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

Early events in the evolution of spider silk genes.

Silk spinning is essential to spider ecology and has had a key role in the expansive diversification of spiders. Silk is composed primarily of proteins called spidroins, which are encoded by a multi-gene family. Spidroins have been studied extensively in the derived clade, Orbiculariae (orb-weavers), from the suborder Araneomorphae ('true spiders'). Orbicularians produce a suite of different silks, and underlying this repertoire is a history of duplication and spidroin gene divergence. A second class of silk proteins, Egg Case Proteins (ECPs), is known only from the orbicularian species, Lactrodectus hesperus (Western black widow). In L. hesperus, ECPs bond with tubuliform spidroins to form egg case silk fibers. Because most of the phylogenetic diversity of spiders has not been sampled for their silk genes, there is limited understanding of spidroin gene family history and the prevalence of ECPs. Silk genes have not been reported from the suborder Mesothelae (segmented spiders), which diverged from all other spiders >380 million years ago, and sampling from Mygalomorphae (tarantulas, trapdoor spiders) and basal araneomorph lineages is sparse. In comparison to orbicularians, mesotheles and mygalomorphs have a simpler silk biology and thus are hypothesized to have less diversity of silk genes. Here, we present cDNAs synthesized from the silk glands of six mygalomorph species, a mesothele, and a non-orbicularian araneomorph, and uncover a surprisingly rich silk gene diversity. In particular, we find ECP homologs in the mesothele, suggesting that ECPs were present in the common ancestor of extant spiders, and originally were not specialized to complex with tubuliform spidroins. Furthermore, gene-tree/species-tree reconciliation analysis reveals that numerous spidroin gene duplications occurred after the split between Mesothelae and Opisthothelae (Mygalomorphae plus Araneomorphae). We use the spidroin gene tree to reconstruct the evolution of amino acid compositions of spidroins that perform different ecological functions

Early Events in the Evolution of Spider Silk Genes

<div><p>Silk spinning is essential to spider ecology and has had a key role in the expansive diversification of spiders. Silk is composed primarily of proteins called spidroins, which are encoded by a multi-gene family. Spidroins have been studied extensively in the derived clade, Orbiculariae (orb-weavers), from the suborder Araneomorphae (‘true spiders’). Orbicularians produce a suite of different silks, and underlying this repertoire is a history of duplication and spidroin gene divergence. A second class of silk proteins, Egg Case Proteins (ECPs), is known only from the orbicularian species, <em>Lactrodectus hesperus</em> (Western black widow). In <em>L. hesperus,</em> ECPs bond with tubuliform spidroins to form egg case silk fibers. Because most of the phylogenetic diversity of spiders has not been sampled for their silk genes, there is limited understanding of spidroin gene family history and the prevalence of ECPs. Silk genes have not been reported from the suborder Mesothelae (segmented spiders), which diverged from all other spiders >380 million years ago, and sampling from Mygalomorphae (tarantulas, trapdoor spiders) and basal araneomorph lineages is sparse. In comparison to orbicularians, mesotheles and mygalomorphs have a simpler silk biology and thus are hypothesized to have less diversity of silk genes. Here, we present cDNAs synthesized from the silk glands of six mygalomorph species, a mesothele, and a non-orbicularian araneomorph, and uncover a surprisingly rich silk gene diversity. In particular, we find ECP homologs in the mesothele, suggesting that ECPs were present in the common ancestor of extant spiders, and originally were not specialized to complex with tubuliform spidroins. Furthermore, gene-tree/species-tree reconciliation analysis reveals that numerous spidroin gene duplications occurred after the split between Mesothelae and Opisthothelae (Mygalomorphae plus Araneomorphae). We use the spidroin gene tree to reconstruct the evolution of amino acid compositions of spidroins that perform different ecological functions.</p> </div

Spidroin gene tree with inferred duplication events.

<p>Spidroin gene tree is based on a ML analysis of the carboxy-terminal encoding region with gaps coded as binary characters and monophyly of some groups constrained (see Methods). Numbers next to nodes and terminals correspond to numbers in supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038084#pone.0038084.s002" target="_blank">Tables S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038084#pone.0038084.s003" target="_blank">S2</a> showing support values, alternate rootings, and continuous character data. Spidroins are colored according to the taxonomic group from which they were characterized: purple = Mesothelae, blue = Mygalomorphae, green = Araneomorphae. Gray squares indicate duplication events inferred by reconciliation. Hash marks on branch indicate arbitrary shortening of branch for figure quality purposes. Brackets indicate clades with the following abbreviations: AcSp = Aciniform, TuSp = Tubuliform, PySp = Pyriform, MaSp = Major ampullate, MiSp = Minor ampullate, Flag = Flagelliform.</p

Majority rule consensus of ensemble repeats within spidroins.

<p>Ensemble repeats are tandemly arrayed. Amino acid sequences with single letter abbreviations are shown. Alanine (red), serine (blue), and glycine (green) are highlighted. Single amino acids repeated in tandem are underlined. Repeat lengths are given in parentheses.</p

Alignment of Egg Case Proteins (ECPs) and Egg Case Protein-like proteins (ECPLs).

<p>A) Schematic of alignment of <i>Latrodectus hesperus</i> ECPs and <i>Liphistius malayanus</i> ECPLs. B) Alignment of amino acid sequences, abbreviated using single letters. Only partial <i>Latrodectus</i> (Latr) ECPs are shown as <i>Liphistius</i> (Liph) ECPLs lack the extended repetitive region. Alignment columns were highlighted using GeneDoc <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038084#pone.0038084-Nicholas1" target="_blank">[67]</a> according to physiochemical properties (Text color/Shade color: Proline Blue/Red; Glycine Green/Red; Tiny Blue/Yellow; Small Green/Yellow; Positive Red/Blue; Negative Green/Blue; Charged White/Blue; Amphoteric Red/Green; Polar Black/Green; Aliphatic Red/Gray; Aromatic Blue/Gray; Hydrophobic White/Black). Upper-case single letters occur above alignment positions showing 100% amino acid conservation, while lower case single letters occur above positions showing >50% conservation.</p

Phylogeny for spider groups analyzed in this study.

<p>Phylogeny is based on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038084#pone.0038084-Coddington1" target="_blank">[2]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038084#pone.0038084-Hedin1" target="_blank">[48]</a>.</p

Alignment of DNA sequences for <i>Liphistius</i> fib1 repeats.

<p>Amino acid translation and DNA consensus sequences are above repeat sequences. Dots indicate identity to the consensus sequence. Non-synonymous and synonymous differences from the consensus are indicated by upper and lower case letters, respectively.</p

Heat map of percent compositions of alanine, glycine, and serine from spidroin repetitive regions.

<p>Cladogram adjacent to heat map shows relationships as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038084#pone-0038084-g003" target="_blank">Figure 3</a>. <i>Hexura</i> fib1 was omitted since no repetitive region sequence was obtained for that cDNA. Here, red indicates levels furthest below the mean, while white indicates levels furthest above the mean. Histograms on columns also show relative composition levels of the three amino acids across spidroins. Spidroin colors and abbreviations for clade names are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038084#pone-0038084-g003" target="_blank">Figure 3</a>. Numbers at nodes correspond to information in supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038084#pone.0038084.s002" target="_blank">Tables S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038084#pone.0038084.s003" target="_blank">S2</a>.</p