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

Abstract

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.

Figure 1

Schematic of the protein encoded by the complete Argiope argentata aciniform spidroin 1 gene (GenBank KJ206620). Predicted protein is 4,479 aa. Conserved spidroin N- (orange) and C- terminal (blue) domains (shaded boxes) flank 20 iterated repeats (numbered boxes). Boxes are drawn to scale and standardized to the 204 aa length of the first 19 repeats. Arrows point to corresponding amino acid sequences for each domain. Exemplar repeat 11 has 100% identity to the majority rule consensus of the repeat sequences. Alanine (red), serine (purple), and glycine (green) are shaded to emphasize the abundance of those amino acids.

Figure 2

Maximum likelihood tree of concatenated N- and C-terminal coding regions from 29 published spidroins and Aar_AcSp1 from this study (accession numbers in Additional file 2: Table S2). Box highlights aciniform clade, with Argiope argentata AcSp1 further indicated in red. Vertical bars identify clades by silk type. Bootstrap values greater than 70% are shown. Abbreviations defined in Additional file 2: Table S2. Scale bar represents substitutions per site.

Figure 3

Concerted evolution and selection on the repetitive region of AcSp1. (A) Iterated AcSp1 repeats show intra-specific homogenization in the family Araneidae. Midpoint-rooted maximum likelihood tree of AcSp1 DNA repeats (R) from A. amoena (Aam; purple), A. argentata (Aar; orange), A. trifasciata (At; green), and Araneus ventricosus (Av; black). Repeats are numbered from 5’ to 3’. Bootstrap values for species-specific groups are shown. Scale bar indicates substitutions per site. (B) Functional constraint on repeat sequence. Graph of the pairwise identities of consensus AcSp1 repeat sequences from two Argiope species (A. argentata and A. amoena) and Araneus ventricosus to that of Argiope trifasciata. Bars show 100% (green), 66% (yellow), or 33% (red) identity at each position, helical domains found by NMR and DANGLE analyses of A. trifasciata AcSp1 repeat sequence shown in schematic under the graph (helix redrawn from [25]).

Figure 4

Multiple AcSp1 loci in Argiope. Maximum likelihood nucleotide trees of PCR amplified sequences from the (A) repetitive, (B) N-terminal, and (C) C-terminal coding regions of AcSp1 from three Argiope species, A. argentata (Aarg; orange), A. aurantia (Aau; blue), and A. trifasciata (At; green). Araneus ventricosus (Av; black) sequence (GenBank HQ008714) was used to root repeat and C-terminal trees. N-terminal tree is midpoint-rooted (B). For each variant, the adjacent table row indicates the status of that variant in individual spiders. Each individual per species was assigned a number that appears in the corresponding row if a variant was detected. Bootstrap values of 100% are shown. * denotes outgroup or published A. trifasciata sequence (GenBank AY426339), scale bar represents substitutions per site. Accession numbers for sequence generated in this study are given in Additional file 2: Table S4.