 (Giroti andBrescovit 2017).
While the life history of the nocturnal Ariadna in Australia is not well understood, it appears that females remain in their retreats most of their lives. It is believed that mature males leave their retreats in search of females (Beatty 1970). The copulatory behaviour of two species from Uruguay was documented by Prandi (1990), neither of these species bore apophyses on leg I of the males and although it has been hypothesised that the apophyses and spines on leg I of some male Ariadna species are used during mating (Grismado 2008), their precise function is unknown.
Here we describe two new species of Ariadna from South Australia to progress taxonomic research in this poorly studied group in Australia. It is the first Australian study of this genus in which comparatively large number of male and female specimens were available for each species allowing the assessment of intraspecific variation of potentially diagnostic characters. In addition, we explore the functional role of the spines and apophyses on the first leg of males.

Materials and methods
In order to assess the strength of morphological features for specific diagnoses of Australian Ariadna, intra-and inter-species variation were analysed using 90 specimens collected on Kangaroo Island, by J. Marsh between 2016 and 2018. South Australian Museum specimens were screened for additional conspecifics of the Kangaroo Island species. Descriptions of external morphological features followed Grismado (2008) and were made from single specimens (holotypes and paratypes). Measurements were taken using the holotypes and paratypes. The pattern of leg spination is described following standard notation for Araneae, with slight modifications as used by Grismado (2008). Where there are special structures, for example the preening combs on the metatarsi of leg IV, or the spines on leg I of some males, these are described separately. Following Grismado (2008) and Giroti and Brescovit (2018) the spination of leg III was not described due to variability. Terminology to describe the structures of the internal genitalia follows Giroti and Brescovit (2018). Dissection of the female genitalia followed methods employed by Platnick et al. (1999) and Grismado (2008). Due to the destructive nature of dissection, descriptions of the internal genitalia of females were based upon conspecific specimens collected from the same locality as the type specimens.
Examination and description of specimens were made using a Zeiss Stemi 305 stereomicroscope. Images were made using a Leica MZ16A microscope and Leica DFC 500 camera with AutoMontage Pro Version 5.2. All measurements of specimens are given in millimetres (mm). Maps were produced by mapping the GPS co-ordinates given on the specimen data labels on to a map of South Australia, GPS co-ordinates were made using projection WGS84. Where there were no co-ordinates on museum specimens Google Earth was used to determine them.
In order to examine the function of the spines and apophyses of leg I of male Ariadna tangara sp. n., seven different coupling events were observed, using three males and five females. Females were left in their rearing container and the male introduced to the female. Mating occurred on all occasions apart from one. On two occasions, involving different individuals mating occurred when the female was in situ in a tube web. The remaining five occasions occurred where both the male and female were free roaming. Mating images were obtained by placing a mating pair in to 70% ethanol and then in to a freezer. Images were then taken using a Zeiss Stemi 508 stereo microscope with a Zeiss Axiocam 105 colour digital camera.

Remarks.
Ariadna is distinguished from other segestriid genera by having a straight to slightly recurved posterior eye row ( (Beatty 1970). It is distinguished from Citharoceps by the lack of stridulatory organ, females lacking a median flap on the interpulmonary fold and by the length of the labium-sternum junction, which is longer than the length of the endite-sternum junction in Citharoceps (Giroti and Brescovit 2018). Etymology. The specific epithet is an adjective (Latin, clavata -striped) referring to the striped abdominal markings of both the male and female.

Note. Holotypes of
Diagnosis. Males and females of A. clavata sp. n. are distinguished from A. segmentata by the curvature of the posterior eye row, which is strongly procurved in A. segmentata (Hickman, 1967, fig. 63) and which is slightly recurved in A. clavata (Fig. 2C). Males of A. clavata differ from A. segmentata by the long, curved and distally hooked embolus (Fig. 1G, I), while the embolus of A. segmentata is shorter and stouter (Hickman 1967, fig. 65). The pedipalp bulb of A. clavata differs from that of A. muscosa by being roughly pyramidal in shape, while that of A. muscosa is spheroidal (Fig. 1G, I; Hickman 1929, fig. 3a). Males and females of A. clavata differ from A. muscosa by the number of teeth on the tarsal claws of the first legs; A. muscosa has 7 or 8 teeth, with the inferior claw bare, A. clavata has 10 or 11 teeth, with a small tooth on the inferior claw (Fig. 2G). Females of A. clavata differ from A. burchelli by the number of teeth on the chelicerae; A. burchelli has one tooth either side of the fang furrow, A. clavata has three teeth on the promargin, one tooth on the retromargin (Fig. 2B, C). Ariadna clavata differs from all remaining Australian Ariadna by the presence of transverse abdominal markings (Fig. 1A).
Female paratype (SAM NN29862). Carapace length 3.8 mm, brown, sternum light brown, with darker patches between coxae; endites yellowish brown, chelicerae brown; legs light brown. Abdomen length 4.6 mm, dorsally pale yellow with 8 dark transverse bands, connected by triangular extensions at the midpoint ( Fig. 2A). Abdomen ventrally yellowish grey with faint longitudinal darker line centrally (Fig. 2B). Carapace dorsal surface sparsely covered with black setae, with stronger and longer black setae projecting forward from the clypeus (Fig. 2C). Carapace elongated and oval, narrowing anteriorly and apically squared; carapace domed so when viewed laterally highest point is mid-way between fovea and posterior eyes; lateral margin of carapace slightly undulating, fovea a shallow indented pit ( Fig. 2A). Sternum oval with precoxal triangles and small, rounder inter-coxal extensions (Fig. 2B). Labium narrowing anteriorly, about ¾ length of maxillae. Posterior eye row slightly recurved, chelicerae hypognathous, covered in strong, long black setae and with basal transverse ridges (Fig. 2C), retromargin with single tooth, promargin with three teeth. Femur I bowed in dorsal view ( Fig. 2A, (1 spine broken), distal retrolateral preening comb with five spines (Fig. 2H). Tarsi with distal ventral hairs, projecting beneath claws, tarsal claws I and II with 11/12 teeth, inferior claw with one small tooth (Fig. 2G). Legs III and IV tarsal claw with six teeth, inferior claw bare. Pedipalps with multiple strong prolateral spines on the tarsus and tibia, and with a single toothless claw. Epigastrium a slightly raised, lightly sclerotized external plate. Internal genitalia: Anterior receptaculum bilobed, dorsal lobe rounded and about 1.5 times as long as ventral lobe. Fig. 3A-C.
Variation (see Suppl. material 1: Table S1): Males (n=21), carapace length: range 2.46-3.64 mm, mean 3.14 mm, standard deviation 0.38 mm. Females (n=23), carapace length: range 2.57-4.34 mm, mean 3.63 mm, standard deviation 0.47 mm. Whilst leg spination showed a large amount of variation, both between and within specimens, some spines showed a higher level of consistency and this was true for both males and females e.g. (1) number of prolateral macrosetae at the apex of the femur I, and (2) the paired macrosetae at the apices of tibiae and metatarsi I and II. The remainder of the spination varied between specimens. The structure of the 'preening' combs on the metatarsus IV was constant in all specimens examined (Figs 1E, 2H).
Distribution. South-eastern South Australia, including the Fleurieu Peninsula, the Mount Lofty Ranges and Kangaroo Island (Fig. 8).
Life history and habitat preferences. The majority of A. clavata specimens were collected from within tube webs located beneath rugose bark of older Euca-lyptus trees. Rather than tree species, the structure of the bark (with specimens found in older, cracked or layered bark), appeared to be of importance. The species appears to be somewhat opportunistic in terms of the location of its retreats, being a variety of holes and crevices, such as borer holes in dead wood (both as standing trees and in logs lying on the ground), in tubular holes in limestone and in burrows in loose soil beneath rocks. The main habitat type was mallee woodland. Specimens were also collected from isolated trees on roadside verges and from a stand of Pinus radiata near D'Estrees Bay (Kangaroo Island). Mature males were collected manually throughout autumn, winter and spring (March through to October). Mature females were present all year.
Ariadna tangara sp. n.   Etymology. The specific name "tangara" refers to the type locality, Tangara Drive, where both the male holotype and the female paratype were collected. It is a noun in apposition.
Diagnosis. Ariadna tangara differs from A. clavata, A. burchelli, A. segmentata and A. muscosa by the lack of abdominal markings. Males of A. tangara differ from all other described Australian male Ariadna spp. by the presence and shape of apophyses and grouped spines on the metatarsus and tibia I (Fig. 4F). Comparison with A. octospinata holotype showed females of A. tangara differed by the size of the preening comb on the fourth metatarsi, which in A. octospinata is about half the length of the tarsus, but is shorter in A. tangara (Fig. 5H) and also by the shape and size of the tooth on the inferior tarsal claw, which is small in A. tangara (Fig. 5G) and long and curved in A. octospinata. Ariadna tangara differ from A. decatetracantha by the presence of a small tooth on the inferior claw, which is bare in A. decatetracantha and by the number of teeth on the main tarsal claws in females, being four in A. decatetracantha and five or six in A. tangara (Fig. 5G, Main 1954, plate II, fig. 3), additionally A. decatetracantha has seven pairs of spines ventrally on metatarsus I, whereas A. tangara has between eight and 12 pairs of spines (Main 1954, plat II, fig. 6). Ariadna tangara differs from A. natalis, A. montana and A. thyrianthina by the number of spines on the apex of femur I, bearing five spines in A. tangara (Fig. 5F), whilst A. natalis has six, A. montana two and three spines in A. thyrianthina. Additionally, A. tangara differs from A. montana by the presence of retrolateral and prolateral spines on tibia I, which are absent in A. montana (Fig. 5E). Ariadna tangara differs from A. major by the number of spines on the prolateral edge of femur I (five spines apically in A. tangara versus two spines in A. major), by the number of teeth on the tarsal claws (eight or nine in A. major versus five or six in A. tangara), and by the absence of a tooth on the inferior tarsal claw of A. major (Fig. 5G).
Spination of females varied, both between specimens and between the left and right legs of individuals. Prolat-   eral and retrolateral spines were present on tibia I of all specimens. The number of spines per specimen varied between two or three prolateral spines, and one or two retrolateral spines. The number of paired ventral spines on tibia I varied between seven and five pairs per specimen. The ventral paired spines on metatarsi I varied between eight and eleven pairs per specimen. The prolateral spines at the apex of femur I was constant (Figs 4E, 5F). The structure of the preening comb showed very little variation (Figs 4D, 5H).
Distribution. South-eastern South Australia, including the Fleurieu Peninsula and Kangaroo Island (Fig. 8).
Life history and habitat preferences. Ariadna tangara has a similar distribution to, and was often found sympatric with A. clavata. Both species were mainly collected from within tube webs located beneath rugose or cracked bark of older trees. As with A. clavata, tree species appeared to be less important than the structure of the bark. Distribution was highly patchy. Hundreds of specimens may be present on one tree, while the species was hard to find on other trees in the locality. Tube-webs of juveniles were often found grouped around those of mature females, with sometimes hundreds of webs belonging to specimens of different ages (as discerned by varying entrance size and specimen size) on a particular tree. Despite a concerted effort to collect males, only two mature males were collected manually, both in winter (June). One of the mature males was collected from under bark in a tube web adjacent to a female web. During captivity, two males matured in May (both collected in December the previous year) constructed tube-webs similar to those of the females, but with less dense weaving. Mature females were collected across the year. Females with eggs or with spiderlings were collected in December through to February. Eggs were not enclosed in an egg sac, but located on the bottom of a thick tube-web, with the female in situ. Once emerged, the spiderlings remain with the female prior to dispersing.
Function of the modified first legs in males. In captivity, Ariadna tangara males employed two distinct coupling tactics, depending on whether the female was in a tubular retreat or not. Where the female was free roaming (n=5), the male approached the female whereupon she adopted a defensive posture with fore legs raised. He hooked the front coxae/trochanters with AP2, the RGS hooked on to the female's front femora and with AP2 wedged against the lateral edges of the female's carapace (n=7) (Fig. 7A, B). The male then turned the female on to her back and mated with her in that position (n=5) (Fig. 7A, C). Where the female was occupying a tubular retreat (n=2), the male placed his front legs on the entrance of the retreat and vibrated his body rapidly (n=2). The female emerged slowly, with front legs raised. Once the female's front legs were out of the retreat the male gripped her front coxae/trochanters as described above, but mated with the female upright, whilst her abdomen was still in the retreat. Most often the male mated with both emboli inserted in the epigyne concurrently.

Discussion
As a genus, there has been little agreement on species-delimiting traits and thus classification of various Ariadna spp. is fraught with complications and challenges for morphologically based taxonomic identification. This is especially so for females, which form the majority of the currently described Australian Ariadna (Table 1). In agreement with the findings of Beatty (1970) and Grismado (2008) we found intra-specific variation in spination of females to be high, both in different individuals of the same species and for legs on different sides of the same specimen. We also found variation between females of different species to be relatively low, considering the amounts of intra-species variation. The type specimen of A. octospinata was morphologically similar to the type specimen of A. tangara sp. n., with the exception of the structure of the preening comb on the fourth metatarsus, which was significantly longer, relative to the tarsus, in A. octospinata. On the other hand, the type specimen of A. montana was morphologically distinct to that of A. tangara sp. n. for most of the key diagnostic macrosetal groups used here, namely the pattern of spination dorsally on femur I, the presence/absence of retrolateral spines on tibia I and the structure of the preening comb on metatarsi IV. The number of paired spines ventrally on tibiae and metatarsi I for the type specimens of A. octospinata, A. montana and A. tangara differed little and were within the range of intra-species variation we recorded for these spines. These ventral paired spines have been identified as being useful diagnostically for American Ariadna (Beatty 1970), however our findings suggest caution is required when using them diagnostically, especially when a small number of specimens are examined, and where intra-species variation cannot be adequately assessed. We showed significant differences between the internal genitalia of females of A. clavata and A. tangara, particularly in the structure of the anterior receptacula. These findings support those of Grismado (2008) and Giroti and Brescovit (2018), that the structure of the anterior receptaculum is diagnostically important and give further strength to the usefulness of the character taxonomically. This highlights the importance of the inclusion of descriptions on internal female genitalia in future taxonomic work on Australian Ariadna.
Male Ariadna examined in this study proved to be more taxonomically informative than females. There was little variation in leg spination of conspecific males for some key spine groups of the tibia and metatarsus of the first leg, however, further research using a wider range of specimens from different localities or populations is needed to characterise the geographical or population-level variation, and ultimately the variation that could be expected from a range of species across a known geographical range. Male pedipalps differed between species, both in the shape of the embolus, the shape of the pedipalp bulb and the shape and relative size of the pedipalp tibia and femur. For both males and females, the number of teeth on the tarsal claws and the presence and shape of the tooth on the inferior claw; the number of spines and their arrangement in the preening comb of metatarsi IV; and the curvature of the eyes did not vary, outside of a defined range, and are therefore strong characters for species identification.
In agreement with the hypothesis of Grismado (2008), we showed that the apophyses of the metatarsus of leg I and the retrolateral grouped spines of the tibia of leg I in males, had an important function in gripping the female during mating. The apophyses and opposing spines effectively held the female's carapace and prevented her from moving away from the male, or from killing him. This is an important finding as, excepting the study by Prandi (1990), there has been little documentation of the mating behaviour of Ariadna, and the function of the apophyses, which are found on males of many species of Ariadna internationally (for example Beatty 1970, Grismado 2008, Giroti and Brescovit 2018, was unknown.
As discussed in the introduction, the taxonomy of Australian Ariadna is in an early state, with many historical taxonomic papers missing out crucial diagnostic features. The findings from this work point towards the necessity of a revision of the genus in Australia and the redescriptions of species using modern taxonomic techniques, including the internal genital structures of females.