The spermatozoa of P. phalangioides are characterized by several unique features, as pointed out in detail by Alberti and Weinmann 20. Apart from additional details, we provide information on the relationship between sperm and secretions in the male genital system.

Early stages of spermatogenesis are characterized by a large spherical nucleus surrounded by a manchette of microtubules, an acrosomal vacuole on its anterior pole and an axonemal basis, which migrates into an indentation at the posterior pole of the nucleus. In this early stage, the manchette of microtubules and the nuclear surface possesses a unique arrangement. As seen in cross-sections, the microtubules are not densely packed and the nuclear envelope shows a slightly wave-like outline (Fig. 5a). The quadrangular acrosomal vacuole is tightly connected to the cell membrane, which is indented in this region (Fig. 5a). Within the cytoplasm annulate lamellae are present (Fig. 5a).

During spermiogenesis the chromatin condenses and the nucleus strongly elongates (Fig. 5b). A thin helical band of nuclear material covered by microtubules surrounds the nucleus over its entire length (Figs. 5b,c). The acrosomal vacuole elongates during spermiogenesis, and finally possesses a cylindrical shape (Fig. 5d). A considerable amount of cytoplasm accumulates at the posterior end of the spermatids, forming a big droplet (Figs. 5b,d).

Within the lumen of the testis, only coiled spermatozoa are found embedded in a secretion matrix, which is likely produced by the extensive somatic cells (Figs. 5e,f). The extensions of the somatic cells surround the cysts of spermatids. Apically, the somatic cells bear long microvilli and contain many vesicles, indicative of strong secretory activity (Fig. 5f). Several secretion droplets are visible between the spermatozoa (Figs. 5e,f). The large secretion droplets seem to be produced through fusion of smaller droplets, as indicated by the irregular ring of loose material around the electron-dense center of the droplet (Fig. 5f, arrows).

Within the vas deferens the spermatozoa are densely packed and embedded in a homogenous secretion matrix (Fig. 6a). Each single spermatozoon is surrounded by a secretion sheath forming a so-called cleistosperm (Figs. 6a,b). The secretion sheath is produced within the vas deferens since it is absent in the testis (Fig. 5e). Within the secretion matrix three different kinds of secretions are present (Fig. 6). The first type of secretion consists of small droplets which are densely distributed and characterized by a bright center and a dark border. The second kind is a large electron-dense secretion droplet with a very irregular shape. These droplets often partially surround the spermatozoa (Fig. 6a). The third and rarest type of secretion is a droplet which appears less electron-dense. It is characterized by a heterogeneous content and an irregular surface to which particles may adhere or are partly embedded (Fig. 6c). The epithelium of the vas deferens is flat and bears microvilli in its apical region. A muscle layer surrounds the vas deferens (Fig. 6b).

The male genital system of P. phalangioides is characterized by thick tube-like testes and thin convoluted vasa deferentia which fuse distally to form the ductus ejaculatorius. This organization is reported from many species of different families, e.g., Cybaeidae, Theridiidae and Agelenidae, and seems to be the general condition in araneomorph spiders (see background). However, theraphosid spiders show no distinct separation between testis and vas deferens [14; PM personal observation] and in theraphosid as well as mesothelid spiders the testes themselves are convoluted [1422; PM personal observation]. In some species, e.g., the cybaeid Argyroneta aquatica, the testes are curved but distinct from the vasa deferentia 11. Petrunkevitch 23 suggested that the general organization of the male genital system is not useful for phylogenetic consideration, but the few data available indicate a potential for systematic interpretation. For example, in all theridiid spiders studied thus far, the vasa deferentia lead into a roundish vesicula seminalis (=ductus ejaculatorius?), the storage site of the mature spermatozoa, which is connected via an unpaired duct with the genital opening [13; PM personal observation]. Considering the information available to date, it is at least misleading to take the theraphosid type of male genital system as representative for spiders 2425.

In P. phalangioides, spermiogenesis starts several weeks before the last molt and continues in the adults. In the subadult males approximately four weeks before the final molt, the lumina of the testes are very thin and only a small amount of the dense secretion matrix is observable. The secretory activity increases within the last two weeks of the subadult stage. In adults, the lumina are wide and full of secretion and mature spermatozoa. The two different functions of the testis – the production of sperm cells and different kinds of secretion (see below), are evident. Several authors suggested that these secretions are products of degenerated spermatids and the cytoplasmic droplet discarded at the end of spermiogenesis 202627. However, rough endoplasmic reticulum, Golgi bodies and vesicles in the somatic cells demonstrate that the epithelium itself produces the secretions as was suggested by Alberti et al. 28.

In the subadult stages, the vasa deferentia possess a very extensive lumen in which the mature spermatozoa and secretion from the testes accumulate continuously. The thick epithelium of the vasa deferentia also shows secretory activity. It may be responsible for the formation of the secretion sheath surrounding each spermatozoon, as suggested for other spider species 202829303132. In adults, several kinds of secretion are present in the vasa deferentia, whereas in young subadult stages only a dense secretion matrix is present. Thus, it is evident that secretory activity increases in subadults when approaching the final molt. Furthermore, in adult male spiders the epithelium of the vasa deferentia is very thin and secretory activity is low as was stated for the cybaeid Argyroneta aquatica 11.

In adult males of P. phalangioides cleistospermia and secretions are transferred into the palpal copulatory organ soon after the final molt. Sperm uptake by the male can occur repeatedly after each mating and may require a continuous production of sperm cells. In fact, P. phalangioides males mate several times 33 and refill their pedipalp sperm stores after each mating [GU unpublished observation]. To do so successfully, cellar spider males begin sperm production in the subadult phase and continue production throughout adulthood, as demonstrated by our study. In contrast, studies on short lived spider males with only a single mating opportunity showed that males do not refill their sperm stores and probably cease to produce sperm after the final molt 1, instead using their resources for rigorous mate selection [Pauly A, Uhl G, Schneider JM unpublished data].

Three different kinds of secretion droplets could be identified in the dense secretion matrices of the testes and vasa deferentia of P. phalangioides. According to Romeis 36, the Goldner staining method results in a green coloration of acidic mucosubstances and in red coloration of proteinaceous substances. It cannot be ruled out that the seemingly different kinds of secretion represent different steps of a single secretory pathway. However, since we also found different secretion droplets within the palpal organ, we conclude that different secretory pathways result in a variety of secretions with potentially different functions. Morphologically, the secretions are clearly different from those found in two other spider species, a mesothelid spider 30 and the related pholcid spider Holocnemus pluchei 34. The obvious difference in seminal secretory products in closely related species suggests that there is rapid divergence of seminal fluid substances that may originate from the process of sexual selection. It has been assumed that the rapid evolution of reproductive proteins is a motor in the speciation process 35.

In spiders, sperm and the seminal secretions are not directly transferred into the female genital tract, but taken up into the palpal organ before mating. Within the pedipalps, glandular epithelia adjacent to the sperm storage site seem to be widespread and also occur in P. phalangioides and other spider species [3738; Rose W, unpublished data]. It has been assumed that their products serve to release the sperm into the female genital tract during copulation or to serve as a protective matrix for sperm storage within the male palp [e.g., 373940]. During mating, sperm are transferred to the storage site within the female together with the secretions originating from both genital tract and pedipalp. Finally, inside the female cellar spider, sperm and male secretions encounter secretion that is produced by the female. The only non-morphological investigation existing to date identified proteinaceous substances and glyco- and lipoprotein components in the sperm storage site of virgin female P. phalangioides using gel-electrophoretic methods 41.

For spiders, a nutritive and protective function (desiccation and pathogens) has mainly been attributed to either male or female secretions since the sperm cells are often stored for extended periods of time [e.g., 4142434445], but secretions may also serve as lubricants and promote physical uptake or release of sperm inside the male palp (see above). Female secretory products have been suggested to control sperm activation 18464748. However, male secretions may also play an important role in this process.

It has been known since the study of Alberti and Weinmann 20 that P. phalangioides possesses very aberrant spermatozoa with several unique characters (e.g., an elongated proximal centriole and a central nuclear canal containing the acrosomal filament), but the most conspicuous character is the band of nuclear material spirally surrounding the nucleus. As shown in the present study, the first signs of this peculiar transformation of the helical band are present in very early spermatid stages: microtubules are loosely arranged around the nucleus resulting in a wave-like outline of the nuclear envelope. The functional implications of the band are still unknown. It may influence the mobility of the spermatozoa or play a role during fertilization. The phylogenetic value of the peculiar spermatozoa of P. phalangioides remains uncertain as well, as the sperm ultrastructure of only one further pholcid spider, H. pluchei, is known 3449. The spermatozoa of these two species however do markedly differ in accord with the present ideas on systematic relationships within the Pholcidae 50. On the other hand, a helical band of nuclear material is also present in Spermophora senoculata [PM personal observation], a species closely related to P. phalangioides 50. It therefore seems that sperm characteristics may be successfully applied successfully in phylogenetic considerations.

Males of P. phalangioides were collected in houses and cellars within the city of Freiburg i.Br., Germany at an early juvenile stage and reared in the laboratory in individual containers on a diet of fruitflies. During the last intermolt interval, males were classified into two developmental stages according to the external morphology of their developing palpal organs. Young subadult males (stage 1, N = 5) were fixed approximately four weeks before the final molt and older subadult males (stage 2, N = 5) were fixed about two week before the final molt. Adult males (N = 5) were examined one to two days after their final molt.

Male specimens were fixed in Bouin solution according to Gregory 51, dehydrated in a graded series of ethanol or isopropanol and embedded in Paraplast. Sections (7 μm) were made with a Reichert 1150 Autocut microtome, stained according to Goldner 52, and mounted with Eukitt medium. Examination was performed with an Olympus BX 60 and pictures were taken with an Olympus DP 10.

Elder male specimens (N = 2) were dissected in 0.1 M phosphate buffer supplemented with 1.8% sucrose. The isolated genital system was fixed in 2.5% glutaraldehyde in the same buffer followed by postfixation in buffered 2% osmium tetroxide. After washing, the tissue pieces were dehydrated in graded ethanols and embedded in Spurr's resin 53. Ultrathin sections (50 nm) were made with a Leica ultramicrotome and stained with uranyl acetate and lead citrate 54. Examination was performed with a Zeiss EM 10A electron microscope.

The isolated genital system of elder males (N = 3) was split open in a droplet of phosphate buffer (see above) with thin needles on glass coverslips covered with 1% poly-L-lysin. After 10 min sedimentation the adhering material was fixed in 2.5% glutaraldehyde buffer for 1 h at 4°C. Samples were then rinsed in buffer and post-fixed in buffered 1% osmium tetroxide, dehydrated in ethanol, dried in a Balzers CPD 030 critical point dryer, coated with gold in a Balzers MED 010 sputtering device and examined in a Philips XL20 scanning electron microscope.