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Modular Evolution of Egg Case Silk Genes Across Orb-Weaving Spider Superfamilies

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Modular Evolution of Egg Case Silk Genes Across Orb-Weaving Spider Superfamilies Modular evolution of egg case silk genes across orb-weaving spider superfamilies Jessica E. Garb* and Cheryl Y. Hayashi Department of Biology, University of California, Riverside, CA 92521 Edited by M. T. Clegg, University of California, Irvine, CA, and approved June 13, 2005 (received for review March 25, 2005) Spider silk proteins (fibroins) are renowned for their extraordinary many presumed members of the spidroin gene family remain mechanical properties and biomimetic potential. Despite extensive uncharacterized at the nucleotide level. evolutionary, ecological, and industrial interest in these fibroins, Spiders construct egg cases, protective cocoons where eggs only a fraction of the known silk types have been characterized at undergo development, primarily from silk fibers synthesized by the the molecular level. Here we report cDNA and genomic sequences tubuliform (cylindrical) silk glands, and tubuliform fibers are ex- of the fibroin TuSp1, which appears to be the major component of clusively used for egg case construction (6, 11). Tubuliform glands tubuliform gland silk, a fiber exclusively synthesized by female are found only in female spiders, and silk synthesis is initiated in spiders for egg case construction. We obtained TuSp1 sequences these glands at sexual maturation (6, 12). Guerette et al. (4) cloned from 12 spider species that represent the extremes of phylogenetic a putative component of egg case silk (ADF-2) expressed in diversity within the Orbicularia (orb-weaver superfamilies, Arane- tubuliform glands of the orb-weaving spider Araneus diadematus. oidea and Deinopoidea) and finer scale sampling within genera. Examination of ADF-2, in comparison with additional spidroins, TuSp1 encodes tandem arrays of an Ϸ200-aa-long repeat unit and subsequently identified it as a member of the major ampullate individual repeats are readily aligned, even among species that spidroin 1 (MaSp1) silk ortholog group (7), used in dragline fibers diverged >125 million years ago. Analyses of these repeats across (13, 14). Recently, Hu et al. (15) reported another putative com- species reveal the strong influence of concerted evolution, result- ponent of egg case silk (ECP-1) expressed in silk glands of the black ing in intragenic homogenization. However, deinopoid TuSp1 widow spider, Latrodectus hesperus. However, the translated se- repeats also contain insertions of coding, minisatellite-like se- quence of neither ADF-2 nor ECP-1 could account for the amino quences, an apparent result of replication slippage and nonrecip- acid composition of tubuliform silk fibers or the gland’s contents (4, rocal recombination. Phylogenetic analyses of 37 spider fibroin 11, 15, 16, 17), indicating that the dominant silk expressed by sequences support the monophyly of TuSp1 within the spider tubuliform glands must be a different, unidentified protein (4). fibroin gene family, consistent with a single origin of this ortholog Here we report cDNA and genomic sequences of a spider silk group. The diversity of taxa and silks examined here confirms that gene that exhibits features consistent with its protein product being repetitive architecture is a general feature of this gene family. a major component of tubuliform silk fibers. The tubuliform gene Moreover, we show that TuSp1 provides a clear example of (TuSp1) shares the distinctive molecular architecture characteristic of members of the spidroin gene family. We found the TuSp1 modular evolution across a range of phylogenetic levels. transcript in high copy number in tubuliform silk gland cDNA libraries constructed from two araneoid species, the garden spider concerted evolution ͉ fibroin ͉ gene family ͉ spider silk ͉ TuSp1 Argiope argentata (Araneidae) and the black widow spider L. hesperus (Theridiidae), as well as in the total silk gland cDNA defining feature of the diverse order Araneae (Ͼ37,000 libraries made from two species of the divergent Deinopoidea, a Adescribed species) (1) is the ability to spin multiple task- feather-legged spider, Uloborus diversus (Uloboridae), and an ogre- specific silks. Spiders use different combinations of silk proteins faced spider, Deinopis spinosa (Deinopidae). Genomic fragments of (fibroins) to construct structures for reproduction, prey capture, the tubuliform silk gene were sequenced from Argiope argentata and and locomotion (2, 3). Silk fibers are assembled from one or more L. hesperus in addition to eight other araneoid species. In view of fibroins that are expressed in abdominal glands connected to the the diversity of species from which we obtained TuSp1 sequences, spinnerets (4). Silk gland morphology varies extensively across we investigated the evolution of the gene’s modular organization species. For example, members of the infraorder Mygalomorphae and the phylogenetic relationship of TuSp1 to other members of the possess one or two types of globular silk glands (5, 6). In contrast, spidroin gene family. an individual araneoid spider (ecribellate orb-web weaver) has seven morphologically distinct types of silk glands (3), each of which Materials and Methods synthesizes different suites of silk proteins (4, 7). Spider Collection and Dissections. We obtained Argiope argentata The amino acid sequences of spider fibroins (spidroins) share a (A.ar.) from Encinitas, CA; Deinopis spinosa (D.s.) from Gaines- number of distinctive features. Iterations of four simple amino acid ville, FL; and L. hesperus (L.he.) and U. diversus (U.d.) from motifs characterize the majority of sequenced spider silks: (i) Riverside, CA. Tubuliform glands were dissected from A.ar. and polyalanine (An), (ii) alternating glycine and alanine [(GA) n], (iii) L.he. and frozen in liquid nitrogen. Total silk glands (tubuliform GGX (X ϭ subset of residues), and (iv) GPGXn. Previous studies and other silk glands) dissected from U.d. and D.s. were similarly indicate that these motifs correspond to distinct structural modules frozen. Taxa used for PCR amplification were obtained from the [e.g., An and (GA)n form crystalline ␤-sheets, GPGXn forms EVOLUTION ␤-spirals], and it is hypothesized that different proportions of these modules dictate fiber mechanics (8). Combinations of these amino This paper was submitted directly (Track II) to the PNAS office. acid motifs are organized into a larger unit, termed the ensemble Abbreviations: A.ar., Argiope argentata; D.s., Deinopis spinosa; L.he., Latrodectus hespe- rus; U.d., Uloborus diversus; L.g., Latrodectus geometricus; N.c., Nephila clavipes; P.t., repeat, which is tandem-arrayed throughout the silk sequence (7, 8). Plectreurys tristis; TuSp1, tubuliform spidroin 1; MaSp, major ampullate spidroin; MiSp, In addition to their similar repetitive architecture, all spidroins have minor ampullate spidroin; ML, maximum likelihood. a nonrepetitive C terminus exhibiting length and sequence conser- Data deposition: The sequences reported in this paper have been deposited in the GenBank vation (4, 7, 9, 10). These shared features are consistent with database (accession nos. AY953070–AY953093). spidroins being encoded by members of a gene family that func- *To whom correspondence should be addressed. E-mail: [email protected]. tionally diversified after gene duplication events (4, 7). However, © 2005 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0502473102 PNAS ͉ August 9, 2005 ͉ vol. 102 ͉ no. 32 ͉ 11379–11384 Downloaded by guest on October 1, 2021 following localities: (i) family Araneidae: Argiope aurantia, Colum- TuSp1 sequences and across species, were aligned by CLUSTAL W bus, OH; Cyrtophora moluccensis, Queensland, Australia; Gea (MACVECTOR 7.2, Accelrys, San Diego), refined by eye, and used to heptagon, Loudon, TN; and (ii) family Theridiidae: L.he., Yarnell, align the encoding nucleotides. A neighbor-joining tree of TuSp1 AZ; Lactrodectus mactans, Dominican Republic; Lactrodectus geo- repeats was constructed by using PAUP* (19), based on uncorrected metricus (L.g.), San Diego, CA; Lactrodectus hasselti, Perth, Aus- genetic distance; support was evaluated by 10,000 bootstrap repli- tralia; Lactrodectus tredecimguttatus, Israel; Steatoda grossa, cates. Translations of the silk C termini were combined with Haleakala National Park, HI. published silk cDNA C termini in a phylogenetic analysis of the spider fibroin gene family. C termini were aligned by CLUSTAL W, cDNA Library Construction and Screening. Total RNA was extracted refined by eye, and used to align encoding nucleotide sequences. from silk gland tissue (tubuliform glands or all glands) by homog- The A.ar. TuSp1 genomic C- terminal sequence was identical to the enization in TRIzol (Invitrogen), followed by purification with an A.ar. TuSp1 cDNA sequence at the DNA level; thus, the genomic RNeasy Mini Kit (Qiagen). mRNA was extracted from total RNA sequence was not included in phylogenetic analyses. Heuristic with oligo(dT)25-tagged magnetic beads (Dynal, Brown Deer, WI). parsimony and maximum likelihood (ML) tree searches were cDNA was synthesized by using the SuperScript II Choice protocol conducted in PAUP*. Parsimony searches treated characters as (Invitrogen) with an anchored oligo(dT)18V primer. ChromaSpin unweighted with gaps as a 5th state, and included 1,000 random 1000 columns (Clontech) were used to select cDNA fragments taxon addition replicates; clade support was assessed by 10,000 Ն1,000 bp. cDNA fragments were ligated into EcoRV-digested bootstrap replicates with 100 random taxon addition replicates and (New England Biolabs) pZErO-2 plasmids
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