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.

Facilitating taxonomy and phylogenetics: An informative and cost-effective protocol integrating long amplicon PCRs and third-generation sequencing.

Phylogenetic inference has become a standard technique in integrative taxonomy and systematics, as well as in biogeography and ecology. DNA barcodes are often used for phylogenetic inference, despite being strongly limited due to their low number of informative sites. Also, because current DNA barcodes are based on a fraction of a single, fast-evolving gene, they are highly unsuitable for resolving deeper phylogenetic relationships due to saturation. In recent years, methods that analyse hundreds and thousands of loci at once have improved the resolution of the Tree of Life, but these methods require resources, experience and molecular laboratories that most taxonomists do not have. This paper introduces a PCR-based protocol that produces long amplicons of both slow- and fast-evolving unlinked mitochondrial and nuclear gene regions, which can be sequenced by the affordable and portable ONT MinION platform with low infrastructure or funding requirements. As a proof of concept, we inferred a phylogeny of a sample of 63 spider species from 20 families using our proposed protocol. The results were overall consistent with the results from approaches based on hundreds and thousands of loci, while requiring just a fraction of the cost and labour of such approaches, making our protocol accessible to taxonomists worldwide.

Dynamic evolution of locomotor performance independent of changes in extended phenotype use in spiders.

Many animals use self-built structures (extended phenotypes) to enhance body functions, such as thermoregulation, prey capture or defence. Yet, it is unclear whether the evolution of animal constructions supplements or substitutes body functions-with disparate feedbacks on trait evolution. Here, using brown spiders (Araneae: marronoid clade), we explored if the evolutionary loss and gain of silken webs as extended prey capture devices correlates with alterations in traits known to play an important role in predatory strikes-locomotor performance (sprint speed) and leg spination (expression of capture spines on front legs). We found that in this group high locomotor performance, with running speeds of over 100 body lengths per second, evolved repeatedly-both in web-building and cursorial spiders. There was no correlation with running speed, and leg spination only poorly correlated, relative to the use of extended phenotypes, indicating that web use does not reduce selective pressures on body functions involved in prey capture and defence per se. Consequently, extended prey capture devices serve as supplements rather than substitutions to body traits and may only be beneficial in conjunction with certain life-history traits, possibly explaining the rare evolution and repeated loss of trapping strategies in predatory animals.

Geometric regularity in webs of non-orb-weaving spiders.

Geometric regularity of spider webs has been intensively studied in orb-weaving spiders, although it is not exclusive of orb weavers. Here, we document the geometrically regular, repetitive elements in the webs of the non-orb-weaving groups Leptonetidae and Telemidae for the first time. Similar to orb weavers, we found areas with regularly spaced parallel lines in the webs of Calileptoneta helferi, Sulcia sp., and cf. Pinelema sp. Furthermore, we provide a detailed account of the regular webs of Ochyrocera (Ochyroceratidae). The sections of the web with regularly disposed parallel lines are built as U-shaped modules reminiscent of orb webs. It has been suggested that the regularly spaced parallel lines in the webs of Ochyroceratidae and Psilodercidae may be produced in a single sweep of their posterior lateral spinnerets, which have regularly spaced aciniform gland spigots, perhaps involving expansion of the spinnerets. To test this hypothesis, we compared the spacing between parallel lines with the spacing between spigots, searched for expansible membranes in the spinnerets, and examined the junctions of regularly spaced lines. The distance between parallel lines was 10-20 times the distance between spigots, and we found no expansible membranes, and the intersection of parallel lines are cemented, which opposes the single sweep hypothesis. Furthermore, we found cues of viscid silk in the parallel lines of the psilodercid Althepus and broadened piriform gland spigots that may be responsible of its production. Finally, we evaluated the presence or absence of geometrically regular web elements across the spider tree of life. We found reports of regular webs in 31 spider families, including 20 families that are not orb weavers and hypothesize that the two basic aspects of regularity (parallel lines spaced at regular intervals, and radial lines spaced at regular angles) probably appeared many times in the evolution of spiders.

Fast acrobatic maneuvers enable arboreal spiders to hunt dangerous prey.

Spiders, the most specious taxon of predators, have evolved an astounding range of predatory strategies, including group hunting, specialized silk traps, pheromone-loaded bolas, and aggressive mimicry. Spiders that hunt prey defended with behavioral, mechanical, or chemical means are under additional selection pressure to avoid injury and death. Ants are considered dangerous because they can harm or kill their predators, but some groups of spiders, such as the Theridiidae, have a very high diversification of ant-hunting species and strategies [J. Liu et al., Mol. Phylogenet. Evol. 94, 658-675 (2016)]. Here, we provide detailed behavioral analyses of the highly acrobatic Australian ant-slayer spider, Euryopis umbilicata (Theridiidae), that captures much larger and defended Camponotus ants on vertical tree trunks. The hunting sequence consists of ritualized steps performed within split seconds, resulting in an exceptionally high prey capture success rate.

AnimalTraits - a curated animal trait database for body mass, metabolic rate and brain size.

Trait databases have become important resources for large-scale comparative studies in ecology and evolution. Here we introduce the AnimalTraits database, a curated database of body mass, metabolic rate and brain size, in standardised units, for terrestrial animals. The database has broad taxonomic breadth, including tetrapods, arthropods, molluscs and annelids from almost 2000 species and 1000 genera. All data recorded in the database are sourced from their original empirical publication, and the original metrics and measurements are included with each record. This allows for subsequent data transformations as required. We have included rich metadata to allow users to filter the dataset. The additional R scripts we provide will assist researchers with aggregating standardised observations into species-level trait values. Our goals are to provide this resource without restrictions, to keep the AnimalTraits database current, and to grow the number of relevant traits in the future.

Stabilized Morphological Evolution of Spiders Despite Mosaic Changes in Foraging Ecology.

A prominent question in animal research is how the evolution of morphology and ecology interacts in the generation of phenotypic diversity. Spiders are some of the most abundant arthropod predators in terrestrial ecosystems and exhibit a diversity of foraging styles. It remains unclear how spider body size and proportions relate to foraging style, and if the use of webs as prey capture devices correlates with changes in body characteristics. Here, we present the most extensive data set to date of morphometric and ecological traits in spiders. We used this data set to estimate the change in spider body sizes and shapes over deep time and to test if and how spider phenotypes are correlated with their behavioral ecology. We found that phylogenetic variation of most traits best fitted an Ornstein-Uhlenbeck model, which is a model of stabilizing selection. A prominent exception was body length, whose evolutionary dynamics were best explained with a Brownian Motion (free trait diffusion) model. This was most expressed in the araneoid clade (ecribellate orb-weaving spiders and allies) that showed bimodal trends toward either miniaturization or gigantism. Only few traits differed significantly between ecological guilds, most prominently leg length and thickness, and although a multivariate framework found general differences in traits among ecological guilds, it was not possible to unequivocally associate a set of morphometric traits with the relative ecological mode. Long, thin legs have often evolved with aerial webs and a hanging (suspended) locomotion style, but this trend is not general. Eye size and fang length did not differ between ecological guilds, rejecting the hypothesis that webs reduce the need for visual cue recognition and prey immobilization. For the inference of the ecology of species with unknown behaviors, we propose not to use morphometric traits, but rather consult (micro-)morphological characters, such as the presence of certain podal structures. These results suggest that, in contrast to insects, the evolution of body proportions in spiders is unusually stabilized and ecological adaptations are dominantly realized by behavioral traits and extended phenotypes in this group of predators. This work demonstrates the power of combining recent advances in phylogenomics with trait-based approaches to better understand global functional diversity patterns through space and time. [Animal architecture; Arachnida; Araneae; extended phenotype; functional traits; macroevolution; stabilizing selection.].

Cerotegument microstructure of whip spiders (Amblypygi: Euamblypygi Weygoldt, 1996) reveals characters for systematics from family to species level.

Like other arthropods, whip spiders of the arachnid order Amblypygi Thorell, 1883 protect themselves against external environmental influences. In this taxon, in addition to the epicuticle, the outermost layer of the exoskeleton, a cement layer (cerotegument) with superhydrophobic properties is deposited over certain body parts. Due to the high level of interspecific variation, the cerotegument structure and the morphology of its associated gland openings have been suggested to be informative for whip spider systematics. The first comparative study of the cerotegument is presented herein, based on a survey across 4 families, 16 genera, and 62 species of Euamblypygi Weygoldt, 1996, the suborder comprising all extant whip spiders except the rare monotypic family Paracharontidae Weygoldt, 1996. Results confirmed that the morphology of the colloidal particles and their assembly on cement globules differ considerably among taxa, but that the level of variation differs among lineages. Interspecific variation in cerotegument ultrastructure was highest among species of Neoamblypygi Weygoldt, 1996, making it an informative character in this clade. Evolutionary trends and intraspecific variation in the structure of the amblypygid cerotegument are briefly discussed.

Nutritionally induced nanoscale variations in spider silk structural and mechanical properties.

Spider major ampullate (MA) silk is characterized by high strength and toughness and is adaptable across environments. Experiments depriving spiders of protein have enabled researchers to examine nutritionally induced changes in gene expression, protein structures, and bulk properties of MA silk. However, it has not been elucidated if it varies in a similar way at a nanoscale. Here we used Atomic Force Microscopy (AFM) to simultaneously examine the topographic, structural, and mechanical properties of silks spun by two species of spider, Argiope keyserlingi and Latrodectus hasselti, at a nanoscale when protein fed or deprived. We found height, a measure of localized width, to substantially vary across species and treatments. We also found that Young's modulus, which may be used as an estimate of localized stiffness, decreased with protein deprivation in both species' silk. Our results suggest that nanoscale skin-core structures of A. keyserlingi's MA silk varied significantly across treatments, whereas only slight structural and functional variability was found for L. hasselti's silk. These results largely agreed with examinations of the bulk properties of each species' silk. However, we could not directly attribute the decoupling between protein structures and bulk mechanics in L. hasselti's silk to nanoscale features. Our results advance the understanding of processes inducing skin and core structural variations in spider silks at a nanoscale, which serves to enhance the prospect of developing biomimetic engineering programs.

The World Spider Trait database: a centralized global open repository for curated data on spider traits.

Spiders are a highly diversified group of arthropods and play an important role in terrestrial ecosystems as ubiquitous predators, which makes them a suitable group to test a variety of eco-evolutionary hypotheses. For this purpose, knowledge of a diverse range of species traits is required. Until now, data on spider traits have been scattered across thousands of publications produced for over two centuries and written in diverse languages. To facilitate access to such data, we developed an online database for archiving and accessing spider traits at a global scale. The database has been designed to accommodate a great variety of traits (e.g. ecological, behavioural and morphological) measured at individual, species or higher taxonomic levels. Records are accompanied by extensive metadata (e.g. location and method). The database is curated by an expert team, regularly updated and open to any user. A future goal of the growing database is to include all published and unpublished data on spider traits provided by experts worldwide and to facilitate broad cross-taxon assays in functional ecology and comparative biology. Database URL:https://spidertraits.sci.muni.cz/.

Robust substrate anchorages of silk lines with extensible nano-fibres.

Living systems are built of multiscale-composites: materials formed of components with different properties that are assembled in complex micro- and nano-structures. Such biological multiscale-composites often show outstanding physical properties that are unachieved by artificial materials. A major scientific goal is thus to understand the assembly processes and the relationship between structure and function in order to reproduce them in a new generation of biomimetic high-performance materials. Here, we tested how the assembly of spider silk nano-fibres (i.e. glue coated 0.5 µm thick fibres produced by so-called piriform glands) into different micro-structures correlates with mechanical performance by empirically and numerically exploring the mechanical behaviour of line anchors in an orb weaver, a hunting spider and two ancient web builders. We demonstrate that the anchors of orb weavers exhibit outstanding mechanical robustness with minimal material use by the indirect attachment of the silk line to the substrate through a soft domain ('bridge'). This principle can be used to design new artificial high-performance attachment systems.

Building behavior does not drive rates of phenotypic evolution in spiders.

Do animals set the course for the evolution of their lineage when manipulating their environment? This heavily disputed question is empirically unexplored but critical to interpret phenotypic diversity. Here, we tested whether the macroevolutionary rates of body morphology correlate with the use of built artifacts in a megadiverse clade comprising builders and nonbuilders-spiders. By separating the inferred building-dependent rates from background effects, we found that variation in the evolution of morphology is poorly explained by artifact use. Thus natural selection acting directly on body morphology rather than indirectly via construction behavior is the dominant driver of phenotypic diversity.

Fine structure of the epicuticular secretion coat and associated glands of Pedipalpi and Palpigradi (Arachnida).

Pedipalpi Latreille, 1810 is a poorly studied clade of arachnids comprising the whip spiders (Amblypygi Thorell, 1883), short-tailed whip scorpions (Schizomida Petrunkevitch, 1945) and whip scorpions (Thelyphonida Cambridge, 1872). It has recently been shown that whip spiders coat their exoskeleton with a solid cement layer (cerotegument) that forms elaborate microstructures and turns the cuticle into a super-hydrophobic state. The amblypygid cerotegument provides taxonomic information due to its fine structural diversity, but its presence and variation in the sister groups was previously unknown. The present contribution reports the surface structure of the cuticle in species of Palpigradi, Thelyphonida, and Schizomida to determine if these taxa possess a solid epicuticular secretion coat. Scanning electron microscopy revealed that in addition to Amblypygi only species of Thelyphonida possess solid epicuticular secretion layers. Unlike in Amblypygi, in the Thelyphonida this layer does not usually form microstructures and is less rigidly attached to the underlying cuticle. A species of Typopeltis Pocock, 1894, which exhibited globular structures analogous to the amblypygid cerotegument, was an exception. Glandular structures associated with cement secretions in Amblypygi and Thelyphonida were considered homologous due to similar structure. Solid epicuticular secretion coats were absent from Schizomida, which is interpreted as a secondary loss despite the presence of slit-like glandular openings that appear to produce such epicuticular secretions. The micro-whip scorpion order Palpigradi Thorell, 1900 exhibited markedly different cuticular surface structures and lacked solid epicuticular secretions, consistent with the hypothesis that this order is not closely related to Pedipalpi. These results enhance the knowledge of the small, enigmatic orders of Arachnida.

Locomotion and kinematics of arachnids.

A basic feature of animals is the capability to move and disperse. Arachnids are one of the oldest lineages of terrestrial animals and characterized by an octopodal locomotor apparatus with hydraulic limb extension. Their locomotion repertoire includes running, climbing, jumping, but also swimming, diving, abseiling, rolling, gliding and -passively- even flying. Studying the unique locomotor functions and movement ecology of arachnids is important for an integrative understanding of the ecology and evolution of this diverse and ubiquitous animal group. Beyond biology, arachnid locomotion is inspiring robotic engineers. The aim of this special issue is to display the state of the interdisciplinary research on arachnid locomotion, linking physiology and biomechanics with ecology, ethology and evolutionary biology. It comprises five reviews and ten original research reports covering diverse topics, ranging from the neurophysiology of arachnid movement, the allometry and sexual dimorphism of running kinematics, the effect of autotomy or heavy body parts on locomotor efficiency, and the evolution of silk-spinning choreography, to the biophysics of ballooning and ballistic webs. This closes a significant gap in the literature on animal biomechanics.

Evolution of Silk Anchor Structure as the Joint Effect of Spinning Behavior and Spinneret Morphology.

Spider web anchors are attachment structures composed of the bi-phasic glue-fiber secretion from the piriform silk glands. The mechanical performance of the anchors strongly correlates with the structural assembly of the silk lines, which makes spider silk anchors an ideal system to study the biomechanical function of extended phenotypes and its evolution. It was proposed that silk anchor function guided the evolution of spider web architectures, but its fine-structural variation and whether its evolution was rather determined by changes of the shape of the spinneret tip or in the innate spinning choreography remained unresolved. Here, we comparatively studied the micro-structure of silk anchors across the spider tree of life, and set it in relation to spinneret morphology, spinning behavior and the ecology of the spider. We identified a number of apomorphies in the structure of silk anchors that may positively affect anchor function: (1) bundled dragline, (2) dragline envelope, and (3) dragline suspension ("bridge"). All these characters were apomorphic and evolved repeatedly in multiple lineages, supporting the notion that they are adaptive. The occurrence of these structural features can be explained with changes in the shape and mobility of the spinneret tip, the spinning behavior, or both. Spinneret shapes generally varied less than their fine-tuned movements, indicating that changes in construction behavior play a more important role in the evolution of silk anchor assembly. However, the morphology of the spinning apparatus is also a major constraint to the evolution of the spinning choreography. These results highlight the changes in behavior as the proximate and in morphology as the ultimate causes of extended phenotype evolution. Further, this research provides a roadmap for future bioprospecting research to design high-performance instant line anchors.

Evolutionary kinematics of spinneret movements for rapid silk thread anchorage in spiders.

Many organisms secrete structural materials from their bodies to enhance protection, foraging or signalling. The function of such secretion products can be further extended by their assembly into complex structures, so-called extended phenotypes, such as shells, nests and biofilms. Understanding the variation in the efficacy of such assembly processes could help to explain why extended phenotypes are common on some lineages and rare in others. Here, I comparatively studied the assembly of sticky silk fibres into thread anchorages by the innate 'printing' behaviour in 92 species of spiders from 45 families, representing the so-far largest comparative study of construction-related motion patterns. I found a global evolutionary trend towards a faster production of silk thread anchorages, in both web builders and hunting spiders. The slowest producers of silk anchors belong to a clade with an ancestral configuration of respiratory organs, suggesting that a major constraint to the evolution of spinning speed is the efficiency of oxygen uptake. Motion patterns were found to contain a high phylogenetic signal, but did not correlate with spinning speeds. These results help to explain the variation in diversity and ecological success among the spider fauna and showcase the value of comparative kinematics in biodiversity studies.

Limits of piriform silk adhesion-similar effects of substrate surface polarity on silk anchor performance in two spider species with disparate microhabitat use.

It has been suggested that physical interactions between biological and environmental surfaces may constrain ecological niche spaces. However, the mechanistic understanding of niche formation is frequently limited by the lack of information on the function and variation of these interactions. Here, we hypothesised that two closely related species of orb-web spiders have evolved different adhesion performance of web attachment (i.e. piriform silk) facilitating the occupation of contrasting microhabitats: plants versus rocks. Contrary to our prediction, we found that piriform silk adhesion was equally affected by surface chemistry in both species, with maximal adhesion on surfaces with high surface polarity and an average adhesion loss of 70-75% on low polar surfaces. Spiders did not respond to adhesion losses by increasing the anchor size, despite the repeated failure to attach their web to low polar surfaces. In a natural setting, poor adhesion on low polar surfaces may be mitigated by behavioural means, like the preference to place anchors on corrugated surface features such as leaf edges, or the spinning of multiple anchorages and formation of a bundled anchor line. Thus, microhabitat choice for web-building spiders may be governed by structural properties rather than surface chemistry. These results suggest that the repeatedly demonstrated effects of surface chemistry on bio-adhesion may be ecologically less important than assumed and that the role of behaviour in the evolution of bio-adhesion performance has been underestimated.

Evolution of aerial spider webs coincided with repeated structural optimization of silk anchorages.

Physical structures built by animals challenge our understanding of biological processes and inspire the development of smart materials and green architecture. It is thus indispensable to understand the drivers, constraints, and dynamics that lead to the emergence and modification of building behavior. Here, we demonstrate that spider web diversification repeatedly followed strikingly similar evolutionary trajectories, guided by physical constraints. We found that the evolution of suspended webs that intercept flying prey coincided with small changes in silk anchoring behavior with considerable effects on the robustness of web attachment. The use of nanofiber based capture threads (cribellate silk) conflicts with the behavioral enhancement of web attachment, and the repeated loss of this trait was frequently followed by physical improvements of web anchor structure. These findings suggest that the evolution of building behavior may be constrained by major physical traits limiting its role in rapid adaptation to a changing environment.

Amblypygid-fungal interactions: The whip spider exoskeleton as a substrate for fungal growth.

Fungi and arthropods represent some of the most diverse organisms on our planet, yet the ecological relationships between them remain largely unknown. In animals, fungal growth on body surfaces is often hazardous and is known to cause mortality. In contrast, here we report the presence of an apparently non-harmful mycobiome on the cuticle of whip spiders (Arachnida: Amblypygi). The associations are not species-specific and involve a diversity of fungal species, including cosmopolitan and local decomposers as well as entomopathogens. We discuss the ecology of the detected fungal species and hypothesize that the thick epicuticular secretion coat of whip spiders (the cerotegument) promotes fungal growth. It is possible that this relationship is beneficial towards the host if it leads to parasite control or chemical camouflage. Our findings, which are the first from this arthropod lineage, indicate that non-pathogenic interactions between arthropods and fungi may be much more widespread than predicted and call for more studies in this area.

Zooplankters' nightmare: The fast and efficient catching basket of larval phantom midges (Diptera: Chaoborus).

Filter feeding zooplankton are a crucial component of limnic food webs. Copepods and cladocerans are important prey organisms for first-level predators like the common and abundant larvae of phantom midges (Chaoborus sp.). The latter possess a complex catching basket built of head appendages specialized to capture small crustaceans. The predator-prey-relationship of Chaoborus (Diptera, Nematocera) and Daphnia (Crustacea, Cladocera) has been studied in particular detail owing to the daphniids' ability to react upon the threat of predation with inducible defenses. Daphnia pulex expresses so-called 'neckteeth' in the presence of Chaoborus larvae that are discussed as a defensive trait that interferes with the larval head appendages and their effectiveness has been shown in several studies. Nonetheless, mode of function of these neckteeth is not understood and the hypothesis that they interfere with the predator's head appendages still has to be confirmed. To clarify the role of neckteeth in Daphnia, an understanding of the Chaoborus capture apparatus is essential. Here, we present a detailed three-dimensional analysis of Chaoborus obscuripes' larval head morphology as well as a kinematic analysis of the attack motion, which revealed an impressive strike velocity (14 ms to prey contact). The movement of the larvae's head appendages is reconstructed in the three-dimensional space using a combination of high-speed videography, micro-computed tomography and computer animation. Furthermore, we provide predation trial data to distinguish between pre- and post-attack defensive effects in D. pulex. Our findings suggest a combination of pre- and post-attack defenses with an average effectiveness of 50% each. With this study, we quantitatively describe prey capture kinematics of C. obscuripes and take a further step to reveal the neckteeth' mode of function in D. pulex.