External female genitalia
In early-subadult females (7 days after the moult to subadult stage), the genital area is slightly elevated forming a pale bulge (Fig. 1a). Approximately six days before the moult to the adult stage, this protuberance is darker (Fig. 1b). The dark coloured area indicates that the epigynal plate is formed and sclerotized underneath the exoskeleton already before the moult. In this stage, there is no access to the genitalia since the epigyne is still covered by body cuticle of the late-subadult females (Figs. 1b, 2a). During the final moult, the cuticle is shed, and the arched, dark brown, sclerotized epigynal plate with a slit-like access to the atrium and connected spermathecae is exposed (Figs. 1c, 2c). On each side of the atrium there is a broad entrance to a copulatory duct each leading to a bi-lobed spermatheca.
When matings occur with late-subadult females, the males disrupt the cuticle in the genital area by which the males gain access to the underlying epigyne and the connected spermathecae (Figs. 2 b, 3a; video S1). The disrupted cuticle is apparent in Fig. 2b.
Additional file 3: Video S1. Immature mating in Latrodectus geometricus. Male contacts female external genitalia (i.e. epigyne) with his chelicerae and copulatory organs; male bites through the cuticle covering the epigyne, exposing the atrium with the copulatory openings; male inserts one of his copulatory organs and transfers sperm; male withdraws the copulatory organ.
When females that mated as late-subadults (N = 15) eventually moult to the adult stage, their shed cuticle shows a rupture in the genital area. The underlying genital structures are not affected by the moulting process. When matings occur with adult females, their external genitalia do not differ before and after copulation (Fig. 1c versus 3B).
Both late-subadult and adult females possess copulatory ducts, spermathecae and fertilization ducts (Fig. 4; but see below for more subtle differences between the two stages). Each copulatory duct spirals from the copulatory opening towards and around a spermatheca and connects to it (Fig. 4). As can be seen from the histological sections, the copulatory duct is not a closed tube but a spiralling fold consisting of a broad, functional duct part and a connecting part (Figs. 5a, c, e, 6b, c, e, f, 7a, d, 8b, c, e, f). The duct part hosts the male embolus during copulation. The connections between the functional ducts are hidden between their coils (Figs. 4, 9a, b). Each spermatheca is dumb-bell-shaped consisting of an anterior and posterior lobe connected by a narrow middle region  (Figs. 2, 4, 9a, b). The copulatory duct connects to the anterior lobe of the spermatheca close to the middle region (Fig. 2). Before the posterior lobe, the spermathecal cuticle forms a short, sclerotized fertilization duct (Fig. 4). The fertilization ducts connect to the medially-located common fertilization duct (Fig. 6c, f) which leads to the uterus externus (Fig. 6c). Muscle fibres extend from the common duct to the epigynal plate (Fig. 6c, f).
Maturation of female genitalia
In early-subadult females, the cuticle of the copulatory ducts and spermathecae is not yet formed, however, the course of the copulatory ducts and the shape of the spermathecae are already outlined by an invaginated epidermal epithelium (also termed hypodermis) consisting of elongated cells that stain in dark violet with Azan (Fig. 5a, b) (see also [21, 22]). The lumina they enclose are the prospective lumina of the copulatory ducts and spermathecae. A substance staining in light blue is abundant in the spermathecal lumen (Fig. 5b), and also occurs in the lumina of the copulatory ducts, where it appears contracted and more fibrous (Fig. 5a, b).
In late-subadult females, the hypodermal cells show a thin cuticle towards the lumina of both ducts and spermathecae. The cuticle stains bright red along the ducts and orange along the spermathecae (Fig. 5c, d), suggesting different degrees of sclerotization. The spermathecal lumen is filled with a light-blue substance (Fig. 5c, d) with occasional red droplets.
In adult females, the epidermis surrounding the spermathecae appears less prismatic, more vacuolised and stains paler violet than in both subadult stages (Fig. 5b, d versus 5F). Compared to the epidermis of the copulatory duct, the narrow connection to the spermathecae is highly sclerotized and shows an epithelium of densely arranged and elongated cells (Fig. 6e) similar to late-subadult females (Fig. 6b). The cuticle of the spermatheca is much thicker in adult females compared to late-subadult females and exhibits numerous pores (Figs. 5f, 6d). The pores host epithelial cells that seems to discharge a red staining substance into the bright blue secretion of the spermathecal lumen (Figs. 5f, 6d). Bright blue secretion also occurs in smaller amounts in parts of copulatory ducts (Fig. 6e, f).
After mating, in all late-subadult (N = 5) and adult females (N = 8), at least one spermatheca contained spermatozoa. Typically, the anterior lobe - to which the copulatory duct connects (Fig. 2) - and the middle region of a spermatheca contained large quantities of sperm, while the amount of sperm in the posterior lobe - to which the fertilization duct connects (Fig. 4) - varied considerably (see below). In spermathecae that did not contain sperm or only a small amount of sperm in the anterior lobe, spermatozoa were found neither in the middle region nor in the posterior lobe of the spermathecae.
Large amounts of sperm were found embedded in a red-orange substance in both late-subadult and adult females after mating (Figs. 7b, e, 8a, d). In five adult females (N = 8) a small amount of blue substance was also present in the anterior lobe of one or both spermathecae. In four late-subadult (N = 5, 80%) and five adult females (N = 8, 62.5%) both spermathecae contained sperm. In one of the adult females, however, the anterior lobe of one spermatheca contained only a few spermatozoa embedded in blue substance with a few red droplets. When no sperm was found, a blue substance with few red droplets was present, as typical for virgin females.
A tip of the embolus was found in two late-subadult (N = 5) and three adult females (N = 8) at the entrances to the anterior lobes of both spermathecae, both of which contained sperm (Fig. 7c, f). Whenever embolus tips were present in spermathecae, they always occurred together with sperm but embolus tops did not always occur when sperm was present. The embolus tips were always located in the narrow connection between the copulatory duct and the spermatheca, and extended into the lumen of the anterior lobe (Figs. 7c, f, 9a).
If sperm was present in the anterior lobe, the middle narrow region of the spermathecae also contained spermatozoa as well as small amounts of red secretion (Fig. 8b, d). These materials either filled the entire region or were surrounded by blue substance. In one spermatheca of one adult female, however, the narrow region contained predominantly bright blue substance with few red droplets and no sperm, while in another adult female the narrow region of one spermatheca contained sperm within bright blue substance.
Irrespective of the developmental stage at mating, the posterior lobe of the spermathecae of mated females was mostly partly or entirely filled with spermatozoa and associated red substance as well as blue substance (Fig. 8f). In three late-subadult females (N = 5, 60%) sperm was present in large amount in the posterior lobe of one (N = 1) or both (N = 2) spermathecae. In four adult females (N = 8, 50%) the sperm in the posterior lobe was present in one (N = 2) or both (N = 2) spermathecae.
The areas of the anterior lobe and the posterior lobe of the spermathecae did not differ between late-subadult (N = 11) and adult females (N = 14) (Wilcoxon signed-ranked test; anterior lobe, W = 46.00, P = 0.10; Fig. S1A; posterior lobe, W = 61.00, P = 0.40; Fig. S1B). There was also no significant difference between females of different developmental stage and mating status in the areas of anterior or posterior lobes (Kruskal Wallis test; anterior lobe: χ2 = 3.21, P = 0.36, Fig. S1A; posterior lobe: χ2 = 2.31, P = 0.51, Fig. S1B).
Spermathecal cuticle thickness
The spermathecal cuticle was significantly thinner in late-subadult females (N = 11) than in adult females (N = 14) both in the anterior (ANOVA: F = 156.00, P < 0.01; Fig. 10a) and posterior lobe (ANOVA: F = 112.98, P < 0.01; Fig. 10b). Overall, the cuticle thickness differed among the females of different developmental stages and mating statuses (ANOVA: anterior lobe F = 67.76, P < 0.01; posterior lobe ANOVA: F = 44.79, P < 0.01). As to the anterior lobe, the cuticle was thinner in virgin late-subadult females, intermediately thick in mated late-subadult females and the thickest in adult females (Table S1; Fig. 10). As to posterior lobes, the cuticle was thinner in late-subadult females compared to adult females with no difference between mating status in both developmental stages (Table S1; Fig. 10).
Spermathecal epithelium thickness
In comparison to adult females, the epithelium of late-subadult females was significantly thicker in the anterior (ANOVA: F = 6.98, P = 0.01; Fig. 11a) but not in the posterior lobe (ANOVA: F = 2.31, P = 0.14; Fig. 11b). When separating the females into virgin and mated females of the two developmental stages there was no significant difference in epithelium thickness, neither in the anterior (ANOVA: F = 2.19, P = 0.12; Fig. 11a) nor in the posterior lobe (ANOVA: F = 0.79, P = 0.52, Fig. 11b).
Successful matings occurred in 53 of 60 (88.3%) trials with late-subadult and 37 of 42 (88.1%) trials with adult females, which was not significantly different (Chi2 test: χ2(1) < 0.01, P = 0.97).
Embolus breakage in males
The probability of breaking off the tip or the whole embolus during mating did not differ significantly between males that mated with late-subadult females (35 of 50; 70%) and males that mated with adult females (24 of 31; 77.4%) (Chi2 test: χ2(1) = 0.53, P = 0.47). Mostly, males were missing only the tip of the embolus (Fig. 9a, d). In 14.3% (5 of 35) of males mated to late-subadult females and 29.2% (7 of 24) of males mated to adult females, a larger piece or the entire embolus was missing (Fig. 9b). The proportion of a small or large part of the embolus breaking off did not differ significantly between males mated to late-subadult and adult females (Chi2 test: χ2(1) = 1.95, N = 59, P = 0.16). However, significantly more males lost both emboli (tips or larger parts) when mating with late-subadult females (23 of 35; 65.7%), compared to males mating with adult females (8 of 24; 33.3%) (Chi2 test: χ2(1) = 5.99, P = 0.01, Fig. 12a).
Males that mated with late-subadult females were similarly likely to deposit the broken off embolus in the genitalia of the female. Plugging success was 85.7% (30 of 35) in late-subadult matings compared to 66.7% in matings with adult females (16 of 24; Chi2 test: χ2(1) = 3.01, P = 0.08).
Plugs in the female genitalia
Overall, one or two embolus pieces were found in the macerated genitalia of 53 out of 90 (58.8%) females. In 92.5% of cases (49 of 53), the parts consisted of a tip of the embolus and in 7.5% of cases of a larger part of the embolus (4 of 53). Plugs occurred in 62.3% of late-subadult females (33 of 53) and 54.1% of adult females (20 of 37); the occurrence of mating plugs did not significantly differ between the developmental stages of the female (Chi2 test: χ2(1) = 0.61, P = 0.44; Fig. 12b). The plugs were located at the entrance to the spermatheca in 93.9% of subadult females (31 of 33) and in 95% of adult females (19 of 20), and in the remaining cases inside the copulatory duct. There was no significant difference in plug position between the groups (Fisher’s exact test: P = 1.00). Females that mated in the subadult stage had both spermathecae plugged with significantly higher probability (21 of 33; 63.6%) than adult females (7 of 20; 35.0%) (Chi2 test: χ2(1) = 4.10, N = 53, P = 0.04; Fig. 12b).