Open Access

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

  • Jannelle M. Vienneau-Hathaway1,
  • Elizabeth R. Brassfield1,
  • Amanda Kelly Lane1,
  • Matthew A. Collin2,
  • Sandra M. Correa-Garhwal2,
  • Thomas H. Clarke1, 2,
  • Evelyn E. Schwager3,
  • Jessica E. Garb3,
  • Cheryl Y. Hayashi2 and
  • Nadia A. Ayoub1Email author
BMC Evolutionary BiologyBMC series – open, inclusive and trusted201717:78

DOI: 10.1186/s12862-017-0927-x

Received: 24 August 2016

Accepted: 24 February 2017

Published: 14 March 2017

Abstract

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 β-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.

Keywords

Theridiidae Latrodectus Steatoda Silk glands Gene expression Silk proteins Spidroin gene family

Background

Orb-web and cobweb weaving spiders in the superfamily Araneoidea use silk fibers and glues for a variety of functions, including prey wrapping, prey capture, egg casings, and web framing [1]. Each task-specific silk is made up of a unique combination of proteins produced in one of seven different gland types [2, 3]. Despite making architecturally distinct prey-capture webs (Fig. 1), orb-web and cobweb weaving araneoid spiders possess homologous gland types, most of which produce silks with homologous functions. For example, major ampullate glands synthesize dragline fibers, tubuliform glands express proteins that form outer egg-casing fibers, and pyriform gland protein products are used to attach other fiber types to substrates by all araneoid spiders [1, 4]. Other gland types are homologous in morphology, but have diverged in some functions, such as minor ampullate glands which produce silk used as scaffolding fibers by most araneoids, but additionally functions to form auxiliary spirals in the orb-web versus contributing to prey-wrapping in cobweb weavers [5].
Fig. 1

Relationships among spider species in this study. See Table 1 for species abbreviations and family status. We focused on comparing minor ampullate silks between species that build cobwebs, a disordered three-dimensional web that typically catches walking prey (top graphic) and species that build orb-webs, a two dimensional wagon-wheel shaped web that catches flying prey (bottom graphic, from http://cliparts.co/spiders-web-clip-art). Relationships are based on a compilation of published spider phylogenies (see Methods). The red elbow indicates that one study [38] found the relationship shown in the figure, while another [39] switched the placement of the families to which these species belong

Major ampullate silk has been the primary focus of silk research because it is relatively easy to harvest and has tensile strength rivaling that of steel [6, 7]. However, all silk fibers have impressive and unique material properties [8]. For instance, minor ampullate silk of the orb-web weavers Argiope trifasciata, Argiope argentata, and Nephila clavipes have higher extensibility and lower strength than major ampullate silks from the same species, yet are equally tough [812]. Additionally, minor and major ampullate silks display different physical properties when wet. Supercontraction in water occurs with A. trifasciata and Nephila inaurata major ampullate silks, which allows them to become more extensible while maintaining their high strength [13]. However, when minor ampullate silks from the same species are exposed to water, supercontraction does not occur and the mechanical properties do not change [13]. Despite having consistent behaviors within species whether wet or dry, mechanical properties of minor ampullate silks varied more among species than did the properties of major ampullate silks [14, 15]. The consistency of minor ampullate silks’ mechanical properties when wet or dry, as well as their increased extensibility relative to draglines and greater variability among species, could allow for different applications of minor ampullate silk that would not be feasible for major ampullate silk. The material properties of minor ampullate silks made by cobweb weavers have yet to be measured, but given the different functions of this silk in cobweb weavers compared to in orb-web weavers, we predict divergence in the mechanical performance of these fibers.

The variation in physical properties between major and minor ampullate silks and among minor ampullate silks of different species likely results from sequence differences among their component proteins, termed spidroins (spider fibroins). Spidroins are extremely large (>200 kDa) and are made almost entirely of a highly repetitive region, which is flanked by short, conserved carboxy (C)- and amino (N)-terminal regions [1621]. The repetitive region of many spidroins contain numerous subrepeats of short amino acid sequences, called motifs [2]. Motifs found frequently in spidroin repetitive regions include GGX (where X refers to a subset of amino acids), (GA)n where n ≥ 2, An where n ≥ 4, and GPG [2, 18]. An and (GA)n form β-sheets, GGX forms 31-helices, and GPG forms β-spirals [2, 14, 18, 2226]. Secondary structures of β-sheets likely confer strength, and β-spirals contribute to elasticity [18].

Major ampullate silks of all araneoid spiders examined thus far are made up of two proteins, MaSp1 and MaSp2, which are distinguished by a high GPG content in the latter but not the former [27, 28]. Similarly, N. clavipes minor ampullate silk is made up of two proteins, MiSp1 and MiSp2, although they are not as distinctly different from each other as MaSp1 and MaSp2 [29]. All described minor ampullate spidroins (generalized as MiSp) have consensus repeats that are primarily made up of An, (GA)n and GGX motifs disrupted by serine and threonine-rich “spacer” regions [2, 14, 18, 29, 30]. The functions of the spacer regions are not well understood, but artificially made recombinant proteins composed solely of spacer regions tend to form oligomers and only recombinant MiSp proteins that include at least one spacer, repetitive region, and the C-terminal domain can form fibers [31]. Both MaSp1 and MiSp are predicted to be composed of β-sheets connected by helical regions and amorphous chains [2, 14, 18]. The higher tensile strength of major ampullate silk may result from longer β-sheets made up of An repeats, in contrast to shorter β-sheets made up of An and (GA)n repeats in minor ampullate silk [14].

The large size and repetitiveness of spidroins make spidroin-encoding genes extremely difficult to sequence completely. The only full-length sequence of MiSp known is for Araneus ventricosus [30]. Furthermore, even partial MiSp sequences have been characterized from only a few orb-web weavers [29, 30]. The full length sequence shows that MiSp repeat units are not as similar to each other as are the repeats of MaSp1 or MaSp2 repeats; and that A. ventricosus MiSp possesses exactly two spacers that interrupt the glycine and alanine rich motifs [30]. The generality of these features for other MiSp-encoding sequences needs to be tested.

The only known sequences of MiSp in a cobweb weaver are partial sequences from Latrodectus hesperus [5, 32]. The known repetitive regions are similar between L. hesperus and orb-web weavers’ MiSp, with the main difference being length of spacers [30, 32]. However, because cobweb weavers use minor ampullate silks for prey wrapping, while orb-web weavers use them in the auxiliary spiral of their web [5], we predict different selective pressures on cobweb weaving MiSp that could consequently alter secondary structure and physical properties.

Here, we characterize complete or nearly complete MiSp sequences from five cobweb weaving spider species: L. hesperus, L. tredecimguttatus, L. geometricus, Steatoda grossa, and Parasteatoda tepidariorum (Theridiidae). We also measured mechanical properties of minor ampullate silks in L. hesperus, L. geometricus, and S. grossa. Our goals were to determine the extent of variation in MiSp sequences and minor ampullate silk properties within cobweb weaving spiders, compare this variation to that found in orb-web weaving spiders, and identify any relationship between sequence and material property variation across both groups. We found evidence for at least two MiSp loci in each cobweb weaving species, and that one copy is more highly expressed than the other in minor ampullate glands in L. hesperus and L. geometricus. Phylogenetic analyses suggested extensive gene duplication and concerted evolution of MiSp-encoding loci contributed to variation in MiSp sequences. Finally, we found significant differences in mechanical properties among minor ampullate silks that were partially explained by variation in MiSp sequences.

Results

Multiple loci encode MiSp in cobweb weavers

We employed a series of molecular experiments to identify MiSp-encoding sequences in our five species of cobweb weavers. Additional file 1: Figure S1 provides an overview of these various methods.

L. hesperus

Shotgun sequencing of an L. hesperus MiSp-positive fosmid genomic clone resulted in the assembly of three contiguous sequences (contigs). One ~13 kilobase (kb) contig contained a complete open reading frame (ORF) predicted to encode MiSp (6549 base pairs, bp) and 4689 bp upstream and 2183 bp downstream flanking sequence. Amplification and cloning of L. hesperus genomic DNA with primers designed from the 3’UTR of a MiSp cDNA and an N-terminal encoding cDNA from this species resulted in an almost complete ORF (5568 bp) and 85 bp of 3’UTR, which was 98.8% identical to the published MiSp cDNA (HM752571) over a 983 bp alignment and 97.7% identical to the published MiSp N-terminal cDNA (HM752570) over a 903 bp alignment. In contrast to the similarity between the amplified gene and the cDNAs, the fosmid clone and the amplified gene were distinct. Pairwise differences between the fosmid clone and the amplified gene were 4.5% at the N-terminal encoding end (975 bp alignment) and 20.3% at the C-terminal/ 3’ UTR end (960 bp alignment). The repetitive regions were too divergent to align. We designated the fosmid clone as Lh MiSp variant 1 (Lh MiSp_v1) and the amplified gene as Lh MiSp variant 2 (Lh MiSp_v2; see Table 1 for accession numbers).
Table 1

Spidroins included in phylogenetic analyses of N- and C-terminal sequences

Spidroin name

Species

Family

N-term accession

C-term accession

Bc fibroin1

Bothriocyrtum californicum

Ctenizidae

HM752562

EU117162

Kh MaSp1

Kukulcania hibernalis

Filistatidae

HM752563

 

Dc MaSp

Diguetia canities

Diguetidae

HM752564

HM752565

Dc MaSp-like

Diguetia canities

Diguetidae

HM752566

HM752567

Ds MaSp2

Deinopis spinosa

Deinopidae

HM752568

DQ399328, DQ399329a

Aap MaSp

Agelenopsis aperta

Agelenidae

HM752573

AAT08436

Ud MiSp

Uloborus diversus

Uloboridae

HM752574

ABD61597

Mg MiSp

Metepeira grandiosa

Araneidae

HM752575

HM752569

Lh MiSp

Latrodectus hesperus

Theridiidae

HM752570

HM752571

Aap TuSp1

Agelenopsis aperta

Agelenidae

HM752576

HM752572

Aa TuSp1

Argiope argentata

Araneidae

HM752577

AY953071

Ab TuSp1

Argiope bruennichi

Araneidae

AB242144

AB242144

Nct TuSp1

Nephila clavata

Nephilidae

AB218974

AB218973

Lh TuSp1

Latrodectus hesperus

Theridiidae

DQ379383

AY953070

At MaSp2

Argiope trifasciata

Araneidae

DQ059136S1

DQ059136S2

Nc MaSp2

Nephila clavipes

Nephilidae

EU599240

AY654297

Ni MaSp2

Nephila inaurata madagascariensis

Nephilidae

DQ059135

AF350278

Nc MaSp1a

Nephila clavipes

Nephilidae

EU599238

AY654292

Nc MaSp1b

Nephila clavipes

Nephilidae

EU599239

AY654291

Lh MaSp1

Latrodectus hesperus

Theridiidae

EF595246

EF595246

Lh MaSp2

Latrodectus hesperus

Theridiidae

EF595245

EF595245

Lg MaSp1

Latrodectus geometricus

Theridiidae

DQ059133S1b

DQ059134

Ea MaSp

Euprosthenops australis

Pisauridae

AM259067

AJ973155

Nc Flag

Nephila clavipes

Nephilidae

AF027972b

AF027973

Ni Flag

Nephila inaurata madagascariensis

Nephilidae

AF218623S1

AF218623S2

Av Flag

Araneus ventricosus

Araneidae

AY945306

AY587193

Lg MaSp2

Latrodectus geometricus

Theridiidae

EU177657

AF350275

Ds fibroin1a

Deinopis spinosa

Deinopidae

JX978170

DQ399326

Lh AcSp1

Latrodectus hesperus

Theridiidae

JX978171

Lg AcSp1

Latrodectus geometricus

Theridiidae

JX978180

JX978181

Lh MiSp_v1

Latrodectus hesperus

Theridiidae

KX584003

Lh MiSp_v2

Latrodectus hesperus

Theridiidae

KX584020

Lg MiSp_v1

Latrodectus geometricus

Theridiidae

KX584023

Lg MiSp_v2

Latrodectus geometricus

Theridiidae

KX584024

Lt MiSp_v1

Latrodectus tredecimguttatus

Theridiidae

KX584027

Lt MiSp_v2

Latrodectus tredecimguttatus

Theridiidae

KX584026

Sg MiSp_pseudo

Steatoda grossa

Theridiidae

KX584019

Sg MiSp

Steatoda grossa

Theridiidae

KX584035

KX584021

Pt MiSp

Parasteatoda tepidariorum

Theridiidae

KX584055

KX584022

Ncr PySp

Nephilengys cruentata

Nephilidae

 

GU062417

Nc PySp

Nephila clavipes

Nephilidae

 

GQ980330

Nc PySp2

Nephila clavipes

Nephilidae

 

HM020705

Lh PySp

Latrodectus hesperus

Theridiidae

 

FJ973621

Ud AcSp1

Uloborus diversus

Uloboridae

 

DQ399333

Na MiSp

Nephila antipodiana

Nephilidae

 

DQ399324

Nc MiSp1

Nephila clavipes

Nephilidae

 

AF027735

Ncr MiSp

Nephilengys cruentata

Nephilidae

 

EF638447

Ds MiSp

Deinopis spinosa

Deinopidae

 

DQ399324

Ad MiSp

Araneus diadematus

Araneidae

 

U47853

Av MiSp

Araneus ventricosus

Araneidae

 

JX513956.1

Pb MiSp

Parawixia bistriata

Araneidae

 

GQ275358

Ad MaSp1

Araneus diadematus

Araneidae

 

U47854

Ad MaSp2a

Araneus diadematus

Araneidae

 

U47855

Ad MaSp2b

Araneus diadematus

Araneidae

 

U47856

Ncr MaSp_like

Nephilengys cruentata

Nephilidae

 

EF638446

Na MaSp1

Nephila antipodiana

Nephilidae

 

DQ338461

Ud MaSp1

Uloborus diversus

Uloboridae

 

DQ399331

Ud MaSp2a

Uloborus diversus

Uloboridae

 

DQ399334

Ud MaSp2b

Uloborus diversus

Uloboridae

 

DQ399335

Ncr Flag

Nephilengys cruentata

Nephilidae

 

EF638444

Ncr TuSp

Nephilengys cruentata

Nephilidae

 

EF638445

Ds TuSp1

Deinopis spinosa

Deinopidae

 

AY953073

Ud TuSp1

Uloborus diversus

Uloboridae

 

AY953072

Na TuSp1

Nephila antipodiana

Nephilidae

 

DQ089048

aDQ399329 contains the Ds MaSp2a C-terminal sequence and DQ399328 the Ds MaSp2b C-terminal sequence

bDQ059133S1 and AF027972 were edited to include corrections outlined by [73]

Primer sets designed to be specific to Lh MiSp_v1 versus Lh MiSp_v2 successfully amplified genomic DNA of three single individuals. Variant-specific PCR products were 99.3% identical to the targeted variant, but only 86.3% identical to each other on average at the 616 bp C-terminal/ 3’UTR region. Sequence chromatograms of Lh MiSp_v1 and MiSp_v2 amplifications exhibited double peaks in both directions in at least one individual (10 double peaks in one of three individuals amplified with Lh MiSp_v1 specific primers and one double peak in all three individuals amplified with Lh MiSp_v2 specific primers). The pattern of double peaks in one individual but not others for the Lh MiSp_v1 specific PCR product is consistent with allelic variation for Lh MiSp_v1 and confirms that Lh MiSp_v1 and Lh MiSp_v2 represent separate genomic loci.

L. tredecimguttatus and L. geometricus

Cloning and end sequencing of nearly complete MiSp PCR products from genomic DNA of single individuals of L. tredecimguttatus (Lt) and L. geometricus (Lg) resulted in 11 unique Lg sequences (11 clones) and 11 unique Lt sequences (12 clones). Within each of these species, end sequences clustered into two distinct groups (Fig. 2; Additional file 1: Figure S2). Average pairwise differences within a cluster were low for N-terminal and adjacent repetitive encoding sequences: 0.9% (0–1.2%) and 2.0% (1.2–3.5%) for Lt; 0.7% (0–1.6%) and 1.0% (0.2–1.5%) for Lg. Average pairwise differences between clusters for the same region were much higher: 6.4% (4.5–7.6%) for Lt and 11.6% (5.3–14%) for Lg. A similar pattern was found for C-terminal and adjacent repetitive encoding sequences, except divergence among sequences was higher; average pairwise distances within clusters were 0.7% (0–1.5%) and 8% (4.5–15%) for Lt, and 2.8 (0–9.1%) and 6.0 (0.2–12.6%) for Lg; versus average pairwise distances between clusters were 10.2% (2.3–19.5%) for Lt and 52% (42.1–56.6%) for Lg.
Fig. 2

Neighbor joining tree for N-terminal MiSp encoding sequences demonstrates variation within and among species. Variants are shown for L. hesperus (Lh) and TOPO clones from L. tredecimguttatus (Lt), L. geometricus (Lg), and S. grossa (Sg). Units are number of substitutions. Latrodectus clones are arbitrarily numbered, except that Lt M1-7 and Lg M1-5 resulted from amplification with primers designed from L. hesperus, while higher clone numbers resulted from amplification with species-specific primers. S. grossa TOPO clones resulted from two separate PCR reactions, which are indicated here as “A#” and “B#”. Completely sequenced clones are indicated by bolded names. See Table 1 and Additional file 1: Table S4 for accession numbers. Boxes indicate distinct clusters identified in neighbor joining trees based on N-terminal sequences including adjacent repetitive sequences and in C-terminal and adjacent repetitive region encoding sequences (Additional file 1: Figures S2 and S3)

Because these species are diploid, we expect no more than two alleles per locus within a single individual. Thus each species may possess as many as six loci encoding MiSp (11 variants identified in each species). However, because some PCR or cloning error could produce slight variants among sequences (despite the use of a proofreading enzyme) we conservatively considered the two distinct clusters to represent separate loci and completely sequenced one representative from each cluster (Fig. 2). We designated the completely sequenced clones from L. tredecimguttatus as Lt MiSp_v1 and Lt MiSp_v2, and the completely sequenced clones from L. geometricus as Lg MiSp_v1 and Lg MiSp_v2. Designation of MiSp variants with 1 or 2 is not intended to imply orthology among species, but simply reflects different genomic loci within species.

S. grossa

TOPO cloning and end sequencing of an ~5000 bp S. grossa PCR product that was generated with primers targeting complete MiSp resulted in ten identical clones from a single individual. When translated, these ~800 bp end sequences aligned well with our Latrodectus MiSp sequences, but contained numerous stop codons. This variant is likely a non-functional pseudogene, designated Sg MiSp_pseudo.

PCR amplification using primers designed from our ~3 kb C-terminal encoding S. grossa MiSp cDNA (designated Sg MiSp) failed to find any further variants. In contrast, TOPO cloning and sequencing of 20 N-terminal and adjacent repetitive MiSp encoding PCR products for a single S. grossa individual resulted in 20 unique sequences, each different from our ~500 bp N-terminal encoding cDNA. The clones (designated by clone numbers) grouped into three clusters (Additional file 1: Figure S3); within a cluster pairwise differences among sequences were <0.9% on average (0.13–1.5%) while between clusters pairwise differences were much greater (average pairwise distance = 10%). Nucleotide differences within a cluster could reflect alleles or multiple loci, but could also be attributable to PCR error because amplification was performed with Taq polymerase alone, which does not have proofreading capability, in contrast to the high fidelity polymerases used to amplify the Latrodectus species (see Methods). Conservatively, the three clusters represent three true variants. Thus there is a minimum of two loci that encode MiSp in S. grossa.

P. tepidariorum

We found partial length N and C-terminal MiSp-encoding cDNAs from P. tepidariorum (designated Pt MiSp, see Table 1 for accessions) that were nearly identical (>98%) to the 5’ and 3’ ends, respectively, of an Augustus-predicted gene model on Scaffold 853 in the publicly available genome. This gene model was predicted to span 10292 bp, but included four gaps (4353 bp of assembled sequence, 5939 bp of unknown). Characterization of a TOPO clone containing an ~2.3 kb PCR product for P. tepidariorum MiSp (KX584004) resulted in a sequence that was 100% identical to the 104 bp of overlap with the C-terminal encoding region of Scaffold 853 and filled 2236 bp of the last gap. We refer to the combination of our TOPO clone with Scaffold 853 as Pt MiSp variant 1 (Pt MiSp_v1). The fully sequenced TOPO clone included a 1175 bp long intron with canonical donor and acceptor sites identified by aligning with our C-terminal encoding cDNA using SPIDEY [33]. We also found BLASTN alignments of this 2.3 kb TOPO clone to three other scaffolds (954, 3280, and 3758) that may represent additional MiSp-encoding loci. The C-terminal encoding region of our TOPO clone aligned with 91% identity on Scaffold 954. The intron sequence aligned with 95% identity to Scaffold 3280 and 99% identity to Scaffold 3758. The repeat region flanking the intron (including 100 bases of intron) aligned with 89% identity to Scaffold 3280 and 90% identity to Scaffold 3758. The presence of two C-terminal encoding sequences that differ by 9% and are placed on separate scaffolds suggests that at least two loci encode MiSp in P. tepidariorum.

MiSp loci are expressed at different rates in cobweb weaving spiders

In L. geometricus and L. hesperus, both completely sequenced loci (MiSp_v1 and MiSp_v2) are highly transcribed in minor ampullate glands (2.4–44.7% of all reads in minor ampullate gland RNA-seq libraries aligned to MiSp_v1 or MiSp_v2) and transcribed at low levels in other tissue types (e.g. <1% of all reads in major ampullate gland RNA-seq libraries aligned to MiSp_v1 or MiSp_v2, Table 2). MiSp_v2 transcripts are more abundant than MiSp_v1 in both L. geometricus and in L. hesperus minor ampullate glands (Table 2). The ratio was consistent for all tissue types in L. hesperus, however, ratios were much more variable in L. geometricus between libraries of minor ampullate glands and even more so among tissues (Table 2).
Table 2

MiSp transcript abundance based on read counts in minor ampullate, major ampullate, and total silk gland RNA-seq libraries

 

L. geometricus

L. hesperus

RNA-seq Library

Total readsa

MiSp_v2

MiSp_v1

Ratioc

Ratio 3’d

Total readsb

MiSp_v2

MiSp_v1

Ratioc

Ratio 3’d

Minor 1

4832818

275164

57912

4.8

2.6

11679960

5289870

1487650

3.6

3.6

Minor 2

20640831

3496061

564983

6.2

4.2

11148437

4919531

1443331

3.4

3.4

Total Silk

8101748

61016

72774

0.8

0.9

15093424

572185

183028

3.1

4.3

Major 1

13978630

1943

4493

0.4

0.3

6116968

68

24

2.8

2.7

Major 2

13636997

69

9

7.7

5.5

9574596

315

85

3.7

4.4

See Table 1 for accession numbers

a Raw RNA-seq reads are available on NCBI’s SRA database: SRR1539569

b Raw RNA-seq reads are available on NCBI’s SRA database: SRR1539570

c Ratio of MiSp_v2 to MiSp_v1 based on counts of reads aligned to the entire sequences

d Ratio of MiSp_v2 to MiSp_v1 based on counts of reads aligned to the 3’-most 500 bases of coding sequences. See Methods for rationale

Variable genomic structure of MiSp

All MiSp genes characterized from Latrodectus lacked introns interrupting coding sequence. While only L. hesperus MiSp_v1 included both the start and stop codons, the other MiSp sequences missed no more than 300 bp of the N or C-terminal coding regions on either end. Our P. tepidariorum MiSp genomic TOPO clone that includes the C-terminal encoding region has an intron of 1175 bp. For comparison, orb-weaver A. ventricosus MiSp has a large, single intron of 5628 bp [30].

Inspection of the L. hesperus fosmid genomic clone revealed a TATA box approximately 50 bases upstream of the full-length L. hesperus MiSp coding region. The nucleotide motif CACG found in all previously characterized spidroin genes was also found in L. hesperus MiSp, 11 bases upstream of the TATA box. The P. tepidariorum sequence on Scaffold 853 in the i5k genome possessed a TATA box 63 bases upstream of the MiSp coding region and a CACG motif 10 bases upstream of the TATA box. The A. ventricosus sequence has a TATA box 63 bases upstream of the full-length MiSp coding region, and a CACG motif 30 bases upstream of the TATA box [30]. Polyadenylation signals were found 110 bases downstream of the stop codon for L. hesperus MiSp_v1, 242 bases downstream of the stop for P. tepidariorum MiSp_v1, and 82 bases downstream of the stop for A. ventricosus MiSp [30].

MiSp amino acid variation within and among species

Cobweb weaver MiSp length is highly variable within and among species (815 aa to 2751 aa, Additional file 1: Figure S4). For comparison, the completely sequenced L. hesperus MaSp1 is 3129 aa and MaSp2 is 3779 aa, and A. ventricosus MiSp is 1,766 aa [30]. Glycine, alanine, and serine are the three most abundant amino acids for all MiSp variants, showing similar composition percentages to L. hesperus MaSp1 and MaSp2. Serine is found in both the spacer regions and the repeat regions, while glycine and alanine are predominantly found in the repeat region only. Proline is found in very low abundance for both L. hesperus MiSp variants, both L. tredecimguttatus MiSp variants, L. geometricus MiSp_v2, and P. tepidariorum MiSp_v1, but is more abundant in L. geometricus MiSp_v1 and is the fourth most abundant amino acid in S. grossa MiSp, surpassing L. hesperus MaSp2 proline composition (Additional file 1: Figure S4). Codon usage is variable among MiSp loci and species, but there is an overall bias toward A or T in the third position of the codon, especially for alanine and glycine (Additional file 1: Table S1).

Despite gross similarities in MiSp amino acid composition among species, arrangement of amino acids into motifs is more variable. L. hesperus MiSp variants, L. tredecimguttatus MiSp variants, L. geometricus MiSp_v2, and P. tepidariorum MiSp_v1 contain mostly (GA)n and GGX motifs, with GPG being nonexistent. However, GPG is prevalent in both L. geometricus MiSp_v1 and S. grossa MiSp (Fig. 3). In contrast to MaSp1 and MaSp2, which have ensemble repeat units ~30–50 aa long that are nearly identical, there are multiple shorter, more variable ensemble repeats that describe MiSp sequences (Additional file 1: Table S2). Tandem repeats range in length from 7 to 569 aa, repeated in the protein anywhere from 2 to 28 times, and with amino acid sequence identity ranging from 81% to 97% (Additional file 1: Table S2). Modularity of ensemble repeats is highly variable among and within species, with L. hesperus MiSp_v1, L. tredecimguttatus MiSp_v2, L. geometricus MiSp_v1, and S. grossa MiSp having a higher order repeat unit (>160 aa repeated at least once) and L. hesperus MiSp_v2, L. tredecimguttatus MiSp_v1, L. geometricus MiSp_v2, and P. tepidariorum MiSp_v1 having multiple short repetitive units only (24 to 30 aa repeated >10 times).
Fig. 3

Arrangement of amino acid motifs in MiSp sequences of cobweb and orb-web weaving spiders. Latrodectus and S. grossa accession numbers are in Table 1. Lh, L. hesperus; Lt, L. tredecimguttatus; Lg, L. geometricus; Sg, S. grossa; Pt, P. tepidariorum based on combining the incompletely assembled MiSp-encoding region from Scaffold 853 of the i5K genome with an ~2.3 kb TOPO-cloned PCR product, KX584004; Av, A. ventricosus, JX513956.1 [30]. Lengths of missing amino acid sequences in Pt MiSp_v1 are based on the length of gaps in Scaffold 853 of the i5K genome, assuming no introns are present within those gaps. The last gap was predicted to be 1782 bp long (594 aa if no intron) in Scaffold 853, but our TOPO clone added 2236 bp, including a 1175 bp intron, which was longer than the predicted gap. The repetitive encoding region at the 3’ end of our TOPO clone did not overlap with the encoding region present in the i5K genome prior to the last gap

The number and arrangement of spacers is also highly variable among cobweb weaver MiSp. Some MiSp variants lack spacers while others have up to seven spacers. Spacers are varied in their distribution within a monomer, ranging from 100 aa to 400 aa away from each other in MiSp proteins with more than one spacer (Fig. 3). Spacers are easily distinguished from the repetitive motifs in their amino acid composition (rich in threonine, serine, and valine), near perfect repetition within a MiSp protein, and similar length across species (Fig. 4). Furthermore, with the exception of S. grossa MiSp, spacers are much more hydrophilic than the repetitive region (Additional file 1: Figure S5).
Fig. 4

Comparison of theridiid (top) and orb-web weaver (bottom) MiSp spacers. Theridiid MiSp spacers are aligned for L. hesperus (Lh), L. tredecimguttatus (Lt), L. geometricus (Lg), S. grossa (Sg), and P. tepidariorum (Pt). Orb-web weaver MiSp spacers aligned for A. ventricosus (Av), A. diadematus (Ad), Argiope argentata (Aar), Nephila antipodiana (Na), N. clavipes (Nc), Nephilengys cruentata (Ncr), and Deinopis spinosa (Ds) following [30]. See Table 1 and Additional file 1: Table S4 for accession numbers. Note that the sequence of spacers in the Pt MiSp N and C-terminal encoding cDNAs shown here are identical to spacers found in Pt MiSp_v1

Material properties of minor ampullate spidroins correlate with MiSp sequence variation

We found significant differences in each of four tensile properties of minor ampullate silk fibers measured for three cobweb weaving species (Fig. 5). These properties were tensile strength, the amount of force applied to break a single silk fiber, accounting for the diameter of the fiber; extensibility, the proportion of the original fiber length that the fiber was extended at breakage; stiffness or Young’s modulus, the initial slope of the force-extension curve (initial resistance to force); and toughness, the energy required to break the fiber (see Methods for details). Post-hoc Tukey’s test revealed that minor ampullate silk from L. geometricus had the lowest tensile strength (Fig. 5). Minor ampullate silk from S. grossa was significantly more extensible and less stiff than the other two species (Fig. 5).
Fig. 5

Minor ampullate silk fiber tensile properties differ among three cobweb weaving species. Boxplots show differences among L. hesperus (Lh), L. geometricus (Lg), and S. grossa (Sg) for a) strength (MANOVA, F2,147 = 8.9, p < 0.001); b) toughness (MANOVA, F2,147 = 6.8, p = 0.002); c) extensibility (MANOVA, F2,147 = 13.3, p < 0.001); and d) Young’s Modulus (MANOVA, F2,147 = 12.9, p < 0.001). Open circles are outliers that exceed the range of the interquartile distance by 1.5 times. Different letters (e.g. A vs B) above boxplots indicate significant pairwise differences according to Tukey’s Test using a harmonic mean sample size of 50 and α = 0.05

Minor ampullate silks from the cobweb weavers are generally weaker, less stiff, and less tough than orb-web weavers, but more extensible (Table 3). We found a positive, but non-significant, relationship between the proportion of the GPG motif in MiSp and the extensibility of minor ampullate silk fibers among the five species with both published MiSp sequences and material properties (adjusted R 2  = 0.53, p = 0.10; Additional file 1: Figure S6). However, when accounting for phylogenetic relationships and evolutionary distances among species using phylogenetic independent contrasts, we found a significant positive relationship between GPG proportion and extensibility (adjusted R 2  = 0.92, p = 0.006), due to the extensive change in GPG and extensibility on the relatively short S. grossa branch (see Additional file 1: Figure S6 for the effect of varying branch lengths). This pattern of higher GPG content contributing to extensibility parallels patterns found for major ampullate silks, although for major ampullate silks higher GPG content results from higher expression of MaSp2, rather than sequence differences among orthologs [2, 14, 18, 2225].
Table 3

Properties of minor ampullate silk for cobweb and orb-web weaving species

Species

Diameter (μm)

Young’s Modulus (GPa)

Strength (MPa)

Extensibility (ln mm/mm)

Toughness (J/cm3)

GPG (%)

Latrodectus hesperus

1.12 ± 0.47

3.9 ± 2.9

245.4 ± 120.5

0.57 ± 0.02

66.7 ± 46.4

0.07 g

Latrodectus geometricus

1.09 ± 0.29

2.6 ± 1.3

174.4 ± 17.6

0.54 ± 0.02

43.6 ± 20.1

3.48 h

Steatoda grossa

1.07 ± 0.26

2.1 ± 0.7

251.3 ± 106.2

0.74 ± 0.02

57.3 ± 20.7

12.79 i

Nephila clavipes a

2.5 ± 0.2

3.0 ± 0.6

346 ± 53

0.30 ± 0.15

 

0 j

Nephila clavipes b

2.5

 

960 ± 50

0.25 ± 0.03

 

0 j

Nephila inaurata c

1.8 ± 0.1

11.2 ± 0.7

1500 ± 200

0.46 ± 0.05

300 ± 50

 

Argiope trifasciata d,e

0.69 ± 0.10

8.9 ± 0.05

751.5 ± 40

0.44 ± 0.04

150 ± 12

 

Argiope trifasciata c

1.8 ± 0.1

10 ± 0.4

1040 ± 6

0.45 ± 0.02

240 ± 20

 

Argiope argentata e

1.1 ± 0.7

10.6 ± 1.2

923 ± 154

0.33 ± 0.03

137 ± 22

 

Araneus diadematus f

   

0.29

 

0 k

Araneus gemmoides b

2.0

 

1400 ± 100

0.22 ± 0.07

  

Material properties collected from a[10], b[15], c[13], d[9], e[8], f[74]

Proportion of MiSp that is made up of the motif GPG was averaged across sequences described for each species: g Lh MiSp_v1, MiSp_v2; h Lg MiSp_v1, MiSp_v2; i Sg MiSp: C-terminal encoding cDNA (KX584021); j AF027735; k U47853

Phylogenetic relationships of spidroins among orb-web and cobweb weaving species suggest multiple duplication events

Spidroin N- and C-terminal domains and encoding nucleotides demonstrate MiSp sequences found in eight species form a monophyletic group that is usually recovered as sister to a clade of MaSp1 and MaSp2 sequences (Fig. 6, Additional file 1: Figure S7). Theridiid MiSp consistently group together with strong support (Fig. 6, Additional file 1: Figure S7). Within theridiids, MiSp sequences from Latrodectus species group together, S. grossa MiSp variants form a sister clade to Latrodectus MiSp variants, and P. tepidariorum MiSp is sister to Latrodectus and Steatoda MiSp variants. Within Latrodectus, L. geometricus MiSp variants form a sister clade to L. hesperus and L. tredecimguttatus MiSp variants; these patterns are congruent with species relationships [34, 35] (Fig. 6). However, MiSp variants group together within a species, suggesting independent duplication events within each species as inferred by reconciling our gene trees with the species trees in Fig. 1 (Fig. 6). Alternatively, this pattern could be explained by concerted evolution of terminal-encoding regions within each species, which we detail in the Discussion.
Fig. 6

Bayesian tree for combined N- and C-terminal spidroin-encoding nucleotide sequences. See Table 1 for accessions. Bold branches are supported by > 50% bootstrap parsimony and > 95% posterior probability in all analyses of combined N- and C-terminal sequences (see Additional file 1: Figure S7). Asterisks (*) indicate > 95% posterior probability in Bayesian analyses of N- and C-terminal sequences. Units are substitutions per site. The tree is rooted with Bc fibroin1. Duplications inferred by reconciliation with the species trees in Fig. 1 are shown as red dots at nodes. The red dots with yellow outlines indicate that concerted evolution of terminal encoding regions could also explain the observed relationships

Outside of Theridiidae, relationships among MiSp sequences are more ambiguous and do not necessarily reflect species relationships. For instance, molecular and morphological characters strongly support monophyly of the Araneoidea (represented here by Theridiidae, Nephilidae, and Araneidae) to the exclusion of Deinopidae and Uloboridae [3639]. In contrast, N- and C-terminal domains sometimes place deinopid and uloborid MiSp sister to theridiid MiSp, and sometimes place araneid MiSp sequences sister to theridiid MiSp sequences (Fig. 6, Additional file 1: Figure S7). The former placement is consistent with an ancient duplication of a MiSp-encoding locus in the common ancestor of Araneoidea, Deinopidae, and Uloboridae (Fig. 6). C-terminal domains and encoding nucleotides for MiSp from 14 species generally show the same patterns within Theridiidae, but theridiid MiSp sequences tend to group with nephilid and deinopoid MiSp sequences to the exclusion of araneid MiSp sequences, consistent with multiple ancient duplication events (Fig. 7, Additional file 1: Figure S8). Relationships among other spidroins are consistent with previous findings that gland-specific spidroins tend to group together, with the exception of major ampullate spidroins [17, 32, 40]. Overall, we inferred 24–30 duplication events within the spidroin gene family, depending on which C-terminal gene tree was used for reconciliation (Additional file 1: Figure S8).
Fig. 7

Bayesian tree for C-terminal spidroin-encoding nucleotide sequences. See Table 1 for accessions. Bold branches are supported by > 50% bootstrap parsimony and > 95% posterior probability in all analyses of C-terminal sequences (see Additional file 1: Figure S8). Asterisks (*) indicate > 95% posterior probability in Bayesian analyses of C-terminal sequences. Units are substitutions per site. The tree is rooted with Bc fibroin1. Duplications inferred by reconciliation with the species trees in Fig. 1 are shown as red dots at nodes. The red dots with yellow outlines indicate that concerted evolution of terminal encoding regions could also explain the observed relationships

Discussion

Our characterization of complete or almost complete encoding sequences for the minor ampullate spidroin (MiSp) in multiple cobweb weaving spider species (Theridiidae) demonstrate the presence of at least two MiSp loci in each species. The substantial variation of MiSp sequences within and among species and its relationship to variation in material properties has multiple implications for molecular evolution, spider ecology, and biomimetic applications through recombinant DNA technology.

Two MiSp-encoding loci have also been documented in the golden orb-weaver spider, Nephila clavipes (Nephilidae), and therefore the presence of multiple MiSp-encoding loci in spider genomes most likely dates minimally back to the common ancestor of theridiids and nephilids (e.g. Araneoidea). Our reconciliation of gene trees with species trees suggests even older duplication events (Fig. 7). However, the maintenance of two loci appears to involve complex molecular evolutionary processes including intergenic concerted evolution of the loci within species, and multiple gains and losses of individual MiSp copies. Although our reconciliation analyses inferred independent duplication events within each of our cobweb weaving species, the grouping of MiSp loci within species based on the terminal domains (Figs. 6 and 7), could also result from intergenic concerted evolution of the N- and C-terminal encoding regions. We favor the latter hypothesis because of the dramatic differences in the repetitive region within some species (Fig. 3). Concerted evolution of the N and C-terminal encoding regions could occur through non-homologous recombination between the loci, facilitated by their similar sequences, as proposed for the major ampullate spidroin paralogs, MaSp1 and MaSp2, of multiple species [2, 32, 4143]. Within theridiids, the rate of concerted evolution appears to be faster than speciation, since relationships among each species’ pair of MiSp loci reflect species relationships (Figs. 6 and 7). Intergenic concerted evolution of terminal-encoding regions could be favored by selection because these regions are involved in assembly of multiple MiSp monomers into polymers and the conversion of the protein complex from a liquid to a solid [31, 44]. Highly similar terminal regions may be necessary for polymers to form.

Outside of theridiids, relationships among MiSp N and C-terminal domains are not congruent with species relationships. The low posterior probabilities and bootstrap support for these incongruous relationships suggest there is limited phylogenetic signal retained in the MiSp C-termini at the distant time scale of divergence of theridiids from other araneoid families (~170 million years ago, [36, 38, 39]). However, the consistent grouping of Nephila and theridiid MiSp C-terminal domains to the exclusion of araneid MiSp C-terminal domains suggests that nephilid and theridiid MiSp sequences are derived from a different ancient copy than the araneid MiSp sequences, as supported by our reconciliation analyses (Figs. 6 and 7). The loss of functional MiSp loci through multiple single nucleotide mutations is further supported by the presence of a MiSp pseudogene in the S. grossa genome. Many additional losses were inferred by our reconciliation analyses (Additional file 1: Figure S8), but because of incomplete spidroin sampling for most species we do not feel confident in estimating the extent of spidroin gene loss.

We found extensive variation in the MiSp repetitive regions between loci within a genome and among species (Fig. 3). MiSp variants in L. hesperus and L. tredecimguttatus, L. geometricus MiSp_v2, P. tepidariorum MiSp_v1, Nephila MiSp 1 & 2, and araneid MiSp sequences are similar in terms of amino acid motif composition. S. grossa MiSp and L. geometricus MiSp_v1 are especially divergent, with the difference between variants within L. geometricus being extremely striking (Fig. 3). The divergent repeats have an increased proline content, which likely occurred independently in S. grossa MiSp and L. geometricus MiSp_v1, based on gene tree relationships (Figs. 6 & 7). The mutation of alanine or glutamine codons to a proline codon requires only a single base change and the GPG motif is frequently found as GPGA or GPGQ, indicating that it could have evolved from (GA)n or (GQ)n motifs, which are the most common motifs in all other MiSp sequences (Fig. 3). Intragenic concerted evolution could rapidly proliferate these mutations throughout a single gene as has been proposed for other spidroin paralogs [16, 17, 45, 46]. The near identity of spacer sequences within each of our cobweb weaver MiSp variants (Fig. 4) supports the hypothesis that intragenic concerted evolution is common, as also found in orb-web weaver MiSp [30, 31]. However, intragenic concerted evolution must be offset by some other molecular processes, since the remainder of the repetitive regions are not as homogenized as the spacers (Fig. 3). The simple amino acid motifs found in MiSp such as (GA)n are encoded by microsatellite-like sequences and could thus be prone to high rates of slipped strand mispairing. It is also possible that excessive homogenization of MiSp repetitive regions adversely affects its function and is eliminated by selection.

The high proportion of the GPG amino acid motif in S. grossa MiSp correlates with higher extensibility in S. grossa minor ampullate silk fibers (Fig. 5c, Table 3). GPG content is probably not the only predictor of extensibility, however, since Latrodectus minor ampullate fibers were more extensible than orb-web weavers’ even though GPG content for L. hesperus is similar to the orb-web weavers. Furthermore, we did not find a significant difference in extensibility between L. hesperus and L. geometricus minor ampullate silk fibers despite L. geometricus MiSp_v1 having a relatively high percentage of GPG motifs (Fig. 3). The MiSp_v2: MiSp_v1 transcript ratio in L. geometricus suggests that the GPG-rich MiSp_v1 is in lower abundance in L. geometricus (Table 2), which could explain why there is not a significant increase in extensibility of its minor ampullate silk fibers in comparison to L. hesperus. Cobweb weaving spiders use MiSp in their prey wrapping silks [5], and evolution of extensibility-conferring motifs could potentially allow for capturing different prey types by S. grossa compared to other cobweb weavers, although little is known about the ecology or diet of this species. It is also possible that L. geometricus modulates expression of its two MiSp-encoding loci in response to prey availability. Experimentally manipulating prey type has been shown to affect proline content of major ampullate fibers in Nephila, potentially as a result of plastic changes in relative expression levels of the proline-poor MaSp1 and the proline-rich MaSp2 [47, 48]. Variation among individuals in MaSp1 and MaSp2 transcript abundance has also been demonstrated for black widows [49]. Analogous experiments have not been done for minor ampullate fibers. However, the variation in MiSp_v2: MiSp_v1 ratios that we found between L. geometricus individuals (Table 2) suggests plasticity of expression is possible.

Our six complete or nearly complete single exon MiSp-containing clones can also serve as templates for recombinant proteins. Due to the dramatic differences in length and amino acid content of some of the proteins encoded by these loci, it may be possible to spin artificial fibers with custom-made properties. For instance, the GPG-containing L. geometricus MiSp_v1 could be used to make more extensible fibers, while the longer alanine-rich L. hesperus MiSp_v1 may make stiffer fibers.

Conclusions

We found that intragenic concerted evolution within MiSp-encoding genes likely led to rapid proliferation of proline replacements for alanine or glutamine in MiSp protein sequences independently in at least two species. For one species, the proliferation of proline coincides with higher extensibility of minor ampullate silks. This could allow cobweb weavers to access new prey types or the ability to modulate mechanical properties of their silks through altering expression levels of MiSp gene copies. Our multiple nearly complete MiSp sequences also provide various templates for tailored biomimetic applications through recombinant DNA technologies.

Methods

Taxon sampling

Individual L. hesperus were collected in Riverside, California and Tucson, Arizona. L. geometricus individuals were collected in San Diego, California. L. tredecimguttatus and S. grossa were obtained from SpiderPharm (Yarnell, Arizona). P. tepidariorum were obtained from a laboratory culture founded with spiders collected near Cologne, Germany and purchased from SpiderPharm.

Sequencing

Genomic library clone

We screened an L. hesperus genomic library with PCR for clones containing MiSp (see [16] for library construction and screening protocols). Primers used in screening were designed from the C-terminal encoding region of a partial-length L. hesperus MiSp cDNA (HM752571; primers listed in Additional file 1: Table S3). An ~49 kb MiSp containing clone was shotgun sequenced and assembled to 8× coverage by Qiagen (Hilden, Germany).

PCR amplification and cloning of Latrodectus genomic MiSp

We amplified full-length L. hesperus MiSp from genomic DNA using primers designed from the 3’ UTR of the C-terminal encoding MiSp cDNA (HM752571) and the N-terminal region of another partial MiSp cDNA (HM752570). We amplified genomic MiSp from L. tredecimguttatus and L. geometricus first using primers designed from L. hesperus MiSp N and C-terminal cDNAs and then with internal species-specific primers (primers in Additional file 1: Table S3). Genomic PCR was performed with 1 unit of AccuPrimeTM Taq DNA Polymerase High Fidelity (Invitrogen, Carlsbad, CA) according to manufacturer’s instructions. Cycling conditions were 40 cycles of 94 °C for 30 s, 50–60 °C (depending on primer pair) for 45 s, and 68 °C for 10 min.

PCR products were gel-excised and cloned using the TOPO®-TA Cloning® Kit (Invitrogen). Clones were screened by PCR amplification of MiSp N and/or C-terminal encoding region, and MiSp positive clones were sequenced with Sp6, T7, M13F, or M13R universal primers. End sequencing of TOPO clones indicated that, within both L. geometricus and L. tredecimguttatus, clones clustered into two distinct groups (Additional file 1: Figure S2). For both L. tredecimguttatus and L. geometricus, the longest clones representing each cluster were completely sequenced. End sequences and restriction digest patterns of L. hesperus TOPO clones were very similar. Thus, one clone was chosen for complete sequencing. Clones chosen for complete sequencing were subjected to random transposon insertion with the GPS-1 Genome Priming System (New England Biolabs, Ipswich, MA). Position of transposon insertion was mapped with restriction digests and clones were sequenced from both ends of the transposon. Sequences were assembled with Sequencher v. 4.9 (GeneCodes).

Steatoda grossa and P. tepidariorum cDNA libraries

We constructed cDNA libraries from S. grossa total silk glands and P. tepidariorum major and minor ampullate silk glands following procedures detailed in Garb et al. (2010). In brief, total RNA was extracted with TRIzol® (Invitrogen) and the RNeasy Mini Kit (Qiagen, Valencia, CA). mRNA was isolated with oligo-(dT)-tagged magnetic beads (Invitrogen). cDNA was synthesized with SuperScript®III (Invitrogen), fractionated by size with ChromaSpin 1000 columns (Clontech, Mountain View, CA), blunt-end ligated into pZErOTM-2 (Invitrogen), and electroporated into TOP10 Escherichia coli cells (Invitrogen). Approximately 2000 colonies per library were arrayed and stored at −80 °C. The libraries were screened by gel electrophoresis of plasmid DNA [50]. Plasmids with inserts >500 bp were sequenced with T7 or SP6 universal primers. We identified silk protein-encoding sequences by conceptual translations and comparisons to published sequences with BLASTX [51].

An S. grossa cDNA clone (designated Sg MiSp) with a 3 kb insert containing the C-terminal encoding portion of MiSp was completely sequenced by random insertion of transposons with the EZ-Tn5 < Tet > kit (Epicentre). Transposon mapping, sequencing, and assembly were performed as for TOPO clones above.

Amplification and cloning of S. grossa and P. tepidariorum MiSp

We attempted to amplify complete MiSp encoding sequences from S. grossa genomic DNA using primers designed from conserved regions of Latrodectus MiSp N-terminal encoding sequences and from the C-terminal encoding region of the S. grossa MiSp cDNA (Additional file 1: Table S3). S. grossa genomic DNA was amplified with 1 unit of Phusion® High-Fidelity DNA Polymerase (New England Biolabs), 200 μM each dNTP, 0.5 μM each primer, 1X Phusion® GC buffer, and 3% DMSO. Cycling conditions were 40 cycles of 98 °C for 5 s, 58 °C for 15 s, and 72 °C for 2.5 min. PCR products were gel-excised and cloned using the TOPO®-Blunt Cloning® Kit (Invitrogen). End sequencing of these clones revealed numerous premature stop codons and thus clones were not completely sequenced.

In order to obtain longer N-terminal MiSp encoding sequences from S. grossa, we amplified S. grossa genomic DNA using forward primers designed from the conserved region of Latrodectus MiSp N-terminal encoding sequences or from the S. grossa N-terminal MiSp cDNA, and a reverse primer designed from the repetitive region of the S. grossa C-terminal cDNA (Additional file 1: Table S3). Genomic DNA was amplified with 1 unit GoTaq Polymerase (Promega), 1X GoTaq Buffer, 200 μM each dNTP, 0.5 μM each primer, and 1.5 mM MgCl2 with 45 cycles of 30s at 94 °C, 45 s at 54.2 °C or 61 °C, and 1.5 or 2 min at 72 °C. PCR products from the two amplifications were separately gel-excised and cloned using the TOPO®-TA XL PCR Cloning ® Kit (Invitrogen). TOPO clones from each reaction were amplified with M13 forward and reverse primers and sequenced with T7 or SP6 universal primers.

A MiSp gene was identified in the P. tepidariorum genome by BLASTN searches of our N- and C-terminal encoding MiSp cDNA sequences against the genome assembly [52] generated by the i5K initiative [53]. We found alignments of both cDNAs to a region on Scaffold 853 that included an Augustus-predicted gene model (aug3.g7951.t3). Using the genomic sequence, we designed a reverse primer in the C-terminal encoding region and a forward primer in the adjacent repetitive region. Genomic DNA and cDNA were amplified as for S. grossa N-terminal encoding region but with 40 cycles of 30s at 94 °C, 45 s at 60.1 °C and 1 min at 72 °C. PCR products were gel-excised and sequenced. Genomic PCR products had sizes ranging from 250 bp to 2500 bp, while cDNA PCR products had sizes ranging from 250 bp to 1200 bp. Those bands that were too large to directly sequence were TOPO cloned using the TOPO-TA PCR Cloning Kit (Invitrogen) and completely sequenced by random insertion of transposons as for S. grossa TOPO clones above. Completed TOPO clones were compared to the P. tepidariorum genome by BLASTN.

Amino acid sequence characteristics

Amino acid composition and codon usage were calculated with codonW [54]. Kyte-Doolittle hydropathy plots [55] with a window size of seven were generated online [56]. Amino acid motifs GGX, An (n ≥ 4), (GA)n (n ≥ 2), and GPG were identified by word searches in Microsoft Word. Spacer regions were defined manually as regions that were not in the N or C-termini and were low in alanine and glycine and were manually aligned. Two authors (JVH and CYH) independently assigned spacer regions. Tandem repeats were identified by XSTREAM v1.4.8 using default parameters for moderate repeat degeneracy and high significance [57].

Transcript abundance estimation

Transcript abundance was estimated by aligning raw RNA-seq reads [58] (SRR1539569 and SRR1539569 for L. hesperus and L. geometricus respectively) to our full or almost full-length MiSp paralogs found in L. hesperus (Lh MiSp_v1 and MiSp_v2) and L. geometricus (Lg MiSp_v1 and MiSp_v2) using Bowtie 2 with default parameters, which searches for multiple alignments for each read but reports the one best alignment [59]. RNA-seq reads were generated for mRNA isolated from major and minor ampullate glands of ~30 adult females and from all the silk glands (“total silk”) of a single adult female for both L. hesperus and L. geometricus. SAMtools v1.1 was used to report the number of sequence reads matching our reference MiSp sequences [60]. To compare transcript abundance between loci within the same library, we first compared the total counts of reads aligned to the entire reference sequence. We did not correct for gene length because many of our libraries were heavily 3’-prime biased. However, to ensure that differences in gene length did not dramatically affect our estimates of the ratio of one MiSp locus to another, we also compared the counts that aligned to the last 500 bases of coding sequence (Table 2).

Relationships among spidroin paralogs

We added our theridiid and A. ventricosus [30] MiSp N and C-terminal coding sequences to an alignment of 29 spidroin termini analyzed previously (see [17]). N-terminal sequences were not available for many spidroins and thus we added 25 additional spidroin sequences to the C-terminal alignment to more comprehensively represent spidroin gene family diversity in our analyses (Table 1). Additional C-termini of spidroin paralogs were chosen for species for which MiSp has been characterized. The expanded amino acid alignment was manually edited and used to guide the nucleotide alignment in SeaView v. 4.2.7 [61, 62].

We conducted heuristic searches for maximum parsimony (MP) and maximum likelihood (ML) trees based on amino acid and nucleotide alignments in PAUP* v4.0b10 using tree bisection reconnection branch swapping and 1000 (MP) or 10 (ML) replicates of random stepwise addition of taxa [63]. Support for clades recovered in MP analyses was evaluated with 1000 bootstrap pseudoreplicates and 10 random addition sequences per pseudoreplicate.

Bayesian analyses were carried out with MRBAYES v.3.1.2 [64, 65]. Optimal models of evolution were determined for nucleotide sequences with JMODELTEST v0.1.1 [66] and for protein sequences with PROTTEST v2.4 [67] for N and C-termini separately. Combined analysis of nucleotides employed a model partitioned by N and C-termini. Combined analysis of amino acids employed a mixed model, which allowed estimation of the optimal model of protein evolution during the Bayesian analysis. Default priors and Metropolis coupled, Markov-chain, Monte Carlo sampling procedures were executed for two independent runs of 10 million generations each, sampled every 1000th generation. Convergence was assessed every 1000th generation and the posterior distribution was considered adequately sampled if the standard deviation of split frequencies of these two runs was below 0.01. The mygalomorph spidroin, Bothriocyrtum californicum fibroin1 (Bc fibroin1), was set as the outgroup in all analyses.

Gene tree – species tree reconciliation

We inferred gene duplication events by reconciling each of our gene trees with two species trees (Fig. 1) using Notung v. 2.8.1.7 [68]. Species trees were based on published phylogenies as follows. Relationships among families were based on Garrison et al. [38] and Dimitrov et al. [39]. These studies agreed on all family-level relationships except the placement of Deinopidae and Uloboridae. The former placed Deinopidae sister to the RTA-clade (represented here by E. australis and A. aperta) with Uloboridae sister to Deinopidae plus the RTA-clade. The latter switched the placement of Uloboridae and Deinopidae. For relationships among theridiid species we used Liu et al. [35] and Garb et al. [34]. Relationships among nephilid species were based on Kuntner et al. [69]. Relationships among araneid genera were based on Dimitrov et al. [39] with relationships for Argiope species from Cheng and Kuntner [70].

Mechanical properties of minor ampullate fibers

We obtained minor ampullate silk from L. hesperus, L. geometricus, and S. grossa adult females within one week of being received or collected. Individual spiders were anesthetized with CO2 for 2–5 min. We then secured the spider ventral side up to a stereo microscope stage using Scotch ® tape, exposing the spinnerets. We manually pulled silk emanating from the minor ampullate spigots. Single fibers were taped to m-shaped cardstock (2.54 × 7.62 cm), with a single silk fiber suspended across each of the two rectangular notches (1 cm wide × 1.5 cm deep) in a collection card. Fibers were secured onto the cards using cyanoacrylate glue. We collected four to ten minor ampullate fibers from each of seven (L. hesperus and S. grossa) or eight (L. geometricus) individuals per species.

We measured silk fiber diameters using polarized light microscopy (as described in [6]). Tensile tests were conducted with a NanoBionix tensile tester (formerly MTS Systems Corp., Oak Ridge, TN; currently Keysight Technologies, Santa Rosa, CA) [6]. In brief, each m-shaped card was cut in half, such that only one fiber segment was tested at one time. Each silk sample was mounted onto the NanoBionix and extended at a rate of 1% per second until failure. We defined strength as the true stress of the fiber at breakage. True stress is the force applied to the fiber divided by its cross-sectional area calculated from the original cross-sectional area assuming constant volume of the fiber during extension [71]. We defined extensibility as the true strain at breakage. True strain is the natural log of the ratio of the instantaneous length of the fiber to the original gage length of the fiber. We also measured stiffness or Young’s modulus, the initial slope of the true stress-true strain curve, and toughness, the area under the true stress-true strain curve. Force-extension data were visualized using Testworks 4.0 software (MTS Systems Corp.). We tested for differences among species for the four tensile properties using multivariate analysis of variance (MANOVA). We used Tukey’s test for post-hoc pairwise comparisons. All statistical tests were carried out with SPSS 13.0 (SPSS Inc, Chicago, IL).

We compiled published tensile data for minor ampullate silks and fit the relationship between proportion of GPG amino acid motifs and extensibility to linear models implemented in R for each species for which at least one MiSp sequence and minor ampullate fiber tensile data were available. Minor ampullate silk was assumed to be composed only of MiSp and for each species all available MiSp motif percentages were averaged to estimate composition. To account for phylogenetic relationships and evolutionary distances among species we calculated phylogenetic independent contrasts using APE [72] implemented in R. We used estimates of divergence time from three published molecular clock studies [35, 38, 39] for branch lengths (see Additional file 1: Figure S6).

Declarations

Acknowledgements

We thank Stephen Richards and colleagues at Baylor College of Medicine’s Human Genome Sequencing Center for access to the P. tepidariorum genome. We also thank Alistair McGregor, Mario Stanke, and colleagues for annotating this genome. Students in Genetics Lab (BIOL 221) at Washington and Lee University assisted with collecting sequence data.

Funding

This work was supported by the National Science Foundation (IOS-0951086 to NAA, IOS-0951061 to CYH), National Institutes of Health (F32 GM78875-1A to NAA; 1F32GM083661-01 and 1R15GM097714-01 to JEG), Army Research Office (W911NF-06-1-0455 and W911NF-11-1-0299 to CYH), and Washington and Lee University through Lenfest Summer Fellowships to NAA and Summer Research Scholarships to JVH and AKL. ERB was supported in part by a grant to Washington and Lee University from the Howard Hughes Medical Institute through the Precollege and Undergraduate Science Education Program (52007570).

Availability of data and materials

Sequences generated for this study are available in GenBank (Accessions: KX584003 - KX584055). Accessions for previously published sequences are reported in the manuscript text and in Table 1.

Authors’ contributions

CYH and NAA conceived the study. JMV, ERB, JEG, EES, and NAA generated sequence data. AKL, MAC, SMC, and CYH measured tensile properties of minor ampullate silk fibers. JMV, ERB, MAC, THC, and NAA analyzed the data. JMV and NAA wrote the manuscript. All authors edited the manuscript and read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Not applicable.

Ethics approval and consent to participate

Not applicable.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Department of Biology, Washington and Lee University
(2)
Department of Biology, University of California
(3)
Department of Biological Sciences, University of Massachusetts

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