Insight into the adaptive role of arachnid genome-wide duplication through chromosome-level genome assembly of the Western black widow spider.

Although spiders are one of the most diverse groups of arthropods, the genetic architecture of their evolutionary adaptations is largely unknown. Specifically, ancient genome-wide duplication occurring during arachnid evolution ~450 mya resulted in a vast assembly of gene families, yet the extent to which selection has shaped this variation is understudied. To aid in comparative genome sequence analyses, we provide a chromosome-level genome of the Western black widow spider (Latrodectus hesperus)-a focus due to its silk properties, venom applications, and as a model for urban adaptation. We used long-read and Hi-C sequencing data, combined with transcriptomes, to assemble 14 chromosomes in a 1.46 Gb genome, with 38,393 genes annotated, and a BUSCO score of 95.3%. Our analyses identified high repetitive gene content and heterozygosity, consistent with other spider genomes, which has led to challenges in genome characterization. Our comparative evolutionary analyses of eight genomes available for species within the Araneoidea group (orb weavers and their descendants) identified 1,827 single-copy orthologs. Of these, 155 exhibit significant positive selection primarily associated with developmental genes, and with traits linked to sensory perception. These results support the hypothesis that several traits unique to spiders emerged from the adaptive evolution of ohnologs-or retained ancestrally duplicated genes-from ancient genome-wide duplication. These comparative spider genome analyses can serve as a model to understand how positive selection continually shapes ancestral duplications in generating novel traits today within and between diverse taxonomic groups.

The house spider genome reveals an ancient whole-genome duplication during arachnid evolution.

BACKGROUND: The duplication of genes can occur through various mechanisms and is thought to make a major contribution to the evolutionary diversification of organisms. There is increasing evidence for a large-scale duplication of genes in some chelicerate lineages including two rounds of whole genome duplication (WGD) in horseshoe crabs. To investigate this further, we sequenced and analyzed the genome of the common house spider Parasteatoda tepidariorum. RESULTS: We found pervasive duplication of both coding and non-coding genes in this spider, including two clusters of Hox genes. Analysis of synteny conservation across the P. tepidariorum genome suggests that there has been an ancient WGD in spiders. Comparison with the genomes of other chelicerates, including that of the newly sequenced bark scorpion Centruroides sculpturatus, suggests that this event occurred in the common ancestor of spiders and scorpions, and is probably independent of the WGDs in horseshoe crabs. Furthermore, characterization of the sequence and expression of the Hox paralogs in P. tepidariorum suggests that many have been subject to neo-functionalization and/or sub-functionalization since their duplication. CONCLUSIONS: Our results reveal that spiders and scorpions are likely the descendants of a polyploid ancestor that lived more than 450 MYA. Given the extensive morphological diversity and ecological adaptations found among these animals, rivaling those of vertebrates, our study of the ancient WGD event in Arachnopulmonata provides a new comparative platform to explore common and divergent evolutionary outcomes of polyploidization events across eukaryotes.

Duplication and concerted evolution of MiSp-encoding genes underlie the material properties of minor ampullate silks of cobweb weaving spiders.

BACKGROUND: Orb-web weaving spiders and their relatives use multiple types of task-specific silks. The majority of spider silk studies have focused on the ultra-tough dragline silk synthesized in major ampullate glands, but other silk types have impressive material properties. For instance, minor ampullate silks of orb-web weaving spiders are as tough as draglines, due to their higher extensibility despite lower strength. Differences in material properties between silk types result from differences in their component proteins, particularly members of the spidroin (spider fibroin) gene family. However, the extent to which variation in material properties within a single silk type can be explained by variation in spidroin sequences is unknown. Here, we compare the minor ampullate spidroins (MiSp) of orb-weavers and cobweb weavers. Orb-web weavers use minor ampullate silk to form the auxiliary spiral of the orb-web while cobweb weavers use it to wrap prey, suggesting that selection pressures on minor ampullate spidroins (MiSp) may differ between the two groups. RESULTS: We report complete or nearly complete MiSp sequences from five cobweb weaving spider species and measure material properties of minor ampullate silks in a subset of these species. We also compare MiSp sequences and silk properties of our cobweb weavers to published data for orb-web weavers. We demonstrate that all our cobweb weavers possess multiple MiSp loci and that one locus is more highly expressed in at least two species. We also find that the proportion of ß-spiral-forming amino acid motifs in MiSp positively correlates with minor ampullate silk extensibility across orb-web and cobweb weavers. CONCLUSIONS: MiSp sequences vary dramatically within and among spider species, and have likely been subject to multiple rounds of gene duplication and concerted evolution, which have contributed to the diverse material properties of minor ampullate silks. Our sequences also provide templates for recombinant silk proteins with tailored properties.

Proteomic Evidence for Components of Spider Silk Synthesis from Black Widow Silk Glands and Fibers.

Spider silk research has largely focused on spidroins, proteins that are the primary components of spider silk fibers. Although a number of spidroins have been characterized, other types of proteins associated with silk synthesis are virtually unknown. Previous analyses of tissue-specific RNA-seq libraries identified 647 predicted genes that were differentially expressed in silk glands of the Western black widow, Latrodectus hesperus. Only ∼5% of these silk-gland specific transcripts (SSTs) encode spidroins; although the remaining predicted genes presumably encode other proteins associated with silk production, this is mostly unverified. Here, we used proteomic analysis of multiple silk glands and dragline silk fiber to investigate the translation of the differentially expressed genes. We find 48 proteins encoded by the differentially expressed transcripts in L. hesperus major ampullate, minor ampullate, and tubuliform silk glands and detect 17 SST encoded proteins in major ampullate silk fibers. The observed proteins include known silk-related proteins, but most are uncharacterized, with no annotation. These unannotated proteins likely include novel silk-associated proteins. Major and minor ampullate glands have the highest overlap of identified proteins, consistent with their shared, distinctive ampullate shape and the overlapping functions of major and minor ampullate silks. Our study substantiates and prioritizes predictions from differential expression analysis of spider silk gland transcriptomes.

Intragenic homogenization and multiple copies of prey-wrapping silk genes in Argiope garden spiders.

BACKGROUND: Spider silks are spectacular examples of phenotypic diversity arising from adaptive molecular evolution. An individual spider can produce an array of specialized silks, with the majority of constituent silk proteins encoded by members of the spidroin gene family. Spidroins are dominated by tandem repeats flanked by short, non-repetitive N- and C-terminal coding regions. The remarkable mechanical properties of spider silks have been largely attributed to the repeat sequences. However, the molecular evolutionary processes acting on spidroin terminal and repetitive regions remain unclear due to a paucity of complete gene sequences and sampling of genetic variation among individuals. To better understand spider silk evolution, we characterize a complete aciniform spidroin gene from an Argiope orb-weaving spider and survey aciniform gene fragments from congeneric individuals. RESULTS: We present the complete aciniform spidroin (AcSp1) gene from the silver garden spider Argiope argentata (Aar_AcSp1), and document multiple AcSp1 loci in individual genomes of A. argentata and the congeneric A. trifasciata and A. aurantia. We find that Aar_AcSp1 repeats have >98% pairwise nucleotide identity. By comparing AcSp1 repeat amino acid sequences between Argiope species and with other genera, we identify regions of conservation over vast amounts of evolutionary time. Through a PCR survey of individual A. argentata, A. trifasciata, and A. aurantia genomes, we ascertain that AcSp1 repeats show limited variation between species whereas terminal regions are more divergent. We also find that average dN/dS across codons in the N-terminal, repetitive, and C-terminal encoding regions indicate purifying selection that is strongest in the N-terminal region. CONCLUSIONS: Using the complete A. argentata AcSp1 gene and spidroin genetic variation between individuals, this study clarifies some of the molecular evolutionary processes underlying the spectacular mechanical attributes of aciniform silk. It is likely that intragenic concerted evolution and functional constraints on A. argentata AcSp1 repeats result in extreme repeat homogeneity. The maintenance of multiple AcSp1 encoding loci in Argiope genomes supports the hypothesis that Argiope spiders require rapid and efficient protein production to support their prolific use of aciniform silk for prey-wrapping and web-decorating. In addition, multiple gene copies may represent the early stages of spidroin diversification.

Dramatic expansion of the black widow toxin arsenal uncovered by multi-tissue transcriptomics and venom proteomics.

BACKGROUND: Animal venoms attract enormous interest given their potential for pharmacological discovery and understanding the evolution of natural chemistries. Next-generation transcriptomics and proteomics provide unparalleled, but underexploited, capabilities for venom characterization. We combined multi-tissue RNA-Seq with mass spectrometry and bioinformatic analyses to determine venom gland specific transcripts and venom proteins from the Western black widow spider (Latrodectus hesperus) and investigated their evolution. RESULTS: We estimated expression of 97,217 L. hesperus transcripts in venom glands relative to silk and cephalothorax tissues. We identified 695 venom gland specific transcripts (VSTs), many of which BLAST and GO term analyses indicate may function as toxins or their delivery agents. ~38% of VSTs had BLAST hits, including latrotoxins, inhibitor cystine knot toxins, CRISPs, hyaluronidases, chitinase, and proteases, and 59% of VSTs had predicted protein domains. Latrotoxins are venom toxins that cause massive neurotransmitter release from vertebrate or invertebrate neurons. We discovered ≥ 20 divergent latrotoxin paralogs expressed in L. hesperus venom glands, significantly increasing this biomedically important family. Mass spectrometry of L. hesperus venom identified 49 proteins from VSTs, 24 of which BLAST to toxins. Phylogenetic analyses showed venom gland specific gene family expansions and shifts in tissue expression. CONCLUSIONS: Quantitative expression analyses comparing multiple tissues are necessary to identify venom gland specific transcripts. We present a black widow venom specific exome that uncovers a trove of diverse toxins and associated proteins, suggesting a dynamic evolutionary history. This justifies a reevaluation of the functional activities of black widow venom in light of its emerging complexity.

Multi-tissue transcriptomics of the black widow spider reveals expansions, co-options, and functional processes of the silk gland gene toolkit.

BACKGROUND: Spiders (Order Araneae) are essential predators in every terrestrial ecosystem largely because they have evolved potent arsenals of silk and venom. Spider silks are high performance materials made almost entirely of proteins, and thus represent an ideal system for investigating genome level evolution of novel protein functions. However, genomic level resources remain limited for spiders. RESULTS: We de novo assembled a transcriptome for the Western black widow (Latrodectus hesperus) from deeply sequenced cDNAs of three tissue types. Our multi-tissue assembly contained ~100,000 unique transcripts, of which > 27,000 were annotated by homology. Comparing transcript abundance among the different tissues, we identified 647 silk gland-specific transcripts, including the few known silk fiber components (e.g. six spider fibroins, spidroins). Silk gland specific transcripts are enriched compared to the entire transcriptome in several functions, including protein degradation, inhibition of protein degradation, and oxidation-reduction. Phylogenetic analyses of 37 gene families containing silk gland specific transcripts demonstrated novel gene expansions within silk glands, and multiple co-options of silk specific expression from paralogs expressed in other tissues. CONCLUSIONS: We propose a transcriptional program for the silk glands that involves regulating gland specific synthesis of silk fiber and glue components followed by protecting and processing these components into functional fibers and glues. Our black widow silk gland gene repertoire provides extensive expansion of resources for biomimetic applications of silk in industry and medicine. Furthermore, our multi-tissue transcriptome facilitates evolutionary analysis of arachnid genomes and adaptive protein systems.

Ancient properties of spider silks revealed by the complete gene sequence of the prey-wrapping silk protein (AcSp1).

Spider silk fibers have impressive mechanical properties and are primarily composed of highly repetitive structural proteins (termed spidroins) encoded by a single gene family. Most characterized spidroin genes are incompletely known because of their extreme size (typically >9 kb) and repetitiveness, limiting understanding of the evolutionary processes that gave rise to their unusual gene architectures. The only complete spidroin genes characterized thus far form the dragline in the Western black widow, Latrodectus hesperus. Here, we describe the first complete gene sequence encoding the aciniform spidroin AcSp1, the primary component of spider prey-wrapping fibers. L. hesperus AcSp1 contains a single enormous (∼19 kb) exon. The AcSp1 repeat sequence is exceptionally conserved between two widow species (∼94% identity) and between widows and distantly related orb-weavers (∼30% identity), consistent with a history of strong purifying selection on its amino acid sequence. Furthermore, the 16 repeats (each 371-375 amino acids long) found in black widow AcSp1 are, on average, >99% identical at the nucleotide level. A combination of stabilizing selection on amino acid sequence, selection on silent sites, and intragenic recombination likely explains the extreme homogenization of AcSp1 repeats. In addition, phylogenetic analyses of spidroin paralogs support a gene duplication event occurring concomitantly with specialization of the aciniform glands and the tubuliform glands, which synthesize egg-case silk. With repeats that are dramatically different in length and amino acid composition from dragline spidroins, our L. hesperus AcSp1 expands the knowledge base for developing silk-based biomimetic technologies.

Complex gene expression in the dragline silk producing glands of the Western black widow (Latrodectus hesperus).

BACKGROUND: Orb-web and cob-web weaving spiders spin dragline silk fibers that are among the strongest materials known. Draglines are primarily composed of MaSp1 and MaSp2, two spidroins (spider fibrous proteins) expressed in the major ampullate (MA) silk glands. Prior genetic studies of dragline silk have focused mostly on determining the sequence of these spidroins, leaving other genetic aspects of silk synthesis largely uncharacterized. RESULTS: Here, we used deep sequencing to profile gene expression patterns in the Western black widow, Latrodectus hesperus. We sequenced millions of 3'-anchored "tags" of cDNAs derived either from MA glands or control tissue (cephalothorax) mRNAs, then associated the tags with genes by compiling a reference database from our newly constructed normalized L. hesperus cDNA library and published L. hesperus sequences. We were able to determine transcript abundance and alternative polyadenylation of each of three loci encoding MaSp1. The ratio of MaSp1:MaSp2 transcripts varied between individuals, but on average was similar to the estimated ratio of MaSp1:MaSp2 in dragline fibers. We also identified transcription of TuSp1 in MA glands, another spidroin family member that encodes the primary component of egg-sac silk, synthesized in tubuliform glands. In addition to the spidroin paralogs, we identified 30 genes that are more abundantly represented in MA glands than cephalothoraxes and represent new candidates for involvement in spider silk synthesis. CONCLUSIONS: Modulating expression rates of MaSp1 variants as well as MaSp2 and TuSp1 could lead to differences in mechanical properties of dragline fibers. Many of the newly identified candidate genes likely encode secreted proteins, suggesting they could be incorporated into dragline fibers or assist in protein processing and fiber assembly. Our results demonstrate previously unrecognized transcript complexity in spider silk glands.

The evolutionary history of cribellate orb-weaver capture thread spidroins.

BACKGROUND: Spiders have evolved two types of sticky capture threads: one with wet adhesive spun by ecribellate orb-weavers and another with dry adhesive spun by cribellate spiders. The evolutionary history of cribellate capture threads is especially poorly understood. Here, we use genomic approaches to catalog the spider-specific silk gene family (spidroins) for the cribellate orb-weaver Uloborus diversus. RESULTS: We show that the cribellar spidroin, which forms the puffy fibrils of cribellate threads, has three distinct repeat units, one of which is conserved across cribellate taxa separated by ~ 250 Mya. We also propose candidates for a new silk type, paracribellar spidroins, which connect the puffy fibrils to pseudoflagelliform support lines. Moreover, we describe the complete repeat architecture for the pseudoflagelliform spidroin (Pflag), which contributes to extensibility of pseudoflagelliform axial fibers. CONCLUSIONS: Our finding that Pflag is closely related to Flag, supports homology of the support lines of cribellate and ecribellate capture threads. It further suggests an evolutionary phase following gene duplication, in which both Flag and Pflag were incorporated into the axial lines, with subsequent loss of Flag in uloborids, and increase in expression of Flag in ecribellate orb-weavers, explaining the distinct mechanical properties of the axial lines of these two groups.

Gene expression profiling reveals candidate genes for defining spider silk gland types.

Molecular studies of the secretory glands involved in spider silk production have revealed candidate genes for silk synthesis and a complicated history of spider silk gene evolution. However, differential gene expression profiles of the multiple silk gland types within an individual orb-web weaving spider are lacking. Each of these gland types produces a functionally distinct silk type. Comparison of gene expression among spider silk gland types would provide insight into the genes that define silk glands generally from non-silk gland tissues, and the genes that define silk glands from each other. Here, we perform 3' tag digital gene expression profiling of the seven silk gland types of the silver garden orb weaver Argiope argentata. Five of these gland types produce silks that are non-adhesive fibers, one silk includes both fibers and glue-like adhesives, and one silk is exclusively glue-like. We identify 1275 highly expressed, significantly upregulated, and tissue specific silk gland specific transcripts (SSTs). These SSTs include seven types of spider silk protein encoding genes known as spidroin genes. We find that the fiber-producing major ampullate and minor ampullate silk glands have more similar expression profiles than any other pair of glands. We also find that a subset of the SSTs is enriched for transmembrane transport and oxidoreductases, and that these transcripts highlight differences and similarities among the major ampullate, minor ampullate, and aggregate silk glands. Furthermore, we show that the wet glue-producing aggregate glands have the most unique SSTs, but still share some SSTs with fiber producing glands. Aciniform glands were the only gland type to share a majority of SSTs with other silk gland types, supporting previous hypotheses that duplication of aciniform glands and subsequent divergence of the duplicates gave rise to the multiple silk gland types within an individual spider.

Protein Composition and Associated Material Properties of Cobweb Spiders' Gumfoot Glue Droplets.

The origin of aggregate silk glands and their production of wet adhesive silks is considered a key innovation of the Araneoidea, a superfamily of spiders that build orb-webs and cobwebs. Orb-web weavers place aggregate glue on an extensible capture spiral, whereas cobweb weavers add it to the ends of strong, stiff fibers, called gumfoot lines. Here we describe the material behavior and quantitative proteomics of the aggregate glues of two cobweb weaving species, the western black widow, Latrodectus hesperus, and the common house spider, Parasteatoda tepidariorum. For each species, respectively, we identified 48 and 33 proteins that were significantly more abundant in the portion of the gumfoot line with glue than in its fibers. These proteins were more highly glycosylated and phosphorylated than proteins found in silk fibers without glue, which likely explains aggregate glue stickiness. Most glue-enriched proteins were of anterior aggregate gland origin, supporting the hypothesis that cobweb weavers' posterior aggregate glue is specialized for another function. We found that cobweb weaver glue droplets are stiffer and tougher than the adhesive of most orb-web weaving species. Attributes of gumfoot glue protein composition that likely contribute to this stiffness include the presence of multiple protein families with conserved cysteine residues, a bimodal distribution of isoelectric points, and families with conserved functions in protein aggregation, all of which should contribute to cohesive protein-protein interactions. House spider aggregate droplets were more responsive to humidity changes than black widow droplets, which could be mediated by differences in protein sequence, post-translational modifications, the non-protein components of the glue droplets, and/or the larger amount of aqueous material that surrounds the adhesive cores of their glue droplets.

Untangling spider silk evolution with spidroin terminal domains.

BACKGROUND: Spidroins are a unique family of large, structural proteins that make up the bulk of spider silk fibers. Due to the highly variable nature of their repetitive sequences, spidroin evolutionary relationships have principally been determined from their non-repetitive carboxy (C)-terminal domains, though they offer limited character data. The few known spidroin amino (N)-terminal domains have been difficult to obtain, but potentially contain critical phylogenetic information for reconstructing the diversification of spider silks. Here we used silk gland expression data (ESTs) from highly divergent species to evaluate the functional significance and phylogenetic utility of spidroin N-terminal domains. RESULTS: We report 11 additional spidroin N-termini found by sequencing approximately 1,900 silk gland cDNAs from nine spider species that shared a common ancestor > 240 million years ago. In contrast to their hyper-variable repetitive regions, spidroin N-terminal domains have retained striking similarities in sequence identity, predicted secondary structure, and hydrophobicity. Through separate and combined phylogenetic analyses of N-terminal domains and their corresponding C-termini, we find that combined analysis produces the most resolved trees and that N-termini contribute more support and less conflict than the C-termini. These analyses show that paralogs largely group by silk gland type, except for the major ampullate spidroins. Moreover, spidroin structural motifs associated with superior tensile strength arose early in the history of this gene family, whereas a motif conferring greater extensibility convergently evolved in two distantly related paralogs. CONCLUSIONS: A non-repetitive N-terminal domain appears to be a universal attribute of spidroin proteins, likely retained from the origin of spider silk production. Since this time, spidroin N-termini have maintained several features, consistent with this domain playing a key role in silk assembly. Phylogenetic analyses of the conserved N- and C-terminal domains illustrate dramatic radiation of the spidroin gene family, involving extensive duplications, shifts in expression patterns and extreme diversification of repetitive structural sequences that endow spider silks with an unparalleled range of mechanical properties.

The common house spider, Parasteatoda tepidariorum, maintains silk gene expression on sub-optimal diet.

Cobweb weaving spiders and their relatives spin multiple task-specific fiber types. The unique material properties of each silk type result from differences in amino acid sequence and structure of their component proteins, primarily spidroins (spider fibrous proteins). Amino acid content and gene expression measurements of spider silks suggest some spiders change expression patterns of individual protein components in response to environmental cues. We quantified mRNA abundance of three spidroin encoding genes involved in prey capture in the common house spider, Parasteatoda tepidariorum (Theridiidae), fed different diets. After 10 days of acclimation to the lab on a diet of mealworms, spiders were split into three groups: (1) individuals were immediately dissected, (2) spiders were fed high-energy crickets, or (3) spiders were fed low-energy flies, for 1 month. All spiders gained mass during the acclimation period and cricket-fed spiders continued to gain mass, while fly-fed spiders either maintained or lost mass. Using quantitative PCR, we found no significant differences in the absolute or relative abundance of dragline gene transcripts, major ampullate spidroin 1 (MaSp1) and major ampullate spidroin 2 (MaSp2), among groups. In contrast, prey-wrapping minor ampullate spidroin (MiSp) gene transcripts were significantly less abundant in fly-fed than lab-acclimated spiders. However, when measured relative to Actin, cricket-fed spiders showed the lowest expression of MiSp. Our results suggest that house spiders are able to maintain silk production, even in the face of a low-quality diet.

Ovarian Transcriptomic Analyses in the Urban Human Health Pest, the Western Black Widow Spider.

Due to their abundance and ability to invade diverse environments, many arthropods have become pests of economic and health concern, especially in urban areas. Transcriptomic analyses of arthropod ovaries have provided insight into life history variation and fecundity, yet there are few studies in spiders despite their diversity within arthropods. Here, we generated a de novo ovarian transcriptome from 10 individuals of the western black widow spider (Latrodectus hesperus), a human health pest of high abundance in urban areas, to conduct comparative ovarian transcriptomic analyses. Biological processes enriched for metabolism-specifically purine, and thiamine metabolic pathways linked to oocyte development-were significantly abundant in L. hesperus. Functional and pathway annotations revealed overlap among diverse arachnid ovarian transcriptomes for highly-conserved genes and those linked to fecundity, such as oocyte maturation in vitellogenin and vitelline membrane outer layer proteins, hormones, and hormone receptors required for ovary development, and regulation of fertility-related genes. Comparative studies across arachnids are greatly needed to understand the evolutionary similarities of the spider ovary, and here, the identification of ovarian proteins in L. hesperus provides potential for understanding how increased fecundity is linked to the success of this urban pest.

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.

Multiple recombining loci encode MaSp1, the primary constituent of dragline silk, in widow spiders (Latrodectus: Theridiidae).

Spiders spin a functionally diverse array of silk fibers, each composed of one or more unique proteins. Most of these proteins, in turn, are encoded by members of a single gene family thought to have arisen through duplication and divergence of an ancestral silk gene. Because of its remarkable mechanical properties, orb weaver dragline silk, a composite of 2 proteins (MaSp1 and MaSp2), is the best studied. Here, we demonstrate that multiple loci encode MaSp1 in widow spiders (Latrodectus). Because these copies may be the result of more recent duplication events than those leading to the currently recognized silk gene paralogs, they offer insight into the early evolutionary fate of silk gene duplicates. In addition to 3 presumed functional MaSp1 loci in Latrodectus hesperus (Western black widow) and Latrodectus geometricus (brown widow) genomes, we find a MaSp1 pseudogene in L. hesperus, demonstrating the potential for unrecognized extinction of silk gene paralogs. We also document recombination events among L. hesperus MaSp1 loci and between Latrodectus MaSp1 loci and MaSp2. This result supports the hypothesis that concerted evolution occurs not only within an individual silk gene but also among silk gene paralogs. One of the L. geometricus MaSp1 copies encodes a protein that has diverged in amino acid composition and potentially converged on the secondary structure of MaSp2. Based on the presence of multiple MaSp1 loci and the phylogenetic distribution of MaSp1 versus MaSp2, we propose that MaSp2 is derived from an ancestral MaSp1 duplicate. Finally, divergence has occurred in the upstream flanking sequences of the L. hesperus MaSp1 loci, the region most likely to contain regulatory motifs, providing ample opportunity for differential expression. However, the benefits associated with increased protein production may be the primary mechanism maintaining multiple functional MaSp1 copies in widow genomes.

Recent progress and prospects for advancing arachnid genomics.

Arachnids exhibit tremendous species richness and adaptations of biomedical, industrial, and agricultural importance. Yet genomic resources for arachnids are limited, with the first few spider and scorpion genomes becoming accessible in the last four years. We review key insights from these genome projects, and recommend additional genomes for sequencing, emphasizing taxa of greatest value to the scientific community. We suggest greater sampling of spiders whose genomes are understudied but hold important protein recipes for silk and venom production. We further recommend arachnid genomes to address significant evolutionary topics, including the phenotypic impact of genome duplications. A barrier to high-quality arachnid genomes are assemblies based solely on short-read data, which may be overcome by long-range sequencing and other emerging methods.

Genomic perspectives of spider silk genes through target capture sequencing: Conservation of stabilization mechanisms and homology-based structural models of spidroin terminal regions.

A powerful system for studying protein aggregation, particularly rapid self-assembly, is spider silk. Spider silks are proteinaceous and silk proteins are synthesized and stored within silk glands as liquid dope. As needed, liquid dope is near-instantaneously transformed into solid fibers or viscous adhesives. The dominant constituents of silks are spidroins (spider fibroins) and their terminal domains are vital for the tight control of silk self-assembly. To better understand spidroin termini, we used target capture and deep sequencing to identify spidroin gene sequences from six species representing the araneoid families of Araneidae, Nephilidae, and Theridiidae. We obtained 145 terminal regions, of which 103 are newly annotated here, as well as novel variants within nine diverse spidroin types. Our comparative analyses demonstrated the conservation of acidic, basic, and cysteine amino acid residues across spidroin types that had been proposed to be important for monomer stability, dimer formation, and self-assembly from a limited sampling of spidroins. Computational, protein homology modeling revealed areas of spidroin terminal regions that are highly conserved in three-dimensions despite sequence divergence across spidroin types. Analyses of our dense sampling of terminal regions suggest that most spidroins share stabilization mechanisms, dimer formation, and tertiary structure, despite producing functionally distinct materials.

Evolutionary shifts in gene expression decoupled from gene duplication across functionally distinct spider silk glands.

Spider silk synthesis is an emerging model for the evolution of tissue-specific gene expression and the role of gene duplication in functional novelty, but its potential has not been fully realized. Accordingly, we quantified transcript (mRNA) abundance in seven silk gland types and three non-silk gland tissues for three cobweb-weaving spider species. Evolutionary analyses based on expression levels of thousands of homologous transcripts and phylogenetic reconstruction of 605 gene families demonstrated conservation of expression for each gland type among species. Despite serial homology of all silk glands, the expression profiles of the glue-forming aggregate glands were divergent from fiber-forming glands. Also surprising was our finding that shifts in gene expression among silk gland types were not necessarily coupled with gene duplication, even though silk-specific genes belong to multi-paralog gene families. Our results challenge widely accepted models of tissue specialization and significantly advance efforts to replicate silk-based high-performance biomaterials.