First description of the male of Thaida chepu Platnick, 1987 (Araneae, Austrochilidae) with micro-computed tomography of the palpal organ

Abstract The male of the austrochilid spider Thaida chepu Platnick, 1987 is described for the first time. We analyzed the internal anatomy of the palpal organ by using micro-computed tomography to investigate the spermophor as well as the muscles and tendons in the cymbium and tibia in detail. As shown by our data, muscles 29 and 30 originate in the tibia and continue with tendons to the base of the bulb, which resembles the ancestral organization for the male palp of spiders; this condition has not been described for Araneomorphae until now. The 3D reconstruction of the spermophor confirms recent interpretations of the male palp sclerites within Austrochilidae.


Introduction
The family Austrochilidae consists of three genera with a very peculiar distribution. Whereas the genera Austrochilus Gertsch & Zapfe, 1955 (6 species) and Thaida Karsch, 1880 (2 species) are endemic to the forests of Central and Southern Chile and adjacent Argentina, the monotypic genus Hickmania Gertsch, 1958 is endemic to Tasmania (Forster et al. 1987). The Austrochilidae, which together with the family Gradungulidae comprise the superfamily Austrochiloidea, are of high interest for spider systematics since it is ambiguously placed among the early derivative taxa of Araneomorphae (e.g., Griswold et al. 2005). Thus, detailed knowledge of taxon-specific structures such as the genitalia is highly valuable not only for species determination but also for a better understanding of interrelationships among the Araneomorphae. In the present study, we describe the male of Thaida chepu Platnick, 1987 for the first time. Furthermore, we studied the internal anatomy of the male palpal organ using X-ray microtomography (micro-CT) in order to reconstruct the spermophore (sperm duct) and position of the embolus, which was debated in former studies (see Griswold et al. 2005: 17).

Material and methods
We collected a male of T. chepu close to the type locality in a wet lowland mixed forest at Lago Huillinco (Chiloé, Chile) (Fig. 1). The material was examined and documented (extended focal range images) in 80% ethanol using a Zeiss Discovery V20 stereo microscope with a Zeiss MCr camera. Editing of images to adjust brightness, contrast and color was performed using Adobe Photoshop CS4. Measurements (given in millimeters) were obtained from digital images using the IntMess module in the program Zeiss AxioVision 4.8 (Carl Zeiss MicroImaging GmbH, Göttingen, Germany). The style of the description is based on Forster et al. (1987) and Grismado et al. (2003).
For the micro-CT analyses of the male palp, the sample was dehydrated in graded ethanol and stained with a 1% iodine solution for 12 hours. After washing in pure ethanol, the sample was scanned in ethanol with an Xradia Micro-XCT-200 X-ray imaging system (Carl Zeiss X-ray Microscopy Inc., Pleasanton, USA) at 40 kV and 8 W using phase contrast (4.0 scintillator-objective lens unit, 15 s exposure time, 4.15 µm pixel size). The obtained data were processed using the 3D analysis software AMIRA v. 5.4.2 (Visage Imaging, Berlin, Germany). Virtual reconstruction of the spermophore was performed by delineation in each section (segmentation) and a smooth surface was computed using the surface editor. The image stack is stored in MorphDBase under creative commons attribution (CC-BY; ID: P_Michalik_20130729-M-4.1; https://www.morphdbase.de?P_ Michalik_20130729-M-4.1). Diagnosis. Similar to T. peculiaris by the clypeus about three times the diameter of the anterior median eyes (Fig. 3); males distinguished from T. peculiaris by the copulatory bulb, which has the median apophysis longer than the embolus (Figs 5-10; about half the length of the embolus in T. peculiaris, Forster et al. 1987, figs 155-157), a stout and curved process of the subtegulum (slender and straight in T. peculiaris, Forster et al. 1987, figs 155-157), and the bent embolus with a curved tip (embolus of T. peculiaris without distinct tip, Forster et al. 1987, figs 155-157). Diagnosis of the female in Forster et al. (1987).

Discussion
Based on the micro-CT data and manual segmentation of the spermophor we confirm the interpretation of the male palp sclerites especially with regard to the position of the embolus given by Griswold et al. (2005). The spermophor of austrochilines is membranous, with thin cuticle, not evident without preparation (e.g., micro-CT or clearing by clove oil; see also Huber 2004). Moreover and in contrast to the findings of Huber (2004) on T. peculiaris, our micro-CT analysis revealed that no muscles originate in the cymbium (tarsus) of T. chepu. Instead, the muscles 29 and 30 originate in the tibia and are connected by tendons with the bulbal sclerites (Figs 8, 11) -an organization only known from the basal spider genera Liphistius Schiödte, 1849 and Atypus Latreille, 1804 (Huber 2004). Preliminary micro-CT analyses of the palp Video 1. Surface model of the spermophor superimposed on the volume rendering of the male palp. Video available for download in full resolution from http://www.pensoft.net/J_FILES/1/articles/6021/ export.php_files/Michalik_Ramirez_Video_1.avi. of the sister group Hickmaniinae (H. troglodytes) (Lipke, personal communication), histological sections as well as micro-CT analyses of representatives of Gradungulidae (Huber 1994;Michalik et al. 2013) and Hypochilus (Hypochilidae) (Huber 1994) revealed that the muscle 30 originates in the cymbium as typical for araneomorph spiders (Huber 2004). This is especially important since austrochilids are key taxa that might reveal important information to interpret the transition from muscular to hydraulically-controlled copulatory organs (Huber 2004), and towards the evolution of higher Araneomorphae (i.e. Haplogynae and Entelgynae).
As shown here micro-CT data can be used for precise and transparent descriptions (for details on the method and data handling see Faulwetter et al. 2013) as well as revealing internal anatomical characters useful for spider taxonomy, systematics and evolutionary/functional morphology.