Specificity of Loxosceles α clade phospholipase D enzymes for choline-containing lipids: Role of a conserved aromatic cage.

Spider venom GDPD-like phospholipases D (SicTox) have been identified to be one of the major toxins in recluse spider venom. They are divided into two major clades: the α clade and the ß clade. Most α clade toxins present high activity against lipids with choline head groups such as sphingomyelin, while activities in ß clade toxins vary and include preference for substrates containing ethanolamine headgroups (Sicarius terrosus, St_ßIB1). A structural comparison of available structures of phospholipases D (PLDs) reveals a conserved aromatic cage in the α clade. To test the potential influence of the aromatic cage on membrane-lipid specificity we performed molecular dynamics (MD) simulations of the binding of several PLDs onto lipid bilayers containing choline headgroups; two SicTox from the α clade, Loxosceles intermedia αIA1 (Li_αIA) and Loxosceles laeta αIII1 (Ll_αIII1), and one from the ß clade, St_ßIB1. The simulation results reveal that the aromatic cage captures a choline-headgroup and suggest that the cage plays a major role in lipid specificity. We also simulated an engineered St_ßIB1, where we introduced the aromatic cage, and this led to binding with choline-containing lipids. Moreover, a multiple sequence alignment revealed the conservation of the aromatic cage among the α clade PLDs. Here, we confirmed that the i-face of α and ß clade PLDs is involved in their binding to choline and ethanolamine-containing bilayers, respectively. Furthermore, our results suggest a major role in choline lipid recognition of the aromatic cage of the α clade PLDs. The MD simulation results are supported by in vitro liposome binding assay experiments.

Huntsmen of the Caribbean: Multiple tests of the GAARlandia hypothesis.

The origin of the Caribbean biota remains debated, but amassing evidence suggests important roles of both dispersal and vicariance events in the colonization the archipelago. The most prominent vicariance hypothesis is colonization over the GAARlandia land bridge that putatively connected the Greater Antilles to South America around 33 mya. This hypothesis has received support from studies of individual lineages, but its main prediction-the simultaneous colonization of multiple lineages during that time window-requires further unambiguous corroboration. Here, we examine the phylogenetic structure of huntsman spiders (Sparassidae) of the Caribbean. Huntsman spiders are appropriate models for this question, as they are expected to be dispersal limited as substrate and foliage dwelling spiders that rarely balloon, yet are found on some volcanic islands, and thus at least some overwater dispersal must have occurred. We focus on the Caribbean endemic Neostasina, but also include Caribbean Olios, for a deeper biogeographical understanding. We use two mitochondrial and four nuclear markers to reconstruct dated phylogenetic trees and to test taxonomic and biogeographic hypotheses. Our analyses strongly support the monophyly of Neostasina and the polyphyly of Olios, with a new clade endemic to the Caribbean. Both Neostasina and Caribbean Olios occur on the Greater and Lesser Antilles and independently colonized the Caribbean around 36-28 mya. Hypothesis testing in BioGeoBEARS suggests a role of the GAARlandia landbridge in the colonization of both clades. The 'Olios-like' clade, in contrast, is restricted to the southern Lesser Antilles and shows a biogeographic history consistent with colonization from S. America, probably within the last 10 my. Thus, many spider lineages on the Greater Antilles seem to have colonized the Caribbean during a relatively short time span approximately coinciding with the proposed timing of GAARlandia. The synchronous colonization of multiple lineages suggests a temporary land connection. However, the main problem in concluding synchronous events across lineages in this study, as in most others, is the ambiguity in chronogram analyses meaning that many different patterns can be 'consistent' with GAARlandia, thus potentially providing a false positive result. Broad comparative biogeographical studies such as the CarBio project will offer the best opportunity to multiply test shared biogeographic patterns among independent lineages. The current paper contributes evidence from multiple lineages that will contribute to this synthesis.

Evolutionary dynamics of origin and loss in the deep history of phospholipase D toxin genes.

BACKGROUND: Venom-expressed sphingomyelinase D/phospholipase D (SMase D/PLD) enzymes evolved from the ubiquitous glycerophosphoryl diester phosphodiesterases (GDPD). Expression of GDPD-like SMaseD/PLD toxins in both arachnids and bacteria has inspired consideration of the relative contributions of lateral gene transfer and convergent recruitment in the evolutionary history of this lineage. Previous work recognized two distinct lineages, a SicTox-like (ST-like) clade including the arachnid toxins, and an Actinobacterial-toxin like (AT-like) clade including the bacterial toxins and numerous fungal homologs. RESULTS: Here we expand taxon sampling by homology detection to discover new GDPD-like SMase D/PLD homologs. The ST-like clade now includes homologs in a wider variety of arthropods along with a sister group in Cnidaria; the AT-like clade now includes additional fungal phyla and proteobacterial homologs; and we report a third clade expressed in diverse aquatic metazoan taxa, a few single-celled eukaryotes, and a few aquatic proteobacteria. GDPD-like SMaseD/PLDs have an ancient presence in chelicerates within the ST-like family and ctenophores within the Aquatic family. A rooted phylogenetic tree shows that the three clades derived from a basal paraphyletic group of proteobacterial GDPD-like SMase D/PLDs, some of which are on mobile genetic elements. GDPD-like SMase D/PLDs share a signature C-terminal motif and a shortened ßα1 loop, features that distinguish them from GDPDs. The three major clades also have active site loop signatures that distinguish them from GDPDs and from each other. Analysis of molecular phylogenies with respect to organismal relationships reveals a dynamic evolutionary history including both lateral gene transfer and gene duplication/loss. CONCLUSIONS: The GDPD-like SMaseD/PLD enzymes derive from a single ancient ancestor, likely proteobacterial, and radiated into diverse organismal lineages at least in part through lateral gene transfer.

Variable Substrate Preference among Phospholipase D Toxins from Sicariid Spiders.

Venoms of the sicariid spiders contain phospholipase D enzyme toxins that can cause severe dermonecrosis and even death in humans. These enzymes convert sphingolipid and lysolipid substrates to cyclic phosphates by activating a hydroxyl nucleophile present in both classes of lipid. The most medically relevant substrates are thought to be sphingomyelin and/or lysophosphatidylcholine. To better understand the substrate preference of these toxins, we used (31)P NMR to compare the activity of three related but phylogenetically diverse sicariid toxins against a diverse panel of sphingolipid and lysolipid substrates. Two of the three showed significantly faster turnover of sphingolipids over lysolipids, and all three showed a strong preference for positively charged (choline and/or ethanolamine) over neutral (glycerol and serine) headgroups. Strikingly, however, the enzymes vary widely in their preference for choline, the headgroup of both sphingomyelin and lysophosphatidylcholine, versus ethanolamine. An enzyme from Sicarius terrosus showed a strong preference for ethanolamine over choline, whereas two paralogous enzymes from Loxosceles arizonica either preferred choline or showed no significant preference. Intrigued by the novel substrate preference of the Sicarius enzyme, we solved its crystal structure at 2.1 Å resolution. The evolution of variable substrate specificity may help explain the reduced dermonecrotic potential of some natural toxin variants, because mammalian sphingolipids use primarily choline as a positively charged headgroup; it may also be relevant for sicariid predatory behavior, because ethanolamine-containing sphingolipids are common in insect prey.

Islands within islands: Diversification of tailless whip spiders (Amblypygi, Phrynus) in Caribbean caves.

Islands have played a key role in understanding species formation ever since Darwin's work on the Galapagos and Wallace's work in the Malay Archipelago. Like oceanic islands, habitat 'islands', such as mountaintops and caves similarly may drive diversification. Here we examine patterns of diversification in the tailless whip spider genus Phrynus Larmarck, 1809 (Amblypygida: Phrynidae) a system that shows evidence of diversification under the influence of 'islands within islands'. We estimate phylogeographic history and measure genetic diversity among representatives of three nominal Phrynus species from epigean and cave systems of Puerto Rico and nearby islands. Data from five loci (mitochondrial 12S, 16S, Cox1; nuclear H3, 28S) were used to generate phylogenetic hypotheses and to assess species monophyly and phylogeographic relationships. Genetic divergences and population limits were estimated and assessed using the Geneious barcoding plugin and the genealogical sorting index. We find that mtDNA sequence divergences within each of the three Phrynus species range between 15% and 20%. Genetic divergence is structured at three spatial scales: among islands in a manner consistent with the GAARlandia hypothesis, among bedrock formations within Puerto Rico, and among caves within these formations. Every isolated cave system contains a unique mtDNA genetic lineage of Phrynus, with divergence among cave systems far exceeding that within. In some localities epigean specimens nest among cave taxa, in others caves are monophyletic. Remarkably, clades that show up to 20% mtDNA sequence divergence show little or no variation in the nuclear markers. We interpret this pattern as resulting from extreme conservation of our nuclear markers rather than male sex-biased dispersal, based on high conservation of 28S and H3 between our individuals and other amblypygid genera that are restricted to Africa. While this study includes but a tiny fraction of Caribbean caves, our findings suggest Phrynus may be much more diverse than hitherto thought, at least in terms of mtDNA diversity, and that the arthropod fauna of caves may represent a dimension of biodiversity that is yet to be discovered in the Caribbean biodiversity hotspot.

Spit and venom from scytodes spiders: a diverse and distinct cocktail.

Spiders from the family Scytodidae have a unique prey capturing technique: they spit a zig-zagged silken glue to tether prey to a surface. Effectiveness of this sticky mixture is based on a combination of contraction and adhesion, trapping prey until the spider immobilizes it by envenomation and then feeds. We identify components expressed in Scytodes thoracica venom glands using combined transcriptomic and proteomic analyses. These include homologues of toxic proteins astacin metalloproteases and potentially toxic proteins including venom allergen, longistatin, and translationally controlled tumor protein (TCTP). We classify 19 distinct groups of candidate peptide toxins; 13 of these were detected in the venom, making up 35% of the proteome. Six have significant similarity to toxins from spider species spanning mygalomorph and nonhaplogyne araneomorph lineages, suggesting their expression in venom is phylogenetically widespread. Twelve peptide toxin groups have homologues in venom gland transcriptomes of other haplogynes. Of the transcripts, approximately 50% encode glycine-rich peptides that may contribute to sticky fibers in Scytodes spit. Fifty-one percent of the identified venom proteome is a family of proteins that is homologous to sequences from Drosophila sp. and Latrodectus hesperus with uncharacterized function. Characterization of these components holds promise for discovering new functional activity.

ArachnoServer 2.0, an updated online resource for spider toxin sequences and structures.

ArachnoServer (www.arachnoserver.org) is a manually curated database providing information on the sequence, structure and biological activity of protein toxins from spider venoms. These proteins are of interest to a wide range of biologists due to their diverse applications in medicine, neuroscience, pharmacology, drug discovery and agriculture. ArachnoServer currently manages 1078 protein sequences, 759 nucleic acid sequences and 56 protein structures. Key features of ArachnoServer include a molecular target ontology designed specifically for venom toxins, current and historic taxonomic information and a powerful advanced search interface. The following significant improvements have been implemented in version 2.0: (i) the average and monoisotopic molecular masses of both the reduced and oxidized form of each mature toxin are provided; (ii) the advanced search feature now enables searches on the basis of toxin mass, external database accession numbers and publication date in ArachnoServer; (iii) toxins can now be browsed on the basis of their phyletic specificity; (iv) rapid BLAST searches based on the mature toxin sequence can be performed directly from the toxin card; (v) private silos can be requested from research groups engaged in venoms-based research, enabling them to easily manage and securely store data during the process of toxin discovery; and (vi) a detailed user manual is now available.

Protein salvage and repurposing in evolution: Phospholipase D toxins are stabilized by a remodeled scrap of a membrane association domain.

The glycerophosphodiester phosphodiesterase (GDPD)-like SMaseD/PLD domain family, which includes phospholipase D (PLD) toxins in recluse spiders and actinobacteria, evolved anciently in bacteria from the GDPD. The PLD enzymes retained the core (ß/α)8 barrel fold of GDPD, while gaining a signature C-terminal expansion motif and losing a small insertion domain. Using sequence alignments and phylogenetic analysis, we infer that the C-terminal motif derives from a segment of an ancient bacterial PLAT domain. Formally, part of a protein containing a PLAT domain repeat underwent fusion to the C terminus of a GDPD barrel, leading to attachment of a segment of a PLAT domain, followed by a second complete PLAT domain. The complete domain was retained only in some basal homologs, but the PLAT segment was conserved and repurposed as the expansion motif. The PLAT segment corresponds to strands ß7-ß8 of a ß-sandwich, while the expansion motif as represented in spider PLD toxins has been remodeled as an α-helix, a ß-strand, and an ordered loop. The GDPD-PLAT fusion led to two acquisitions in founding the GDPD-like SMaseD/PLD family: (1) a PLAT domain that presumably supported early lipase activity by mediating membrane association, and (2) an expansion motif that putatively stabilized the catalytic domain, possibly compensating for, or permitting, loss of the insertion domain. Of wider significance, messy domain shuffling events can leave behind scraps of domains that can be salvaged, remodeled, and repurposed.

Molecular evolution, functional variation, and proposed nomenclature of the gene family that includes sphingomyelinase D in sicariid spider venoms.

The venom enzyme sphingomyelinase D (SMase D) in the spider family Sicariidae (brown or fiddleback spiders [Loxosceles] and six-eyed sand spiders [Sicarius]) causes dermonecrosis in mammals. SMase D is in a gene family with multiple venom-expressed members that vary in functional specificity. We analyze molecular evolution of this family and variation in SMase D activity among crude venoms using a data set that represents the phylogenetic breadth of Loxosceles and Sicarius. We isolated a total of 190 nonredundant nucleotide sequences encoding 168 nonredundant amino acid sequences of SMase D homologs from 21 species. Bayesian phylogenies support two major clades that we name alpha and beta, within which we define seven and three subclades, respectively. Sequences in the alpha clade are exclusively from New World Loxosceles and Loxosceles rufescens and include published genes for which expression products have SMase D and dermonecrotic activity. The beta clade includes paralogs from New World Loxosceles that have no, or reduced, SMase D and no dermonecrotic activity and also paralogs from Sicarius and African Loxosceles of unknown activity. Gene duplications are frequent, consistent with a birth-and-death model, and there is evidence of purifying selection with episodic positive directional selection. Despite having venom-expressed SMase D homologs, venoms from New World Sicarius have reduced, or no, detectable SMase D activity, and Loxosceles in the Southern African spinulosa group have low SMase D activity. Sequence conservation mapping shows >98% conservation of proposed catalytic residues of the active site and around a plug motif at the opposite end of the TIM barrel, but alpha and beta clades differ in conservation of key residues surrounding the apparent substrate binding pocket. Based on these combined results, we propose an inclusive nomenclature for the gene family, renaming it SicTox, and discuss emerging patterns of functional diversification.

Diversity of Loxosceles spiders in Northwestern Africa and molecular support for cryptic species in the Loxosceles rufescens lineage.

Until recently, Loxosceles rufescens was the only species known from a geographic range including Northern Africa, Mediterranean Europe and the Middle East. Rich Loxosceles diversity in the New World suggests either that L. rufescens is a young lineage or that its diversity is underappreciated. We use a molecular phylogenetic and morphological approach to examine diversity in L. rufescens and other Loxosceles lineages in Northwestern Africa. Molecular analyses of one nuclear and two mitochondrial genes strongly support a monophyletic clade including L. rufescens, the Northern Brazilian L. amazonica and three other divergent Northwestern African lineages, though relationships among them remain unresolved. A genetically divergent Moroccan individual morphologically consistent with L. rufescens was strongly supported as sister to all other putative L. rufescens, consistent with the presence of at least 2 species in this lineage. COI p-distances and population structuring among remaining putative L. rufescens clades further suggest the absence of gene flow between clades and the possibility that they represent multiple species. Morphological characters of preserved Loxosceles collected in a range of African countries provide additional indication that Loxosceles are more diverse and have a deeper history in Africa than has been previously understood.

Heteroonops (Araneae, Oonopidae) spiders from Hispaniola: the discovery of ten new species.

The Caribbean biodiversity hotspot harbors vast reserves of undiscovered species. A large-scale inventory of Caribbean arachnids (CarBio) is uncovering new species across the arachnid tree of life, and allowing inference of the evolutionary history that has generated this diversity. Herein we describe ten new species of Heteroonops (Oonopidae, or goblin spiders), from Hispaniola: H. scapula sp. nov., H. jurassicus sp. nov., H. aylinalegreae sp. nov., H. verruca sp. nov., H. renebarbai sp. nov., H. yuma sp. nov., H. carlosviquezi sp. nov., H. gabrielsantosi sp. nov., H. solanllycarreroae sp. nov. and H. constanza sp. nov. The occurrence of the pantropical type species Heteroonops spinimanus (Simon, 1891) is reported and new localities are given for: H. validus (Bryant, 1948), H. vega (Platnick & Dupérré, 2009) and H. castelloides (Platnick & Dupérré, 2009). Molecular phylogenies indicate substantial genetic divergence separating these taxa. This work adds to evidence that the depth of diversity in the Caribbean biodiversity hotspot is particularly striking for tiny taxa living in leaf litter.

Phylogeography of the widespread Caribbean spiny orb weaver Gasteracantha cancriformis.

BACKGROUND: Modern molecular analyses are often inconsistent with pre-cladistic taxonomic hypotheses, frequently indicating higher richness than morphological taxonomy estimates. Among Caribbean spiders, widespread species are relatively few compared to the prevalence of single island endemics. The taxonomic hypothesis Gasteracantha cancriformis circumscribes a species with profuse variation in size, color and body form. Distributed throughout the Neotropics, G. cancriformis is the only morphological species of Gasteracantha in the New World in this globally distributed genus. METHODS: We inferred phylogenetic relationships across Neotropical populations of Gasteracantha using three target genes. Within the Caribbean, we estimated genetic diversity, population structure, and gene flow among island populations. RESULTS: Our findings revealed a single widespread species of Gasteracantha throughout the Caribbean, G. cancriformis, while suggesting two recently divergent mainland populations that may represent separate species, diverging linages, or geographically isolated demes. The concatenated and COI (Cytochrome c oxidase subunit 1) phylogeny supported a Caribbean clade nested within the New World. Genetic variability was high between island populations for our COI dataset; however, gene flow was also high, especially between large, adjacent islands. We found structured genetic and morphological variation within G. cancriformis island populations; however, this variation does not reflect genealogical relationships. Rather, isolation by distance and local morphological adaptation may explain the observed variation.

Gene content evolution in the arthropods.

BACKGROUND: Arthropods comprise the largest and most diverse phylum on Earth and play vital roles in nearly every ecosystem. Their diversity stems in part from variations on a conserved body plan, resulting from and recorded in adaptive changes in the genome. Dissection of the genomic record of sequence change enables broad questions regarding genome evolution to be addressed, even across hyper-diverse taxa within arthropods. RESULTS: Using 76 whole genome sequences representing 21 orders spanning more than 500 million years of arthropod evolution, we document changes in gene and protein domain content and provide temporal and phylogenetic context for interpreting these innovations. We identify many novel gene families that arose early in the evolution of arthropods and during the diversification of insects into modern orders. We reveal unexpected variation in patterns of DNA methylation across arthropods and examples of gene family and protein domain evolution coincident with the appearance of notable phenotypic and physiological adaptations such as flight, metamorphosis, sociality, and chemoperception. CONCLUSIONS: These analyses demonstrate how large-scale comparative genomics can provide broad new insights into the genotype to phenotype map and generate testable hypotheses about the evolution of animal diversity.

Biogeography of the Caribbean Cyrtognatha spiders.

Island systems provide excellent arenas to test evolutionary hypotheses pertaining to gene flow and diversification of dispersal-limited organisms. Here we focus on an orbweaver spider genus Cyrtognatha (Tetragnathidae) from the Caribbean, with the aims to reconstruct its evolutionary history, examine its biogeographic history in the archipelago, and to estimate the timing and route of Caribbean colonization. Specifically, we test if Cyrtognatha biogeographic history is consistent with an ancient vicariant scenario (the GAARlandia landbridge hypothesis) or overwater dispersal. We reconstructed a species level phylogeny based on one mitochondrial (COI) and one nuclear (28S) marker. We then used this topology to constrain a time-calibrated mtDNA phylogeny, for subsequent biogeographical analyses in BioGeoBEARS of over 100 originally sampled Cyrtognatha individuals, using models with and without a founder event parameter. Our results suggest a radiation of Caribbean Cyrtognatha, containing 11 to 14 species that are exclusively single island endemics. Although biogeographic reconstructions cannot refute a vicariant origin of the Caribbean clade, possibly an artifact of sparse outgroup availability, they indicate timing of colonization that is much too recent for GAARlandia to have played a role. Instead, an overwater colonization to the Caribbean in mid-Miocene better explains the data. From Hispaniola, Cyrtognatha subsequently dispersed to, and diversified on, the other islands of the Greater, and Lesser Antilles. Within the constraints of our island system and data, a model that omits the founder event parameter from biogeographic analysis is less suitable than the equivalent model with a founder event.

Not so Dangerous After All? Venom Composition and Potency of the Pholcid (Daddy Long-Leg) Spider Physocyclus mexicanus.

Pholcid spiders (Araneae: Pholcidae), officially "cellar spiders" but popularly known as "daddy long-legs," are renown for the potential of deadly toxic venom, even though venom composition and potency has never formally been studied. Here we detail the venom composition of male Physocyclus mexicanus using proteomic analyses and venom-gland transcriptomes ("venomics"). We also analyze the venom's potency on insects, and assemble available evidence regarding mammalian toxicity. The majority of the venom (51% of tryptic polypeptides and 62% of unique tryptic peptides) consists of proteins homologous to known venom toxins including enzymes (astacin metalloproteases, serine proteases and metalloendopeptidases, particularly neprilysins) and venom peptide neurotoxins. We identify 17 new groups of peptides (U1-17-PHTX) most of which are homologs of known venom peptides and are predicted to have an inhibitor cysteine knot fold; of these, 13 are confirmed in the proteome. Neprilysins (M13 peptidases), and astacins (M12 peptidases) are the most abundant venom proteins, respectively representing 15 and 11% of the individual proteins and 32 and 20% of the tryptic peptides detected in crude venom. Comparative evidence suggests that the neprilysin gene family is expressed in venoms across a range of spider taxa, but has undergone an expansion in the venoms of pholcids and may play a central functional role in these spiders. Bioassays of crude venoms on crickets resulted in an effective paralytic dose of 3.9 µg/g, which is comparable to that of crude venoms of Plectreurys tristis and other Synspermiata taxa. However, crickets exhibit flaccid paralysis and regions of darkening that are not observed after P. tristis envenomation. Documented bites on humans make clear that while these spiders can bite, the typical result is a mild sting with no long-lasting effects. Together, the evidence we present indicates pholcid venoms are a source of interesting new peptides and proteins, and effects of bites on humans and other mammals are inconsequential.

Phylogenetic relationships of Loxosceles and Sicarius spiders are consistent with Western Gondwanan vicariance.

The modern geographic distribution of the spider family Sicariidae is consistent with an evolutionary origin on Western Gondwana. Both sicariid genera, Loxosceles and Sicarius are diverse in Africa and South/Central America. Loxosceles are also diverse in North America and the West Indies, and have species described from Mediterranean Europe and China. We tested vicariance hypotheses using molecular phylogenetics and molecular dating analyses of 28S, COI, 16S, and NADHI sequences. We recover reciprocal monophyly of African and South American Sicarius, paraphyletic Southern African Loxosceles and monophyletic New World Loxosceles within which an Old World species group that includes L. rufescens is derived. These patterns are consistent with a sicariid common ancestor on Western Gondwana. North American Loxosceles are monophyletic, sister to Caribbean taxa, and resolved in a larger clade with South American Loxosceles. With fossil data this pattern is consistent with colonization of North America via a land bridge predating the modern Isthmus of Panama.

Sexually dimorphic venom proteins in long-jawed orb-weaving spiders (Tetragnatha) comprise novel gene families.

Venom has been associated with the ecological success of many groups of organisms, most notably reptiles, gastropods, and arachnids. In some cases, diversification has been directly linked to tailoring of venoms for dietary specialization. Spiders in particular are known for their diverse venoms and wide range of predatory behaviors, although there is much to learn about scales of variation in venom composition and function. The current study focuses on venom characteristics in different sexes within a species of spider. We chose the genus Tetragnatha (Tetragnathidae) because of its unusual courtship behavior involving interlocking of the venom delivering chelicerae (i.e., the jaws), and several species in the genus are already known to have sexually dimorphic venoms. Here, we use transcriptome and proteome analyses to identify venom components that are dimorphic in Tetragnatha versicolor. We present cDNA sequences including unique, male-specific high molecular weight proteins that have remote, if any, detectable similarity to known venom components in spiders or other venomous lineages and have no detectable homologs in existing databases. While the function of these proteins is not known, their presence in association with the cheliceral locking mechanism during mating together with the presence of prolonged male-male mating attempts in a related, cheliceral-locking species (Doryonychus raptor) lacking the dimorphism suggests potential for a role in sexual communication.

Convergent setal morphology in sand-covering spiders suggests a design principle for particle capture.

Sicarius and Homalonychus are unrelated, desert-dwelling spiders that independently evolved the ability to cover themselves in fine sand particles, making them cryptic against their background. Observations that particles associate with these spiders' setae inspired us to investigate the role of setal microstructure in particle capture and retention. Here we report that Sicarius and Homalonychus convergently evolved numerous high aspect ratio, flexible fibres that we call 'hairlettes' protruding from the setal shaft. We demonstrate that particles attach more densely to regions of Homalonychus with hairlettes than to other regions of the same animal where hairlettes are absent, and document close contact of hairlettes to sand particles that persists after applying force. Mathematical models further suggest that adhesion of hairlettes to sand particles is a sufficient mechanism of particle capture and retention. Together, these data provide the first evidence that hairlettes facilitate sand retention through intermolecular adhesion to particles. Their independent evolutionary origins in Sicarius and Homalonychus suggest that the unique setal structure is adaptive and represents a general biomechanical mechanism for sand capture to cuticle. This discovery has implications for the design of inventions inspired by this system, from camouflage to the management of granular systems.

Sexual dimorphism in venom chemistry in Tetragnatha spiders is not easily explained by adult niche differences.

Spider venom composition typically differs between sexes. This pattern is anecdotally thought to reflect differences in adult feeding biology. We used a phylogenetic approach to compare intersexual venom dimorphism between species that differ in adult niche dimorphism. Male and female venoms were compared within and between related species of Hawaiian Tetragnatha, a mainland congener, and outgroups. In some species of Hawaiian Tetragnatha adult females spin orb-webs and adult males capture prey while wandering, while in other species both males and females capture prey by wandering. We predicted that, if venom sexual dimorphism is primarily explained by differences in adult feeding biology, species in which both sexes forage by wandering would have monomorphic venoms or venoms with reduced dimorphism relative to species with different adult feeding biology. However, we found striking sexual dimorphism in venoms of both wandering and orb-weaving Tetragnatha species with males having high molecular weight components in their venoms that were absent in females, and a reduced concentration of low molecular weight components relative to females. Intersexual differences in venom composition within Tetragnatha were significantly larger than in non-Tetragnatha species. Diet composition was not different between sexes. This striking venom dimorphism is not easily explained by differences in feeding ecology or behavior. Rather, we hypothesize that the dimorphism reflects male-specific components that play a role in mating biology possibly in sexual stimulation, nuptial gifts and/or mate recognition.