Comparative anatomy of the spinneret musculature in cribellate and ecribellate spiders (Araneae)

Silk production is a prominent characteristic of spiders. The silk is extruded through spigots located on the spinnerets, which are single‐ to multimembered paired appendages at the end of the abdomen. Most extant spiders have three pairs of spinnerets, and in between either a cribellum (spinning plate) or a colulus (defunct vestigial organ), dividing these spiders into cribellate and ecribellate species. Previous research has shown that cribellate and ecribellate spiders differ not only in the composition of their spinning apparatus but also in the movements of their spinnerets during silk spinning. The objective of this study was to determine whether the differences in spinneret movements are solely due to variations in spinneret shape or whether they are based on differences in muscular anatomy. This was accomplished by analyzing microcomputed tomography scans of the posterior abdomen of each three cribellate and ecribellate species. It was found that the number of muscles did not generally differ between cribellate and ecribellate species, but varied considerably between the species within each of these two groups. Muscle thickness, particularly of the posterior median spinneret, varied slightly between groups, with cribellate spiders exhibiting more robust muscles, possibly to aid in the combing process during cribellar thread production. Interestingly, the vestigial colulus still possesses muscles, that can be homologized with those of the cribellum. This exploration into spinneret anatomy using microcomputed tomography data reveals that despite being small appendages, the spider spinnerets are equipped with a complex musculature that enables them to perform fine‐scaled maneuvers to construct different fiber‐based materials.

The ability to produce silk is a defining characteristic of spiders.
Unlike to silk-spinning insects, such as Lepidoptera and Hymenoptera, spiders are unique in their ability to produce silk in all life stages and spin it from multiple pairs of spinnerets and different types of silk glands (Foelix, 2011).Spider spinnerets are single to multimembered paired appendages located at the posterior end of the opisthosoma.
They are believed to be modified epipods of successive opisthosomal segments (Damen et al., 2002).The silk is extruded through the spigots located at the tips of each spinneret, with each pair of spinnerets bearing a different set of spigots connected to different types of silk glands (Apstein, 1889).These different glands are used in various situations, such as during locomotion, the construction of different web elements, or during reproduction (Foelix, 2011).
Almost all extant spiders possess three pairs of spinnerets: the anterior lateral spinnerets (ALS), posterior median spinnerets (PMS), and posterior lateral spinnerets (PLS) (Montgomery, 1909).Between the ALS, there may be either a colulus or a cribellum, which are thought to be homologous to the anterior median spinneret pair (Montgomery, 1909).
The cribellum was discovered by Blackwall in 1839 but was not described until 1882 by Bertkau.According to Bertkau (1882), the cribellum is a small transverse, chitinous spinning plate that can sometimes be divided by a median carina.It has numerous tiny spigots that are the openings of multicellular spinning glands (Foelix, 2011).In spider species that have a cribellum (cribellate spiders), the fourth pair of metatarsi bear a comb-like row of specialized setae called the calamistrum (Foelix, 2011).The calamistrum is used to comb out the cribellate fibers to form an adhesive capture thread by brushing over the cribellar spinning field.In contrast, some species have a colulus (ecribellate spiders) located at the same position between the ALS (Menge, 1843).This structure is typically a conical projection or small tubercle, but can also be reduced to only a patch (or pair of patches) of setae.Unlike the cribellum, the colulus does not have any spinning glands.Additionally, ecribellate spiders do not have a calamistrum (Montgomery, 1909).
As a result of these differences, web-building cribellate and ecribellate spiders produce different types of capture threads.In some ecribellate spiders, such as Araneidae, the capture thread consists of two core fibers coated with viscid glue droplets to capture the prey.Cribellate spiders, on the other hand, do not use glue droplets to catch prey but instead produce wool like capture threads consisting of two axial fibers surrounded by masses of nanofibers.
Most of these nanofibers originate from the cribellum, but some also emerge from the paracribellum, which is a structure of elongated spigots on the PMS (Peters, 1983).The axial fibers are spun from a spigot on the PLS (Joel et al., 2015).Upon contact, irregularities of the prey surface become entangled in the brushed-up nanofibers in a velcro-like mechanism.Additionally, due to the numerous nanoscale filaments, cribellar capture threads adhere through van der Waals forces (Hawthorne & Opell, 2003) and interaction with epicuticular waxes on arthropod prey (Bott et al., 2017).
The process of spinning cribellate silk involves a highly conserved sequence of movements in which the ALS is abducted to clear the way for the PMS and PLS, while the PLS perform anteromedial adduction and posterolateral abduction movements (Weissbach et al., 2021).Fine scaled spinneret movements have also been described for the anchoring of silk threads to substrates (Wolff, 2021).In the resting position, the base segment of all spinnerets is partially retracted into the abdomen, and the spinnerets lie next to each other.When the spider begins spinning, all three spinnerets are spread out laterally before the ALS and PMS are folded down ventrally and the abdomen is lowered towards the substrate.The PLS is being spread out dorsolaterally during this process, likely to make space for the ALS and PMS, which perform oscillating movements during which the spinneret tips are rubbed against the substrate surface and against each other.The choreography of these movements is highly species-specific and differs considerably between cribellate and ecribellate spiders: while in cribellate spiders the ALS perform predominantly lateral movements, they are moved more towards the anterior direction or in C-or O-shaped cycles in ecribellate spiders (Wolff, 2021;Wolff et al., 2021).
Another situation in which the spinnerets are moved is during the construction of an egg sac or the wrapping of prey.In the wolf spider genus Lycosa, for example, egg sac construction begins with the spinning of a cushion on which the eggs are laid (McCook, 1884).
The circumference of the cushion is then clasped with the legs, and silk is applied through up-and-down and forward-and-backward movement of the spider's abdomen (McCook, 1884).In other species, the egg sac is moved around with the legs while the silk is being applied (Hajer & Řeháková, 2003).However, in these cases, the exact movements performed by the spinnerets have not been described.
Different behaviors have been also described for prey wrapping.
In the orb weaver Argiope keyserlingi (Araneidae), for example, prey is moved around with the legs while a wreath of silk is pulled from the posterior spinnerets with their fourth leg pair and applied to the prey through simultaneous movement of both legs.In Agelenidae and other families, spiders stand over prey and turn in place to cover it in silk (Barrantes & Eberhard, 2007).The cursorial Hersiliidae face prey with their elongated PLS and run around it in circles while producing silk bands that enclose the prey (Peters, 1967).
Information on the musculature involved in the spinning process is surprisingly limited.Peters (1967) conducted a comparative study of the spinneret musculature in 13 different species of spiders from seven families and compared the spinneret musculature to the musculature of the spider legs that mostly possess flexor muscles only.Furthermore, Peters (1967) found that ecribellate spiders had a less developed musculature than cribellate spiders, particularly in the colulus/cribellum.While the colulus musculature showed a tendency to recede, the cribellum is richly supplied with muscles.Wilson (1962) described the existence of valves in the major and minor ampullate glands of araneid orb weavers, that permit a control of the silk flow.
These valves are controlled by 2-3 muscles, including the valve tensor, duct stabilizer and duct elevator.The valve tensor arises on the ventral body wall and attaches to one side of the valve allowing it to pull on the valve.The position of the duct stabilizer suggests that it is the antagonist of the valve tensor muscle.The valve levator is attached below the valve and pulls the duct into the spinneret.
Previous anatomical studies relied on histological techniques, which are destructive in nature.During such techniques, it is possible that the natural arrangement and position of the muscles may be altered, or that the exact attachment sites or small muscles may be overlooked.To overcome these limitations, we employed a nondestructive approach using microscopic X-ray tomography to determine whether differences in spinneret usage and movements are due to morphological differences in spinneret musculature, such as variations in the number and orientation of muscles controlling each spinneret segment.In particular, we focused on comparing the musculature of cribellate and ecribellate species, to (1) understand if the differences observed in anchor spinning behavior are simply due to mechanical blocking of the mobility of the ALS by the cribellum or if they are due to a different number and orientation of the spinneret muscles; (2) if the hypothesized modification of the cribellum into a vestigial colulus resulted in complete reduction of the attached muscles; and (3) if cribellar thread production requires more or stronger muscles of the spinnerets than the ecribellar silk spinning.

| MATERIALS AND METHODS
The following species were selected to provide a good comparability between cribellate and ecribellate species by including species pairs with similar lifestyles, such as A. keyserlingi (ecribellar orb-weaver) and Philoponella variabilis (cribellar orb-weaver) or close relationships, such as Tegenaria ferruginea and Badumna insignis (both belonging to the marronoid clade of spiders but the first being ecribellar and the second being cribellar; Wheeler et al., 2017).For this study the microcomputed tomography (µCT) datasets of Wolff et al. (2021) were reanalyzed-see for details on the collection data.
Stegodyphus dumicola POCOCK, 1898 (Eresidae): spider that builds three-dimensional substrate-bound webs, often as subsocial colonies, and can be found in the Southwestern regions of Africa.The specimen for this study was taken from a lab stock.

| Ecribellate species
A. keyserlingi KARSCH, 1878 (Araneidae): orb weaving spider that is distributed along the East coast of Australia from Victoria to Queensland.
Tamopsis sp.BAEHR & BAEHR, 1987 (Hersiliidae): spider from Australia that is a specialized tree trunk dweller with highly elongated PLS used in direct prey capture attacks.

| X-ray µCT
Specimens were fixed and stored in 75% ethanol (with exception of S. dumicola which was fixed in Dubosq-Brasil-Solution before being transferred to 75% ethanol).The opisthosoma of each specimen was removed and stained overnight in a solution with 1% iodine in absolute ethanol, followed by critical point drying using a Leica CPD 300 (Leica Microsystems).Dried samples were glued onto plastic sticks and scanned in an Xradia MicroXCT-200 imaging system (Carl Zeiss X-ray Microscopy) with the settings outlined in Table 1.

| Reconstruction and visualization
The tomography projections were reconstructed using XM Reconstructor (Carl Zeiss X-ray Microscopy).The resulting image stacks were segmented with the software Amira 6.4 (Thermo Fisher Scientific).The musculature was color-coded as following: ALS-yellow; PLS-blue; median spinneret-green; cribellum or colulus-pink.The terminology used to name the muscles followed Peters (1967)  The spinning apparatus of the investigated species consisted of three pairs of spinnerets: the anterior lateral (ALS), posterior median (PMS) and the posterior lateral (PLS) (Figure 1a,b).The PMS was always the smallest of the three pairs, and usually, the ALS were the largest.
However, in B. insignis and Tamopsis sp., the PLS were substantially longer than the ALS.
The musculature that led towards or through the spinnerets was connected to the median and lateral opisthosomal musculature (Figure 1c).The muscles controlling the ALS and the cribellum/colulus arose from the apodemes of the 10th body segment, while those controlling the four posterior spinnerets arose from that of the 11th body segment, as well as from lateral tendons.These muscles attached partly on the basal edge of the spinnerets or slightly into them, enabling movement of the spigot fields (as also described in Peters [1967]).
In the cribellate spiders, the cribellum differed in size (relative to the rest of the spinning apparatus), with S. dumicola possessing the largest, followed by P. variabilis and B. insignis.These size differences were also mirrored by differences in muscle thickness.The ecribellate species exhibited some differences in the external morphology of the colulus, possibly representing different stages of cribellum reduction.
The colulus of A. keyserlingi was visibly more reduced than that of T.
ferruginea the latter of which exhibited a broad, bilobed shape similar to a cribellum.In Tamopsis sp.we did not find any remnant colulus musculature even though a small colulus structure is present.
A summary of the spinneret muscles and their presence in the different investigated species is given in Table 2.

| P. variabilis
Cribellum: The cribellum has four pairs of muscles.Two pairs (cr1, cr1.1; Figure 2b) are parallel to each other and arise from the anterior apodeme (10th body segment).From there, they extend towards the ventral side of the body, where cr1 attaches to the ventro-proximal edge of the cribellum while cr1.1 attaches to the dorso-proximal edge.The third and fourth pair (cr2, cr3) arise from the median apodeme (11th body segment) from there cr2 extends towards the ventral edge of the cribellum and cr3 attaches to the dorsal edge (Figure 2b).

ALS:
The ALS are controlled by nine muscle pairs, all of which are quite thick (Figure 2c).Three pairs, v8, v9, and v4.M2 is parallel to m1 and connects to the ventral edge of the spinneret base while also originating from inside the opisthosoma where it connects to t11.There are no muscles leading through the posterior median spinneret towards its apex.A third muscle, M4, originates from the median apodeme (Figure 2a) and attaches to the median base of the spinneret.M1 and m4 are both quite thick and strong muscles, while m2 is relatively small and short.

PLS:
The musculature of the PLS consists of seven muscles per spinneret (Figure 2e).H21 originates from lateral tendons and attaches to the lateral basal edge of the spinneret while h18 connects to the ventrolateral basal edge and also arises alongside h21 from lateral tendons.The antagonist is h17, which connects to the median base of the spinneret and comes from the median apodeme (Figure 2a).There are two more muscles, h21a on the lateral side and h21b on the ventral side of the spinneret, that traverse the proximal spinneret segment from the basal edge to the beginning of the distal segment.H3.2 originates from dorsal tendons and leads towards the dorsomedian edge of the proximal segment.H2.3a is arising at the ventromedian base of the spinneret leading into the spinneret attaching to the base of the distal segment.

| S. dumicola
Cribellum: The musculature of the cribellum consists of four pairs of strong muscles (Figure 3a,b): cr1, cr1.1, cr2, and cr3.The location, direction and attachment points of these muscles are similar to those described for P. variabilis.
ALS: Each ALS of S. dumicola is controlled by five muscles.V3, v3.1, v3.1a, and v11 which exhibit the same location, orientation and attachment points as those described for P. variabilis, but with an overall more robust appearance.However, S. dumicola has one muscle that is absent in P. variabilis: v11.1.This muscle is also known as the compressor mammillae anterioris, which is a control muscle for the dragline flow (Brown, 1939).It originates from the cuticle on the ventral side of the proximal segment of the spinneret around halfway into the segment and goes transversally through the spinneret to theuticlee of the dorsal side of the proximal segment (Figures 3c   and 8c).

PMS:
The PMS are small and only have three pairs of muscles: m1, m2, and m4, as described for P. variabilis (Figure 3d).Again, the musculature of the PMS of S. dumicola appears much stronger, with a great width covering almost the entire base of the spinneret, while in P. variabilis the same muscles have only small attachment points.
PLS: The PLS are around half the volume of the ALS and are controlled by six pairs of muscles.H2.3a connects to the ventral base of the spinneret and connects with the base of the distal segment, passing through the entire proximal segment.H21a connects to h2.3a and follows at an angle towards the median part of the cuticle.
Its antagonist is h2.1, which connects dorsally to the base of the proximal segment and leads dorsally towards the base of the distal segment.There are three muscles connected to the basal edge of each PLS: h3.2, h3, and h17.H3.2 and h3 lead to the dorsal edge of the spinneret originating from dorsal tendons and h17 leads to the ventromedian edge (Figure 3e).

| B. insignis
Cribellum: The cribellum is controlled by mostly similar muscles as described for the species before, with the following differences (Figure 4b).Cr1.1 and cr2 are noticeably short compared to those of S. dumicola and P. variabilis.Cr3 was not found in B. insignis.

PMS:
The PMS have three pairs of muscles, m1, m2, and m4 which have a similar position as described above.M2 has a larger attachment area and is longer compared to the one in P. variabilis (Figure 4d).

PLS:
The PLS are controlled by six pairs of muscles, all of which resemble those of P. variabilis (Figure 4e).

| A. keyserlingi
Colulus: The colulus musculature consists of six long and delicate muscles arranged in three pairs (Figure 5b), which are remarkably similar to three pairs of the cribellar musculature of P. variabilis (Figure 2b).Co1, co1.1, and co2 have the same attachment points and directions as cr1, cr1.1, and cr2 in P. variabilis.

ALS:
The ALS are controlled by 11 pairs of muscles (Figure 5c).
Similar to P. variabilis, we found v3, v3. 1, v4, v4.2, v8, v9, and v11, which have the same attachment points and directions as described above, but the shape of these muscles is slightly different; in particular, v9 and v8 are longer than in P. variabilis and, along with v4.2, slightly thinner.V3.2 originates from the base of the proximal segment, where v9 attaches, and extends slightly upwards into the spinneret where it attaches to the valve of the major ampullate gland duct (Figure 8a).V3.2 is the duct stabilizer muscle described by Wilson (1962).Another muscle associated with the control valve is the duct levator, v3.2a, which also originates from the ventral base of the spinneret.The valve tensor, another small muscle described by Wilson (1962), could not be reconstructed due to the limitations in scan resolution.V3.1 is connected to v3.1a, which emerges from the cuticle close to the basal edge of the spinneret and then joins with v3.1.The antagonist to v4 is v4b that connects to the dorsomedial base of the distal segment, traversing the entire proximal segment and originating in the opisthosoma from dorsal tendons.

PMS:
The PMS are controlled by three pairs of muscles (Figure 5d), two of which are also found in P. variabilis (Figure 5d).
These two muscles are m1 and m2, which are located in the same position as in P. variabilis.The small attachment muscle m0.1.of the control valve of the minor ampullate gland duct (Figure 8b) described by Wilson (1962) was distinguishable, but the other small muscles associated with this valve could not be reconstructed from the scans since the resolution was not high enough.

PLS:
The PLS are controlled by six muscle pairs (Figure 5e).Only four of these are similar to those in P. variabilis: h2.3a, h21, h18 and h17, which are in the same position as described before.h17.1 which connect to the median base of the spinneret and come from the median apodeme (Figure 5a).Inside the spinneret two more muscles of the spinneret leading into the spinneret, where it attaches to the base of the distal segment.

| T. ferruginea
Colulus: The musculature of the colulus is highly reduced compared to that of A. keyserlingi.Only two of the three muscles, co1 and co2, were found, and are very thin (Figure 6b).Their position and attachment points resemble those found in P. variabilis (Figure 2b).

ALS:
The ALS are controlled by seven pairs of muscles, with v8 and v9 appearing much thicker and stronger compared to the other species, while V3 is quite thin, even though it is usually one of the stronger muscles in other species.Furthermore, v3 attaches to v3.1 closer to the ventromedial base of the spinneret than in the other species (Figures 6c   and 8e).

PMS:
The PMS are rather thin and have four pairs of muscles, two of which were found in the other species (m2, m4).The other two are m1a and m2a.M1a originates from the median apodeme (Figure 6a,d), and leads towards the lateral edge of the spinneret base.It is a short but thick muscle.M2a, on the other hand, runs parallel to m4 and originates from the median apodeme, leading towards the ventral base of the spinneret where it attaches close to m2 (Figure 6d).

PLS:
The PLS are controlled by five muscle pairs, all of which resemble those of the species described above (Figure 6e).

| Tamopsis sp.
Colulus: The colulus is small and no musculature could be seen in the scans.

ALS:
The ALS are controlled by eight pairs of muscles, which are thin, and particularly v9, v3, and v8 are relatively short (Figure 7c).
Locations and attachment points of these muscles are similar to A.

PMS:
The PMS have three pairs of muscles, which are all short and thin.M1, m2, and m4 have the same position as the other species (Figure 7d).

PLS:
The PLS are strongly enlarged, especially the distal segment, and have only six pairs of muscles (Figure 7e).H17 and h3.2 attach at the spinneret base and are thick and short.H21a, h21b, and h2.3a have the same direction and attachment points as in the other species.Even though the distal segment is enlarged, we did not find any further muscles engaging its mobility.

| DISCUSSION
We found distinct differences in muscle number, location and relative size between species, but differences seemed to be lineage specific than related to the cribellar/ecribellate configuration of the spinning apparatus.In the following we discuss these differences in the context of ecological function and phylogeny.

| Cribellum/colulus
The cribellum, despite appearing as a flat plate, has recently been shown to be capable of moving at different angles, as well as being protracted and retracted (Weissbach et al., 2021).The function of these movements is not fully understood, but it is likely that the mobility plays a role in forming the serial macrostructures of the cribellar thread in conjunction with the combing movement of the calamistrum, as well as moving the cribellar spinning field into place for the combing.We assume that the strong cribellar muscles observed may be required to withstand the potentially strong forces acting when the calamistrum rubs against the cribellum at a high frequency.Given the muscle positions, protraction of the cribellum into a vertical position for the cribellar capture thread production is caused by cr1.1 and cr3, while cr1 and cr2 retract it into a resting position close to the body surface (Peters, 1967).
Among the cribellate species, we found that differences of the cribellum musculature were well reflected by differences in the relative sizes of the cribellum.In B. insignis, which had the relatively smallest cribellum of the three investigated cribellate species, there were only three of four pairs of muscles and the muscle cr1.1 was relatively short.However, S. dumicola, which had the largest cribellum, had the most strongly developed muscles.This may be, because a larger cribellum has to withstand stronger forces during combing than a small one.
While the cribellum is a functional spinning organ bearing silk gland spigots (with the exception of males of cribellate species in which the spinning function is lost after maturity), the colulus is a vestigial organ exhibiting different states of reduction in different lineages.In A. keyserlingi, it is a small and slightly protruding tubercle while in T. ferruginea it is less reduced, retaining a broad, slightly bilobed shape similar to a cribellum.Surprisingly, the colulus musculature was more strongly reduced in T. ferruginea than in A.
keyserlingi, suggesting that the reduction of the cuticular parts and the attached musculature of this vestigial organ is not strictly coupled.
We found that the colulus has muscles similar to those of the cribellum.Given the comparable attachment sites and position, co1, co1.1, and co2 are most likely homologs to cr1, cr1.1, and cr2.The biggest difference is that muscles of the colulus are less strongly developed and fewer in number.The colulus does not have a known function; however, in video observations with A. keyserlingi it was observed that the colulus moves distinctively during the spinning of thread anchors though it was likely not in contact with any silk (Wolff, 2021).It is possible that the muscular equipment of the colulus is a relict and its movements result from some neural and/or mechanical coupling between the colulus and ALS musculature.
Our results strongly support that the colulus is homologous to the cribellum, which is an overall consensus among most arachnologists (Miller et al., 2010).
In Tamopsis sp. a small colulus but no attached muscles could be found; similar to what was described in other Hersiliidae, like Hersilia sp.(Peters, 1967).However, due to the limited resolution of µCT scans, we cannot exclude that some highly reduced remnants of the colulus musculature are present in this species.

| ALS
In the ALS, the muscles v3, v8, and v9 were present in almost all of the species investigated.Only S. dumicola lacks one of these muscles and other muscles positioned to enable posterior-anterior directed movement.This may be explained by the large cribellum mechanically hindering movement of the ALS into the anterior direction (Wolff et al., 2021(Wolff et al., , 2019)), which would render such muscles dysfunctional.
The movements of the ALS play an important role in the formation of dragline and anchorages as well as in thread connections (Eberhard, 2010;Foelix, 2011).The specific movement patterns of the spinnerets during silk spinning affect the structure and mechanical properties of the product (Wolff et al., 2017(Wolff et al., , 2021(Wolff et al., , 2019)).It was found that in cribellate spiders, the ALS predominantly move laterally, while ecribellate spiders show diverse choreographies, including anterio-lateral and posterior-lateral abductions (Wolff, 2021;Wolff et al., 2019).Lateral movements were especially expressed in S. dumicola (Wolff, 2021;Wolff et al., 2021), and accordingly v8, v11, and v3.1, which are muscles for lateral movements, are well developed in this species.
B. insignis and P. variabilis, like ecribellate spiders, possess muscles for movements in the posterior and anterior direction (v4.2, v9).This matches with the motion motifs observed in the anchor spinning of these two species, where some anterior-posterior component was observed, albeit not as expressed as in most ecribellate species (Wolff, 2021;Wolff et al., 2021).
For the ecribellate species investigated it was expected that they would exhibit muscles permitting anterior-posterior movements as the colulus is usually no obstacle for movement, and the observed motion motifs include a variety of angular movements (Wolff, 2021).
For instance, in A. keyserlingi the ALS follow a sickle-shaped choreography during anchor spinning, with a dominant anteriolateral abduction component (Wolff et al., 2017).Accordingly, this species exhibits strong muscles that permit movements in this direction (v8, v9).
Another remarkable feature are muscles that control the silk flow through the ampullate gland ducts, making it possible not only to control silk fiber diameters and properties, but also to brake during abseiling behaviors without the use of legs (Wilson, 1962).In araneids, such as A. keyserlingi, this is possible by the presence of a valve like structure in the distal duct of the major ampullate glands, which is controlled by three muscles: the valve tensor, the duct stabilizer (v3.2) and the duct levator (v3.2a) (Wilson, 1962).In µCTscans two of these muscles could be recovered, but the delicate duct tensor could not be reconstructed, probably because of the limited resolution.Wilson (1969) also reported a circular valve that is controlled by muscles similar to those found in Araneidae, for Octonoba sinensis (Uloboridae).In our representative of Uloboridae, however, we could neither identify a duct valve nor any of the described control muscles.It remains unclear if this was due nonideal tissue fixation in our specimen or because such apparatus is indeed absent in P. variabilis.In most spider families, both the valves and its associated muscles are seemingly absent, however in three of the investigated species we found a muscle called compressor mammillae anterioris (v11.1).This is a transversal muscle, which may equip these species with an alternative mechanism to stop while lowering down on the dragline (Brown, 1939).It is assumed that if this muscle contracts, it compresses the surrounding tissue, and accordingly the silk gland ducts.Notably, no such muscles were found in Tamopsis sp. and P. variabilis and it remains unclear if or how these species can control dragline extension.

| PMS
The general muscular equipment, including the number and location of muscles, of the PMS did not differ between ecribellate and cribellate species.The muscles m1 and m2, which facilitate movement in anterior and posterior direction, were present in all but one species.In T. ferruginea m1 was absent, but instead m4 may allow posterior movements, while m1a, a muscle that is only present in this species, may enable lateral movements.
The PMS are primarily used to extrude aciniform and (in females) tubuliform silk for building capture threads and cocoons (Foelix, 2011), as well as minor ampullate silk as a component of the dragline (Wolff, 2020), or to build the temporary spiral of orb webs (Foelix, 2011) and bridging lines (Peters, 1990;Wolff et al., 2014).However, it is largely unclear if and how the small PMS perform movements during these spinning behaviors.In Eratigena atrica, Peters (1967) observed that the PMS are spread laterally and perform circular movements while spinning the web sheet.Considering the positioning of the musculature of T. ferruginea this could also be possible in this species.The PMS are also involved in anchoring and dragline initiation behavior in some species, like in the investigated B. insignis and Tamopsis sp.(Wolff et al., 2019).Here, the spinnerets usually perform cyclic posterior-anterior movements (Wolff et al., 2021), which is consistent with the observation that these species lack muscles for lateral movements.
The most notable difference between cribellate and ecribellate spiders is that the musculature of the investigated cribellate species is more strongly developed.Many cribellate spiders have a so-called paracribellum on their PMS in addition to the spigots found in ecribellate spiders.This extends the nanofiber-extracting area of the cribellum and is rubbed against by the calamistrum in a similar way as towards the cribellar spinning field (Joel et al., 2015).It is likely that the PMS of cribellate must withstand significant forces when the calamistrum brushes against the paracribellum to extract paracribellar fibers.In Uloboridae, the PMS are also used for intense prey wrapping, where thousands of fibers are extracted through repeated pulling movements (Eberhard et al., 2006), likely exerting considerable force on the musculature of this spinneret.This may explain the presence of strong muscles on the anterior and posterior margins of the spinnerets.
In Tamopsis sp., the musculature of the posterior median spinneret is short and thin, suggesting reduced use of these spinnerets in this species.This may be explained with Tamopsis sp.
constructing no webs and using the enlarged PLS for prey wrapping.
A. keyserlingi is unique among the investigated species in having a muscle inside its posterior median spinneret.This muscle is the attachment muscle associated to the control valve of the minor ampullate gland duct.In araneids, similar to the major ampullate gland of the ALS, the minor ampullate gland duct also exhibits a control valve although of only about half the size and presumably possessing only two muscles (Wilson, 1962).The second rather delicate muscle could not be reconstructed, possibly because of the limited resolution of the µCT scan.

| PLS
For the PLS, most of the muscles are present in most of the investigated species.Notably, the muscles h17, h18, h21, h3.2 of A. keyserlingi and P.
variabilis seemed stronger than those in other species.Both of these species are orb-weavers and use their PLS during the construction of the capture spiral (Eberhard, 2010;Foelix, 2011).In A. keyserlingi, the PLS are also used in intense prey wrapping, where dense swathes of aciniform silk are pulled out with the hind legs and laid onto the prey (Barrantes & Eberhard, 2007;Herberstein et al., 1998).This may require strong muscles to resist the pulling and brushing forces acting on the spinneret during this behavior.In A. keyserlingi, the PLS only have muscles suitable for lateral movements, which is in stark contrast to the PMS that bear only muscles suitable for anterior-posterior movements.This may be due to the compactness of the spinning apparatus, where the freedom of movement is inhibited by neighboring spinnerets when all spinnerets are moved.Additionally, spiders that routinely perform bridging and ballooning behaviors, such as A. keyserlingi, must be able to widely spread the PLS to expose the PMS for the production of the drifting line (Wolff et al., 2014).
In the investigated cribellate species, the muscle h3.2 was almost always present and positioned with h17 for anterior-posterior movement.This likely plays an important role during cribellar thread spinning, when the spinneret is moved in the direction of the cribellum to contribute to the production of capture thread by extruding silk for the axial fibers and connecting it to the cribellar mat (Joel et al., 2015;Weissbach et al., 2021).The absence of this function in ecribellate spiders may explain, why h3.2 is missing in T.
ferruginea and A. keyserlingi or is positioned less vertically.Only in Tamopsis sp.h3.2 is positioned similarly to the cribellate spiders, possibly to be able to hold the long, slightly curved spinnerets away from the substrate.
Tamopsis sp. is unique in having highly enlarged PLS used for prey capture.Interestingly, this enlargement is not reflected in muscular anatomy.This may be explained by the fact that in this genus wraps its prey by running around it in circles with its PLS facing the prey and covering it in aciniform silk swathes (Wallace, 2003).In contrast to the short musculature found in Tamopsis, Peters (1967) described compact strong muscles for Hersilia sp., another species of the Hersiliidae family.A possible explanation for this could be that Hersilia has more mobile PLS and therefore needs stronger muscles.

| CONCLUSIONS
In conclusion, in contrast to our initial hypothesis that ecribellate and cribellate spiders would differ in the number and position of muscles in their spinning apparatus, we found that this was not generally the case.Both groups showed slight differences in muscle thickness, such as in the PMS.Muscles v3, v8, v9, m1, and m2 were present in almost all investigated species, indicating their involvement in basic spinning patterns and behaviors common to most araneomorph spiders.Even with a limited sample of taxa, we observed remarkable differences in the muscular anatomy of the spinning apparatus.These differences may be related to both the phylogenetic history and ecology of the species.Interestingly, differences in musculature were not always predictable by examining modifications of the external morphology of the spinning apparatus.For example, the cribellum and colulus
2 are connected to the basal edge of the spinneret.V8 attaches laterally to the base while v9 attaches centrally on the ventral side of the spinneret base.They originate parallel to each other from lateral tendons.V4.2, on the other hand, arises from dorsal tendons and attaches dorsally to the spinneret base (Figure2a,c).Next to v8 is v4, which connects dorsolaterally to the base of the distal spinneret segment.V11 arises laterally at the base of the distal spinneret segment.The antagonist to v11 is formed by v3.1 which also connects to the base of the distal segment but on the medial wall of the spinneret.Located partly inside the opisthosoma, v3.1 is connected to v3, which emerges from the median apodeme.P. variabilis also has another muscle pair named v4a2 that arises dorsolaterally from the cuticle of the proximal spinneret segment around halfway through the segment and connects towards the lateral basal edge of the distal segment.PMS:The PMS are the smallest of the three pairs of spinnerets, with each having only three muscles.M1 connects to the dorsal base of the spinneret and arises from the median apodeme (Figure2a,d).
T A B L E 2 List of the muscles found in the investigated species, with attachment points and a summary of the presence and absence of each muscle in the investigated species (+: present, −: absent).ventro-median base of the proximal segment of the spinneret, connecting to v3 Ventro-median edge of the distal segment of the spinneret base of the proximal segment Leads medial into the proximal segment, attaches directly to the control valve of the major ampullate gland duct (valve control muscle) from the cuticle half-way into proximal segment laterally towards base of the distal segment − were found: h2.1, which originates at the dorsolateral base of the proximal spinneret segment leading towards the base of the distal segment, and h2.3a, its antagonist, arising at the ventromedian base T A B L E 2 (Continued) U R E 2 Philoponella variabilis (Uloboridae), reconstructed spinneret musculature.(a) Virtual sagittal section of the opisthosoma with reconstructed musculature of the spinning apparatus (left; anterior apodeme outside of the field of view) and ventral view of the reconstructed musculature of the spinning apparatus.(b) Musculature of the cribellum, lateral and ventral view.(c) Musculature of the anterior lateral spinneret, lateral and ventral view.(d) Musculature of the posterior median spinneret, lateral and ventral view.(e) Musculature of the posterior lateral spinneret, lateral and ventral view.aa, anterior apodeme; cr, cribellum; h, posterior lateral spinneret; m, posterior median spinneret; ma, median apodeme; t, opisthosomal musculature; tm, median opisthosomal muscle; tp, posterior opisthosomal muscle; v, anterior lateral spinneret.F I G U R E 3 Stegodyphus dumicola (Eresidae), reconstructed spinneret musculature.(a) Virtual sagittal section of the opisthosoma with reconstructed musculature of the spinning apparatus (left) and ventral view of the reconstructed musculature of the spinning apparatus.The anterior apodeme and proximal parts of the attaching muscles (e.g., cr1) are incompletely displayed as outside of the scanned volume.(b) Musculature of the cribellum, lateral and ventral view.(c) Musculature of the anterior lateral spinneret, lateral and ventral view.(d) Musculature of the posterior median spinneret, lateral and ventral view.(e) Musculature of the posterior lateral spinneret, lateral and ventral view.cr, cribellum; h, posterior lateral spinneret; m, posterior median spinneret; ma, median apodeme; t, opisthosomal musculature; ta, anterior opisthosomal muscle; tm, median opisthosomal muscle; tp, posterior opisthosomal muscle; v, anterior lateral spinneret.F I G U R E 4 Badumna insignis (Desidae), reconstructed spinneret musculature.(a) Virtual sagittal section of the opisthosoma with reconstructed musculature of the spinning apparatus (left; apodemes could not be reconstructed clearly) and ventral view of the reconstructed musculature of the spinning apparatus.(b) Musculature of the cribellum, lateral and ventral view.(c) Musculature of the anterior lateral spinneret, lateral and ventral view.(d) Musculature of the posterior median spinneret, lateral and ventral view.(e) Musculature of the posterior lateral spinneret, lateral and ventral view.cr, cribellum; h, posterior lateral spinneret; m, posterior median spinneret; t, opisthosomal musculature; tm, median opisthosomal muscle; v, anterior lateral spinneret.F I G U R E 5 Argiope keyserlingi (Araneidae), reconstructed spinneret musculature.(a) Virtual sagittal section of the opisthosoma with reconstructed musculature of the spinning apparatus (left) and ventral view of the reconstructed musculature of the spinning apparatus including cuticle of spinnerets.(b) Musculature of the colulus, lateral, and ventral view.(c) Musculature of the anterior lateral spinneret, lateral, and ventral view.(d) Musculature of the posterior median spinneret, lateral and ventral view.(e) Musculature of the posterior lateral spinneret, lateral, and ventral view.aa, anterior apodeme; co, colulus; h, posterior lateral spinneret; m, posterior median spinneret; ma, median apodeme; t, opisthosomal musculature; ta, anterior opisthosomal muscle; tm, median opisthosomal muscle; tp, posterior opisthosomal muscle; v, anterior lateral spinneret.

F
I G U R E 6 Tegenaria ferruginea (Agelenidae), reconstructed spinneret musculature.(a) Virtual sagittal section of the opisthosoma with reconstructed musculature of the spinning apparatus (left; opisthosomal musculature could not be reconstructed completely) and ventral view of the reconstructed musculature of the spinning apparatus (right).(b) Musculature of the colulus, lateral and ventral view.(c) Musculature of the anterior lateral spinneret, lateral, and ventral view.(d) Musculature of the posterior median spinneret, lateral and ventral view.(e) Musculature of the posterior lateral spinneret, lateral and ventral view.aa, anterior apodeme; co, colulus; h, posterior lateral spinneret; m, posterior median spinneret; ma, median apodeme; v, anterior lateral spinneret.F I G U R E 7 Tamopsis sp.(Hersiliidae), reconstructed spinneret musculature.(a) Virtual sagittal section of the opisthosoma with reconstructed musculature of the spinning apparatus (opisthosomal musculature could not be reconstructed).(b) Ventral view of the reconstructed musculature of the spinning apparatus.(c) Musculature of the anterior lateral spinneret, lateral and ventral view.(d) Musculature of the posterior median spinneret, lateral and ventral view.(e) Musculature of the posterior lateral spinneret, lateral and ventral view.h, posterior spinneret; m, median spinneret; v, anterior spinneret.F I G U R E 8 Virtual sagittal sections through the anterior lateral and posterior median spinneret of different species.(a) Virtual sagittal section of the anterior lateral spinneret of Argiope keyserlingi with view of the control valve of the major ampullate gland duct.(b) Virtual sagittal section of the posterior median spinneret of A. keyserlingi with view of m0.1, associated with the control valve of the minor ampullate gland duct.(c) Virtual sagittal section of the anterior lateral spinneret of Stegodyphus dumicola with view of the compressor mammillae anterioris v11.1.(d) Virtual sagittal section of the anterior lateral spinneret of Badumna insignis with view of the compressor mammillae anterioris v11.1.(e) Virtual sagittal section of the anterior lateral spinneret of Tegenaria ferruginea with view of the compressor mammillae anterioris v11.1.m, posterior median spinneret; v, anterior lateral spinneret.
exhibited similar musculature despite the latter being a vestigial organ with no known function.This may indicate limitations in organ reduction, possibly due to developmental coupling with functional organs.A comparative study with a larger sample of taxa representing different states of cribellum and colulus reduction would provide valuable insights into the evolutionary trajectories of muscular reduction after spinning organ loss of function.