We found different morphological structures that differ between the specimens. These differences appear to be correlated with differences in overall body size. We therefore interpret these changes as coupled to post-embryonic ontogeny. It is important to note that the results are based on a limited amount of specimens which were well enough preserved to be measured; thus no strict statistical tests could be applied. However, especially the ontogenetic changes of the sternum appear to follow a distinct trajectory for all investigated specimens (Fig. 4); hence these changes appear to be real ontogenetic changes.
Chelicerae
Male adults of the mesothelan species Ryuthela nishihirai appear to have smaller chelicerae than conspecific female adults and immatures (Fig. 3f). This observation is coherent with the life history of male and female mesothelan spiders: females can live up to 10 or even 20 years, while males die a few weeks after maturation. Therefore, the adult males do not need as efficient preying abilities as the females. Whether male spiders completely stop eating or if they just focus on smaller prey is not known; both would be possible. According to Schwendinger (pers. comm., cited from Foelix and Erb 2010), males of the group Liphistius hardly capture any prey after reaching adulthood. Furthermore, it is still not clear if adult mesothelan males have venom glands. Haupt (2003) claimed that mesothelans in general have no venom glands (in contrast to opisthothelan spiders), but Foelix and Erb (2010) were able to show the opening of the venom glands on the chelicerae in different species of Liphistius as well as the venom gland itself. Yet, the lack of these structures mentioned by Haupt (2003) might be a misinterpretation from investigating only males (see discussion in Foelix and Erb 2010).
Another possible interpretation for smaller chelicerae in adult males is their position next to the pedipalps. Smaller chelicerae could therefore leave more space for the movements of the pedipalps. As male spiders copulate with their pedipalps, this might ease copulation and act as a selection pressure for the reduction of the size of the chelicerae in adult males, independent of the feeding behaviour of the male spiders.
In general, the smaller chelicerae as well as the longer walking appendages in the male mesothelan spiders could be the result of a heterochronic event only affecting males: the female in principle retains the morphology of the late immature stages, while males indeed add a new morphology at the end of their ontogeny in a peramorphic event, most likely representing a case of hypermorphosis (for details on heterochrony, see Webster and Zelditch 2005). The females therefore retain more plesiomorphic morphological characters, while males possess more derived ones. This kind of sexual dimorphism is so far unknown for the supposed close relatives of spiders, namely, whip spiders (Amblypygi) or whip scorpions (Uropygi = Thelyphonida + Schizomida) (together forming the monophyletic group Megoperculata). Yet, at least the longer walking legs in male adults might already be part of the ground pattern of Araneae as it is known also for several mygalomorph spiders (e.g. Grossi et al. 2016).
While it may seem counterintuitive to have heterochronic events only affecting one sex, this is in fact not unusual. In different lineages of beetles (Coleoptera; fire flies, trilobite larvae), the females have become affected by paedomorphosis, in principle retaining larval morphology, while males gain “normal” adult morphology (e.g. Fu et al. 2012). In isopodan crustaceans of the group Gnathiidae, the female adults largely resemble the larval stages with a rather “typical” isopodan-type morphology. In contrast, the adult males have an aberrant morphology with enlarged heads and mandibles (e.g. Wägele 1987).
However, it would be necessary to evaluate the ontogeny of further opisthothelan spiders and representatives of Amblypygi and Uropygi for such morphological changes to achieve a more reliable character polarisation for reliably identifying heterochrony in the evolution of mesothelan spiders.
Spinnerets
The spinnerets of mesothelan spiders arise from the middle of the opisthosoma in contrast to the spinnerets of opisthothelan spiders, which sit at the posterior end of the opisthosoma. Additionally, the spinnerets of mesothelan spiders consist of more elements than in any opisthothelan spider.
Recently, the spider Chimerarachne yingi Wang et al. 2018, from the Cretaceous was found that retained the plesiomorphic “tail” known from other megoperculatans (Wang et al. 2018; see also Huang et al. 2018). The authors state that in this species, the spinnerets are in a more opisthothelan-like position. Yet, comparing the spinnerets position in C. yingi to that in mesothelan spiders reveals that the relative position is in fact almost identical, only the posterior three segments of the fossil species are more slender than in mesothelan spiders. This corroborates that a non-posterior position of the spinnerets is the ancestral condition for spiders, retained in mesothelan spiders (Fig. 6c).
We could not detect any significant changes of the morphology of the spinnerets during ontogeny. This is probably caused by a variability of the overall length, i.e. the elements or ringlets of the spinnerets appear to be able to partly telescope. This would only be possible if there are no joints, i.e. pivot joints, interconnecting these elements.
Whether the spinnerets originated from joint-bearing appendages has been discussed extensively (though without any focus on the presumed loss of the joints; e.g. Jaworowski 1896; Yoshikura 1955; Marples 1967; Shultz 1987; Damen et al. 2002; Hilbrant 2008; Selden et al. 2008; Pechmann and Prpic 2009; Clarke et al. 2015; Sharma 2017; recently reviewed in Mariano-Martins et al. 2020). Also if an evolutionary derivation from such a joint-bearing appendage is assumed, a still open question remains which parts of the appendage the spinnerets correspond to (see Fig. 6 for the morphology of the corresponding appendages in the evolutionary history towards Mesothelae; see also Haug et al. 2013 for the different appendage parts and appendage evolution).
Already earlier in the evolution towards spiders, the appendage derivatives on the corresponding segments did not possess distal appendage parts that have been suggested as the structure of origin (endopod, epipod); hence an origin from such structures for spinnerets is unlikely. Book lungs were ancestrally present on these segments as highly derived appendages (Fig. 6b), the appendage-like appearance of spinnerets is therefore either a de novo phenomenon or a reversal in the sense of a reactivation of an older morphology. In any case, if we want to draw a comparison to appendage derivatives, the lateral spinnerets could well be understood as correlating to exopods at best (Fig. 6c). The median spinnerets could, however, not easily be understood as endopods, as these structures have long been lost in evolutionary history. Also structurally and from developmental timing such an interpretation is not supported. The late appearance after the appearance of the lateral spinnerets (Yoshikura 1955) and the arising from a single membrane together with the lateral spinnerets (at least in different araneomorphan species; Machado 1944) could even be seen as an indication that these are not axial structures, but abaxial ones (see also anterior median spinnerets in Fig. 3D). Median abaxial structures seem to be present in the embryology of early representatives of spiders during their early ontogeny although they are absent in the adults (Pechmann and Prpic 2009).
Sternum
The rather narrow sternum is characteristic of adult mesothelan spiders (Haupt 2003) and unique among spiders. However, our results indicate that this specific sternum morphology is not yet present at hatching and just develops post-embryonically. In all four investigated species, stage I individuals appear to possess a wide sternum which narrows in the subsequent moults. At a certain state the shape of the sternum apparently remains almost the same when the mesothelan spider moults the next time.
Haupt (1986) counted eight stages in the post-embryonic development of Ryuthela nishihirai. As we sorted the sterna according to the length of the prosomal shield of the specimens and there is a variance in size between spiderlings of the same stage, it is possible that one stage is shown two times and another one is missing. Most probably immature stages II–VI (Fig. 5a–f), male and female subadults (Fig. 5 g and h) and adults (Fig. 5 i and j) are represented in the investigated material.
The possible advantages of a narrow sternum are not easy to evaluate. When a mesothelan spider moults, it must enlarge the burrow. By narrowing the sternum, the basipods (termed coxae in arachnid terminology) of the walking legs are positioned more medially on the prosoma. This might relatively “shorten” the legs and could help the animal to fit into the old burrow for a longer time. However, we could not observe large differences in the relative length of the legs of immatures and adult females.
The arachnid ground pattern contains an endosternum, an internal sclerotisation in the prosoma present in xiphosuridans and most representatives of Arachnida (e.g. Snodgrass 1952). All basipodal (“coxal”) muscles are either attached to the endosternum or to the prosomal shield (Shultz 2007). No muscles of the walking legs are attached to the sternum. In Arachnida, the sternum is not primarily involved in the movement of the walking legs (with the possible exception in Araneomorpha, see Runge and Wirkner 2019). It is passively used to increase the hydrostatic pressure, which acts like an extensor muscle; the latter is missing in the ground pattern of Arachnida (Wilson and Bullock 1973; Stewart and Martin 1974; Anderson and Prestwich 1975; Prestwich 1988; Paul et al. 1989; Shultz 2007). Therefore, the shape of the sternum appears to have only little impact on the locomotion of mesothelan spiders.
Hence, the mechanical consequences of a narrow sternum are not clear, but the development of the sternum in mesothelan spiders could shed some light on the evolution of early spiders. Opisthothelan spiders do not have a narrow sternum, neither as an immature nor as an adult. An outgroup comparison is necessary to shed light on the evolution of the sternum shape. One presumed sister group of Araneae is Amblypygi (Fig. 7, e.g. Paulus 2004 and references therein, though the internal relationships of Megoperculata are not settled yet; see above). Yet, the sternum of Amblypygi is quite different from that of Araneae. It is very small, about as long as broad, and has a long, anteriorly pointing spine, the so-called tritosternum (e.g. Shultz 1999). Hence, Araneae appear to have evolved a larger sternum compared to Amblypygi. Considering the character conditions, there are two possibilities for the evolution of the sternum in Araneae:
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1)
The narrow sternum already exists in the ground pattern of Araneae. Opisthothelae then developed a broader sternum. This could have happened through a heterochronic (in this case paedomorphic) event, resulting in a broader sternum in adult opisthothelans, resembling the condition in immature mesothelans.
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2)
A broad sternum is part of the ground pattern of Araneae, plesiomorphically retained from the last common ancestor of Amblypygi + Araneae. The narrow sternum is apomorphic for Mesothelae and evolved through a heterochronic (in this case peramorphic) event. Opisthothelae retained the ancestral, broader shape of the sternum.
However, the information about the ancestral condition of Amblypygi + Araneae is insufficient in this aspect. The difference in sternum morphology between Araneae and Amblypygi is too large to allow clear conclusions about the possible evolution of the sternum in araneaens.
Also the new fossils from the Cretaceous are inconclusive in this aspect. In the specimens shown in Wang et al. (2018), the sternum is not well preserved. In the specimens presented by Huang et al. (2018), the sternum is in fact different from that of both opisthothelan and mesothelan spiders. It appears narrower than that of opisthothelan spiders, yet not as narrow as in mesothelan spiders. Also, the mesothelan sternum has quite some distance to the basipods of the walking legs, which is not the case in the fossils. While in opisthothelan spiders the sternum appears to be also in closer contact to the basipods (especially in Araneomorpha, Runge and Wirkner 2019), the sternum shows distinct indents corresponding to the insertions of the legs. No such indents are apparent in the fossils. An ideal solution for the complicated situation would be finding fossils that allow the reconstruction of an ontogenetic series of tailed spiders.