Modular evolution of egg case silk genes across orb-weaving spider superfamilies

Abstract

Spider silk proteins (fibroins) are renowned for their extraordinary mechanical properties and biomimetic potential. Despite extensive evolutionary, ecological, and industrial interest in these fibroins, only a fraction of the known silk types have been characterized at the molecular level. Here we report cDNA and genomic sequences of the fibroin TuSp1, which appears to be the major component of tubuliform gland silk, a fiber exclusively synthesized by female spiders for egg case construction. We obtained TuSp1 sequences from 12 spider species that represent the extremes of phylogenetic diversity within the Orbicularia (orb-weaver superfamilies, Araneoidea and Deinopoidea) and finer scale sampling within genera. TuSp1 encodes tandem arrays of an approximately 200-aa-long repeat unit and individual repeats are readily aligned, even among species that diverged >125 million years ago. Analyses of these repeats across species reveal the strong influence of concerted evolution, resulting in intragenic homogenization. However, deinopoid TuSp1 repeats also contain insertions of coding, minisatellite-like sequences, an apparent result of replication slippage and nonreciprocal recombination. Phylogenetic analyses of 37 spider fibroin sequences support the monophyly of TuSp1 within the spider fibroin gene family, consistent with a single origin of this ortholog group. The diversity of taxa and silks examined here confirms that repetitive architecture is a general feature of this gene family. Moreover, we show that TuSp1 provides a clear example of modular evolution across a range of phylogenetic levels.

Fig. 1.

Organization of TuSp1. (A) Schematic view of TuSp1 primary structure. Ovals, individual repeat units numbered starting from the C terminus; arrows, additional sequence toward the N terminus. (B) Alignment of individual repeat units, with unit number and color as in A; gen., repeats obtained by PCR; *, sites in alignment with invariant residue; amino acids colored as follows: A, blue; S, yellow; L, dark purple; G, light purple; V, green; Q, pink; T, light green; numbered carets, positions of expansions; white space, data missing from upstream cDNA sequence or not obtained by PCR; Xs, codons with ambiguous nucleotides; L.m., L. mactans; L.t., L. tredecimguttatus; L.ha., L. hasselti; S.g., S. grossa; A.au., Argiope aurantia; G.h., Gea heptagon.

Fig. 2.

Neighbor-joining tree of repeat units, based on DNA sequences of alignment in Fig. 1B. Numbered and colored ovals, repeat units as labeled in Fig. 1; values at nodes, percent times recovered in 10,000 bootstrap replicates; mid-point rooted; horizontal branch lengths proportional to number of changes; L.m., L. mactans; L.t., L. tredecimguttatus; S.g., S. grossa; A.au., Argiope aurantia; G.h., Gea heptagon.

Fig. 3.

Phylogeny of spidroin gene family: one of two ML trees of C termini (–ln L = 8,378.76). Genomic sequences are labeled with superscript G, and all others are cDNAs. Three circles at branches denote heuristic parsimony bootstrap, ML bootstrap, and clade posterior probabilities support, respectively with coloration indicating percentage values: white, <50; green, 50–69; yellow, 70–89; red, ≥90. Number below branch indicates decay index. The tree is rooted with mygalomorph E.c. fibroin 1; other sequences are from Araneomorphae (sister to Mygalomorphae). GenBank accessions for additional sequences: N.c. MaSp1, U20329; Araneus diadematus ADF1–4, U47853–U47856; N.c. MaSp2, M92913; N.c. MiSp1, AF027735; N.c. MiSp, AF027736; Argiope trifasciata (A.t.) AcSp1, AY426339; N.c. Flag, AF027973; A.t. Flag, AF350264; A.t. MaSp1–2, AF350266–AF350267; Dolomedes tenebrosus (D.t.) fibroin 1, D.t. fibroin 2, E. chisoseus (E.c.) fibroin 1, AF350269–AF350271; L.g. MaSp1, AF350273; L.g. MaSp2, AF350275; P.t. fibroin 1–4, AF350281–AF350284; Agelenopsis aperta (A.ap.) MaSp, AY566305.