Increasing Information Content and Diagnosability in Family-Level Classifications.

Higher-level classifications often must account for monotypic taxa representing depauperate evolutionary lineages and lacking synapomorphies of their better-known, well-defined sister clades. In a ranked (Linnean) or unranked (phylogenetic) classification system, discovering such a depauperate taxon does not necessarily invalidate the rank classification of sister clades. Named higher taxa must be monophyletic to be phylogenetically valid. Ranked taxa above the species level should also maximize information content, diagnosability, and utility (e.g., in biodiversity conservation). In spider classification, families are the highest rank that is systematically catalogued, and incertae sedis is not allowed. Consequently, it is important that family-level taxa be well defined and informative. We revisit the classification problem of Orbipurae, an unranked suprafamilial clade containing the spider families Nephilidae, Phonognathidae, and Araneidae sensu stricto. We argue that, to maximize diagnosability, information content, conservation utility, and practical taxonomic considerations, this "splitting" scheme is superior to its recently proposed alternative, which lumps these families together as Araneidae sensu lato. We propose to redefine Araneidae and recognize a monogeneric spider family, Paraplectanoididae fam. nov. to accommodate the depauperate lineage Paraplectanoides. We present new subgenomic data to stabilize Orbipurae topology which also supports our proposed family-level classification. Our example from spiders demonstrates why classifications must be able to accommodate depauperate evolutionary lineages, for example, Paraplectanoides. Finally, although clade age should not be a criterion to determine rank, other things being equal, comparable ages of similarly ranked taxa do benefit comparative biology. [Classification, family rank, phylogenomics, systematics, monophyly, spider phylogeny.].

Adherence to COVID-19 mitigation measures: The role of sociodemographic and personality factors.

The current COVID-19 pandemic caught the decision makers in many countries sub-optimally prepared to respond. To better cope with similar situations in the future, it is vital to understand the major predictors of health-beneficial behavior and adherence to imposed mitigation measures and guidelines. To tailor the promotion of government-imposed measures, it is important to understand how the sociodemographic background combined with personality traits affect the perception and responsiveness of people. We investigated the perception and adherence to mitigation measures during the pandemic by examining their trends across several sociodemographic categories and personality dimensions. The strongest predictors of confidence in the preventive measures and their implementation were the participants' concern of infection and concern of infecting their loved ones, followed by gender and age. Education, settlement size, field and type of employment, household type, own medical problems, and the age and health of the participants' loved ones had a smaller influence on the perceived guidelines importance and implementation. Adherence to measures was positively related to the participants' score in conscientiousness and, in lesser extent, openness. Agreeableness, energy, and emotional stability correlated positively with adherence to basic guidelines. Study provides information useful for developing and adapting future public health policies and interventions. Supplementary Information: The online version contains supplementary material available at 10.1007/s12144-021-02051-5.

Global Diversification of Anelosimus Spiders Driven by Long-Distance Overwater Dispersal and Neogene Climate Oscillations.

Vicariance and dispersal events, combined with intricate global climatic history, have left an imprint on the spatiotemporal distribution and diversity of many organisms. Anelosimus cobweb spiders (Theridiidae), are organisms ranging in behavior from solitary to highly social, with a cosmopolitan distribution in temperate to tropical areas. Their evolutionary history and the discontinuous distribution of species richness suggest that 1) long-distance overwater dispersal and 2) climate change during the Neogene (23-2.6 Ma), may be major factors in explaining their distribution and diversification. Here, we test these hypotheses, and explicitly test if global Miocene/Pliocene climatic cooling in the last 8 Ma affected Anelosimus radiation in parallel in South America and Madagascar. To do so, we investigate the phylogeny and spatiotemporal biogeography of Anelosimus through a culmination of a 20-year comprehensive global sampling at the species level (69 species, including 84% of the known 75 species worldwide, represented by 268 individuals) using nucleotide data from seven loci (5.5 kb). Our results strongly support the monophyly of Anelosimus with an Oligocene ($\sim $30 Ma) South American origin. Major clades on other continents originate via multiple, long-distance dispersal events, of solitary or subsocial-but not social-lineages, from the Americas. These intercontinental dispersals were to Africa, Madagascar (twice), and SE Asia/Australasia. The early diversification of Anelosimus spiders coincides with a sudden thermal increase in the late Oligocene ($\sim $27-25 Ma), though no causal connection can be made. Our results, however, strongly support the hypothesis that global Neogene climatic cooling in the last 8 Ma drove Anelosimus radiation in parallel in South America and Madagascar, offering a rare empirical evidence for diversification of a socially diverse group driven by an interplay between long-distance dispersal and global Neogene climatic changes. [Cobweb spiders; diversification; global biogeography; long-distance dispersal; molecular phylogenetics; neogene climate changes; sociality; vicariance.].

The jumping spider Saitis barbipes lacks a red photoreceptor to see its own sexually dimorphic red coloration.

Examining the role of color in mate choice without testing what colors the study animal is capable of seeing can lead to ill-posed hypotheses and erroneous conclusions. Here, we test the seemingly reasonable assumption that the sexually dimorphic red coloration of the male jumping spider Saitis barbipes is distinguishable, by females, from adjacent black color patches. Using microspectrophotometry, we find clear evidence for photoreceptor classes with maximal sensitivity in the UV (359 nm) and green (526 nm), inconclusive evidence for a photoreceptor maximally sensitive in the blue (451 nm), and no evidence for a red photoreceptor. No colored filters within the lens or retina could be found to shift green sensitivity to red. To quantify and visualize whether females may nevertheless be capable of discriminating red from black color patches, we take multispectral images of males and calculate photoreceptor excitations and color contrasts between color patches. Red patches would be, at best, barely discriminable from black, and not discriminable from a low-luminance green. Some color patches that appear achromatic to human eyes, such as beige and white, strongly absorb UV wavelengths and would appear as brighter "spider-greens" to S. barbipes than the red color patches. Unexpectedly, we discover an iridescent UV patch that contrasts strongly with the UV-absorbing surfaces dominating the rest of the spider. We propose that red and black coloration may serve identical purposes in sexual signaling, functioning to generate strong achromatic contrast with the visual background. The potential functional significance of red coloration outside of sexual signaling is discussed.

Sex-specific developmental trajectories in an extremely sexually size dimorphic spider.

Adult body size, development time, and growth rates are components of organismal life histories, which crucially influence fitness and are subject to trade-offs. If selection is sex-specific, male and female developments can eventually lead to different optimal sizes. This can be achieved through developmental plasticity and sex-specific developmental trajectories. Spiders present suitable animals to study differences in developmental plasticity and life history trade-offs between the sexes, because of their pronounced sexual dimorphism. Here, we examine variation in life histories in the extremely sexually size dimorphic African hermit spider (Nephilingis cruentata) reared under standardized laboratory conditions. Females average 70 times greater body mass (and greater body size) at maturity than males, which they achieve by developing longer and growing faster. We find a small to moderate amount of variability in life history traits to be caused by family effects, comprising genetic, maternal, and early common environmental effects, suggesting considerable plasticity in life histories. Remarkably, family effects explain a higher variance in male compared to female life histories, implying that female developmental trajectories may be more responsive to environment. We also find sex differences in life history trade-offs and show that males with longer development times grow larger but exhibit shorter adult longevity. Female developmental time also correlates positively with adult body mass, but the trade-offs between female adult mass, reproduction, and longevity are less clear. We discuss the implications of these findings in the light of evolutionary trade-offs between life history traits.

Golden Orbweavers Ignore Biological Rules: Phylogenomic and Comparative Analyses Unravel a Complex Evolution of Sexual Size Dimorphism.

Instances of sexual size dimorphism (SSD) provide the context for rigorous tests of biological rules of size evolution, such as Cope's rule (phyletic size increase), Rensch's rule (allometric patterns of male and female size), as well as male and female body size optima. In certain spider groups, such as the golden orbweavers (Nephilidae), extreme female-biased SSD (eSSD, female:male body length $\ge$2) is the norm. Nephilid genera construct webs of exaggerated proportions, which can be aerial, arboricolous, or intermediate (hybrid). First, we established the backbone phylogeny of Nephilidae using 367 anchored hybrid enrichment markers, then combined these data with classical markers for a reference species-level phylogeny. Second, we used the phylogeny to test Cope and Rensch's rules, sex specific size optima, and the coevolution of web size, type, and features with female and male body size and their ratio, SSD. Male, but not female, size increases significantly over time, and refutes Cope's rule. Allometric analyses reject the converse, Rensch's rule. Male and female body sizes are uncorrelated. Female size evolution is random, but males evolve toward an optimum size (3.2-4.9 mm). Overall, female body size correlates positively with absolute web size. However, intermediate sized females build the largest webs (of the hybrid type), giant female Nephila and Trichonephila build smaller webs (of the aerial type), and the smallest females build the smallest webs (of the arboricolous type). We propose taxonomic changes based on the criteria of clade age, monophyly and exclusivity, classification information content, and diagnosability. Spider families, as currently defined, tend to be between 37 million years old and 98 million years old, and Nephilidae is estimated at 133 Ma (97-146), thus deserving family status. We, therefore, resurrect the family Nephilidae Simon 1894 that contains Clitaetra Simon 1889, the Cretaceous GeratonephilaPoinar and Buckley (2012), Herennia Thorell 1877, IndoetraKuntner 2006, new rank, Nephila Leach 1815, Nephilengys L. Koch 1872, Nephilingis Kuntner 2013, Palaeonephila Wunderlich 2004 from Tertiary Baltic amber, and TrichonephilaDahl 1911, new rank. We propose the new clade Orbipurae to contain Araneidae Clerck 1757, Phonognathidae Simon 1894, new rank, and Nephilidae. Nephilid female gigantism is a phylogenetically ancient phenotype (over 100 Ma), as is eSSD, though their magnitudes vary by lineage.

Optimal foraging, not biogenetic law, predicts spider orb web allometry.

The biogenetic law posits that the ontogeny of an organism recapitulates the pattern of evolutionary changes. Morphological evidence has offered some support for, but also considerable evidence against, the hypothesis. However, biogenetic law in behavior remains underexplored. As physical manifestation of behavior, spider webs offer an interesting model for the study of ontogenetic behavioral changes. In orb-weaving spiders, web symmetry often gets distorted through ontogeny, and these changes have been interpreted to reflect the biogenetic law. Here, we test the biogenetic law hypothesis against the alternative, the optimal foraging hypothesis, by studying the allometry in Leucauge venusta orb webs. These webs range in inclination from vertical through tilted to horizontal; biogenetic law predicts that allometry relates to ontogenetic stage, whereas optimal foraging predicts that allometry relates to gravity. Specifically, pronounced asymmetry should only be seen in vertical webs under optimal foraging theory. We show that, through ontogeny, vertical webs in L. venusta become more asymmetrical in contrast to tilted and horizontal webs. Biogenetic law thus cannot explain L. venusta web allometry, but our results instead support optimization of foraging area in response to spider size.

Spider webs as eDNA samplers: Biodiversity assessment across the tree of life.

The concept of environmental DNA (eDNA) utilizes nucleic acids of organisms directly from the environment. Recent breakthrough studies have successfully detected a wide spectrum of prokaryotic and eukaryotic eDNA from a variety of environments, ranging from ancient to modern, and from terrestrial to aquatic. With their diversity and ubiquity in nature, spider webs might act as powerful biofilters and could thus represent a promising new source of eDNA, but their utility under natural field conditions is severely understudied. Here, we bridge this knowledge gap to establish spider webs as a source of eDNA with far reaching implications. First, we conducted a field study to track specific arthropod targets from different spider webs. We then used high-throughput amplicon sequencing of taxonomic barcodes to investigate the utility of spider web eDNA for biodiversity monitoring of animals, fungi and bacteria. Our results show that genetic remains on spider webs allow the detection of even the smallest target organisms. We also demonstrate that eDNA from spider webs is useful in research of community compositions across the different domains of life, with potentially highly detailed temporal and spatial information.

Characterization of the genome and silk-gland transcriptomes of Darwin's bark spider (Caerostris darwini).

Natural silks crafted by spiders comprise some of the most versatile materials known. Artificial silks-based on the sequences of their natural brethren-replicate some desirable biophysical properties and are increasingly utilized in commercial and medical applications today. To characterize the repertoire of protein sequences giving silks their biophysical properties and to determine the set of expressed genes across each unique silk gland contributing to the formation of natural silks, we report here draft genomic and transcriptomic assemblies of Darwin's bark spider, Caerostris darwini, an orb-weaving spider whose dragline is one of the toughest known biomaterials on Earth. We identify at least 31 putative spidroin genes, with expansion of multiple spidroin gene classes relative to the golden orb-weaver, Trichonephila clavipes. We observed substantial sharing of spidroin repetitive sequence motifs between species as well as new motifs unique to C. darwini. Comparative gene expression analyses across six silk gland isolates in females plus a composite isolate of all silk glands in males demonstrated gland and sex-specific expression of spidroins, facilitating putative assignment of novel spidroin genes to classes. Broad expression of spidroins across silk gland types suggests that silks emanating from a given gland represent composite materials to a greater extent than previously appreciated. We hypothesize that the extraordinary toughness of C. darwini major ampullate dragline silk may relate to the unique protein composition of major ampullate spidroins, combined with the relatively high expression of stretchy flagelliform spidroins whose union into a single fiber may be aided by novel motifs and cassettes that act as molecule-binding helices. Our assemblies extend the catalog of sequences and sets of expressed genes that confer the unique biophysical properties observed in natural silks.

Body Size, Not Personality, Explains Both Male Mating Success and Sexual Cannibalism in a Widow Spider.

Theory suggests that consistent individual variation in behavior relates to fitness, but few studies have empirically examined the role of personalities in mate choice, male-male competition and reproductive success. We observed the Mediterranean black widow, Latrodectus tredecimguttatus, in the individual and mating context, to test how body size measures and two functionally important aggressive behaviors, i.e., male aggression towards rivals and female voracity towards prey, affect mating behaviors, mating success and sexual cannibalism. We specifically selected voracity towards prey in females to test the "aggressive spillover hypothesis", suggesting that more voracious females are more sexually cannibalistic. Both females and males exhibit consistent individual differences in the examined aggressive behaviors. While larger males win contests more often and achieve more copulations, neither male nor female size measures correlate to aggression. Female voracity does not correlate with aggression towards mates and sexual cannibalism, rejecting the "spillover hypothesis". However, occurrence of sexual cannibalism positively relates to longer insertion duration. Furthermore, the smaller the ratio between male and female body length the more likely a female attacked and cannibalized a mate. We show that individual variation in aggression levels plays no direct role in the mating behavior of the Mediterranean black widow. Instead, body size affects male mating success and occurrences of sexual cannibalism in females.

Correlation between protein secondary structure and mechanical performance for the ultra-tough dragline silk of Darwin's bark spider.

The spider major ampullate (MA) silk exhibits high tensile strength and extensibility and is typically a blend of MaSp1 and MaSp2 proteins with the latter comprising glycine-proline-glycine-glycine-X repeating motifs that promote extensibility and supercontraction. The MA silk from Darwin's bark spider (Caerostris darwini) is estimated to be two to three times tougher than the MA silk from other spider species. Previous research suggests that a unique MaSp4 protein incorporates proline into a novel glycine-proline-glycine-proline motif and may explain C. darwini MA silk's extraordinary toughness. However, no direct correlation has been made between the silk's molecular structure and its mechanical properties for C. darwini. Here, we correlate the relative protein secondary structure composition of MA silk from C. darwini and four other spider species with mechanical properties before and after supercontraction to understand the effect of the additional MaSp4 protein. Our results demonstrate that C. darwini MA silk possesses a unique protein composition with a lower ratio of helices (31%) and ß-sheets (20%) than other species. Before supercontraction, toughness, modulus and tensile strength correlate with percentages of ß-sheets, unordered or random coiled regions and ß-turns. However, after supercontraction, only modulus and strain at break correlate with percentages of ß-sheets and ß-turns. Our study highlights that additional information including crystal size and crystal and chain orientation is necessary to build a complete structure-property correlation model.

Mass predicts web asymmetry in Nephila spiders.

The architecture of vertical aerial orb webs may be affected by spider size and gravity or by the available web space, in addition to phylogenetic and/or developmental factors. Vertical orb web asymmetry measured by hub displacement has been shown to increase in bigger and heavier spiders; however, previous studies have mostly focused on adult and subadult spiders or on several size classes with measured size parameters but no mass. Both estimations are suboptimal because (1) adult orb web spiders may not invest heavily in optimal web construction, whereas juveniles do; (2) size class/developmental stage is difficult to estimate in the field and is thus subjective, and (3) mass scales differently to size and is therefore more important in predicting aerial foraging success due to gravity. We studied vertical web asymmetry in a giant orb web spider, Nephila pilipes, across a wide range of size classes/developmental stages and tested the hypothesis that vertical web asymmetry (measured as hub displacement) is affected by gravity. On a sample of 100 webs, we found that hubs were more displaced in heavier and larger juveniles and that spider mass explained vertical web asymmetry better than other measures of spider size (carapace and leg lengths, developmental stage). Quantifying web shape via the ladder index suggested that, unlike in other nephilid taxa, growing Nephila orbs do not become vertically elongated. We conclude that the ontogenetic pattern of progressive vertical web asymmetry in Nephila can be explained by optimal foraging due to gravity, to which the opposing selective force may be high web-building costs in the lower orb. Recent literature finds little support for alternative explanations of ontogenetic orb web allometry such as the size limitation hypothesis and the biogenetic law.

Small size does not confer male agility advantages in a sexually-size dimorphic spider.

Selection pressures leading to extreme, female-biased sexual size dimorphism (SSD) in spiders continue to be debated. It has been proposed that males of sexually size dimorphic spiders could be small because gravity constrains adult agility (locomotor abilities). Accordingly, small males should achieve higher vertical climbing speeds and should be more prone to bridge. The curvilinear model of the gravity hypothesis predicts a negative relationship between vertical climbing speed and male body size only over a threshold of 7.6 mm, 42.5 mg. Because males of most species with extreme SSD fall well below this threshold, the relationship between male size and agility at this scale remains vague. Here, we tested three hypotheses on how male size, mass and age (after maturation) relate to vertical climbing and bridging ability in Nephilingis cruentata, a highly sexually dimorphic orb-weaver with males well below the size threshold. We placed males of different sizes and adult ages in a vertical platform and recorded their climbing speeds. Contrary to the original study testing male bridging ability as binary variable, we measured the duration of the crossing of the bridging thread, as well as its sagging distance. Male body size and mass positively related to the vertical climbing speed and to the distance of the sagging thread during bridging, but had no influence on the bridging duration. The detected positive correlation between male size/mass and vertical climbing speed goes against our first prediction, that small males would have vertical climbing advantage in Nephilingis cruentata, but agrees with the curvilinear model. Against our second prediction, small males were not faster during bridging. Finally, in agreement with our third prediction, threads sagged more in heavier males. These results suggest that small male size confers no agility advantages in Nephilingis cruentata.

The transcriptome of Darwin's bark spider silk glands predicts proteins contributing to dragline silk toughness.

Darwin's bark spider (Caerostris darwini) produces giant orb webs from dragline silk that can be twice as tough as other silks, making it the toughest biological material. This extreme toughness comes from increased extensibility relative to other draglines. We show C. darwini dragline-producing major ampullate (MA) glands highly express a novel silk gene transcript (MaSp4) encoding a protein that diverges markedly from closely related proteins and contains abundant proline, known to confer silk extensibility, in a unique GPGPQ amino acid motif. This suggests C. darwini evolved distinct proteins that may have increased its dragline's toughness, enabling giant webs. Caerostris darwini's MA spinning ducts also appear unusually long, potentially facilitating alignment of silk proteins into extremely tough fibers. Thus, a suite of novel traits from the level of genes to spinning physiology to silk biomechanics are associated with the unique ecology of Darwin's bark spider, presenting innovative designs for engineering biomaterials.

Spider behaviors include oral sexual encounters.

Several clades of spiders whose females evolved giant sizes are known for extreme sexual behaviors such as sexual cannibalism, opportunistic mating, mate-binding, genital mutilation, plugging, and emasculation. However, these behaviors have only been tested in a handful of size dimorphic spiders. Here, we bring another lineage into the picture by reporting on sexual behavior of Darwin's bark spider, Caerostris darwini. This sexually size dimorphic Madagascan species is known for extreme web gigantism and for producing the world's toughest biomaterial. Our field and laboratory study uncovers a rich sexual repertoire that predictably involves cannibalism, genital mutilation, male preference for teneral females, and emasculation. Surprisingly, C. darwini males engage in oral sexual encounters, rarely reported outside mammals. Irrespective of female's age or mating status males salivate onto female genitalia pre-, during, and post-copulation. While its adaptive significance is elusive, oral sexual contact in spiders may signal male quality or reduce sperm competition.

Potential costs of heterospecific sexual interactions in golden orbweb spiders (Nephila spp.).

Though not uncommon in other animals, heterospecific mating is rarely reported in arachnids. We investigated sexual interactions among four closely related and syntopical African golden orbweb spiders, Nephila inaurata, N. fenestrata, N. komaci, and N. senegalensis. In two South African localities, female webs were often inhabited by heterospecific males that sometimes outnumbered conspecifics. Species association of males with females was random in nature. In subsequent laboratory choice experiments, N. inaurata males chose heterospecific females in 30% of trials. We also observed natural mating interactions between N. inaurata males and N. komaci females, and between N. komaci males and N. inaurata females in laboratory experiments. While heterospecific mating in the laboratory never produced offspring, conspecific mating did. We discuss potential ecological and evolutionary consequences of heterospecific mating interactions in Nephila that may be particularly costly to the rarer species.

DNA barcode data accurately assign higher spider taxa.

The use of unique DNA sequences as a method for taxonomic identification is no longer fundamentally controversial, even though debate continues on the best markers, methods, and technology to use. Although both existing databanks such as GenBank and BOLD, as well as reference taxonomies, are imperfect, in best case scenarios "barcodes" (whether single or multiple, organelle or nuclear, loci) clearly are an increasingly fast and inexpensive method of identification, especially as compared to manual identification of unknowns by increasingly rare expert taxonomists. Because most species on Earth are undescribed, a complete reference database at the species level is impractical in the near term. The question therefore arises whether unidentified species can, using DNA barcodes, be accurately assigned to more inclusive groups such as genera and families-taxonomic ranks of putatively monophyletic groups for which the global inventory is more complete and stable. We used a carefully chosen test library of CO1 sequences from 49 families, 313 genera, and 816 species of spiders to assess the accuracy of genus and family-level assignment. We used BLAST queries of each sequence against the entire library and got the top ten hits. The percent sequence identity was reported from these hits (PIdent, range 75-100%). Accurate assignment of higher taxa (PIdent above which errors totaled less than 5%) occurred for genera at PIdent values >95 and families at PIdent values ≥ 91, suggesting these as heuristic thresholds for accurate generic and familial identifications in spiders. Accuracy of identification increases with numbers of species/genus and genera/family in the library; above five genera per family and fifteen species per genus all higher taxon assignments were correct. We propose that using percent sequence identity between conventional barcode sequences may be a feasible and reasonably accurate method to identify animals to family/genus. However, the quality of the underlying database impacts accuracy of results; many outliers in our dataset could be attributed to taxonomic and/or sequencing errors in BOLD and GenBank. It seems that an accurate and complete reference library of families and genera of life could provide accurate higher level taxonomic identifications cheaply and accessibly, within years rather than decades.

Corrigendum: Targeting a portion of central European spider diversity for permanent preservation.

[This corrects the article DOI: 10.3897/BDJ.1.e980.].

Targeting a portion of central European spider diversity for permanent preservation.

Given the limited success of past and current conservation efforts, an alternative approach is to preserve tissues and genomes of targeted organisms in cryobanks to make them accessible for future generations. Our pilot preservation project aimed to obtain, expertly identify, and permanently preserve a quarter of the known spider species diversity shared between Slovenia and Switzerland, estimated at 275 species. We here report on the faunistic part of this project, which resulted in 324 species (227 in Slovenia, 143 in Switzerland) for which identification was reasonably established. This material is now preserved in cryobanks, is being processed for DNA barcoding, and is available for genomic studies.

Mating plugs in polyandrous giants: which sex produces them, when, how and why?

BACKGROUND: Males usually produce mating plugs to reduce sperm competition. However, females can conceivably also produce mating plugs in order to prevent unwanted, superfluous and energetically costly matings. In spiders-appropriate models for testing plugging biology hypotheses-mating plugs may consist of male genital parts and/or of amorphous covers consisting of glandular or sperm secretions. In the giant wood spider Nephila pilipes, a highly sexually dimorphic and polygamous species, males are known to produce ineffective embolic plugs through genital damage, but nothing is known about the origin and function of additional conspicuous amorphous plugs (AP) covering female genitals. METHODOLOGY: We tested alternative hypotheses of the nature and function of AP in N. pilipes by staging mating trials with varying degrees of polyandry. No APs were ever formed during mating trials, which rules out the possibility of male AP formation. Instead, those females that oviposited produced the AP from a liquid secreted during egg sac formation. Polyandrous females were more likely to lay eggs and to produce the AP, as were those that mated longer and with more total insertions. Our further tests revealed that, in spite of being a side product of egg sac production, AP, when hardened, prevented any subsequent copulation. CONCLUSIONS: We conclude that in the giant wood spider (Nephila pilipes), the amorphous mating plugs are not produced by the males, that repeated copulations (most likely polyandrous) are necessary for egg fertilization and AP formation, and that the AP represents a female adaptation to sexual conflict through prevention of unwanted, excessive copulations. Considering the largely unknown origin of amorphous plugs in spiders, we predict that a similar pattern might be detected in other clades, which would help elucidate the evolutionary interplay of various selection pressures responsible for the origin and maintenance of mating plugs.