Phylogene and identified spidrioins of the spiders in this study.

The phylogenetic status of the spiders was marked with different colors. the green line represents Mesothelae, the blue line represents Mygalomorphae, and the red and purple line represents Araneomorphae, specific species are marked at the end of the branch. An asterisk (*) indicates that an event with a significant impact is likely to occur, the ruler represents when the event occurred, the specific time scale is shown on the X axis in millions of years. Each colored box represents a silk type, the gray boxes represent unidentified spidroins, and numbers in each box indicate the total number of occurrences of each silk type.

Analysis of silk protein structure of Liphistius sp.

(A) Structural model analysis of the silk protein of Liphistius sp. from Thailand. (B) Alignment of amino acids in the C-terminal domain of spider silk protein sequences in multiple spider species, red arrows indicate the location of two ancient spider silk proteins. Ajur, A. juruensis; Pmur, P. murinus; Csig, C. signata; Snig, S. nigrolineata; Stib, S. tibialis; Lhes, L. hesperus; Ncla, N. clavipes; Aven, A. ventricosus.

Bayesian Inference tree of spidroin amino-terminal regions.

The colored icons represent different spidroin type, and the specific colour is used for each type. Colored icons surround spidroins categorized as flagelliform spidroin (Flag); aggregate spidroin (AgSp); pyriform spidroin (PySp); tubuliform spidroin (TuSp); aciniform spidroin (AcSp); major ampullate spidroin (MaSp); or minor ampullate spidroin (MiSp). At the end of the gene tree are various spider spidroin, which are represented by different colors and can be roughly divided into three categories: the green line represents Mesothelae, the blue line represents Mygalomorphae, the red and purple line represents Araneomorphae. Red dots indicate nodes with bootstrap values > 80%, Scale bar is substitutions per site. The primitive group Mesothelae and Mygalomorphae sequenced in this study are marked with photos. (A) Bayesian tree of spidroins N-terminal regions. (B) Bayesian tree of spidroins C-terminal regions.

Bayesian Inference tree of spidroin repeat unit regions.

The colored icons represent different spidroin type, and the specific colour is used for each type. Colored icons surround spidroins categorized as flagelliform spidroin (Flag); aggregate spidroin (AgSp); pyriform spidroin (PySp); tubuliform spidroin (TuSp); aciniform spidroin (AcSp); major ampullate spidroin (MaSp); or minor ampullate spidroin (MiSp). At the end of the gene tree are various spider spidroin, which are represented by different colors and can be roughly divided into three categories: the green line represents Mesothelae, the blue line represents Mygalomorphae, the red and purple line represents Araneomorphae. Red dots indicate nodes with bootstrap values > 80%, Scale bar is substitutions per site. The primitive group Mesothelae and Mygalomorphae sequenced in this study are marked with photos. Calculating the amino acid frequency from the repeating domain in spidroins and displaying it in a heatmap, the size of the circle and the depth of the color represent the frequency.

Evolutionary patterns of AS-rich and GSGA-rich ancient spidroin.

Amino acid sequence pattern structure of spider silk is depicted, with different colored circles representing different mechanical properties of motif, the specific type motif has been annotated in the lower left corner of the figure. (A) The AS-rich spider silk sequence structure of Mesothelae and Mygalomorphae, with photos marking the primitive groups Mesothelae and Mygalomorphae sequenced in this study. (B) Tubuliform silk protein and pyriform silk protein patterns in Araneomorphae. (C) The pattern structure of GSGA-rich spidroin.

Analysis of important capture silk protein Flag sequence features.

(A) Schematic diagrams of cob weaver (including repeat motif from L. elegans), two orb weavers (including repeat motif from N. pilipes and A. ventricosus) and RTA specialized clade spider (H. davidbowie). Each repeat is composed of different motifs which are shown as oval module labeled with different colors. Spacer, spacer region. NTD, N-terminal domain. CTD, C-terminal domain. (B) The collinearity of the upstream and downstream of A. bruennichi Flag gene in the ancient species of Araneomorphae (S. dumicola), the cobweb spider (L. elegans) and RTA specialized clade spider (D. plantarius) genome, the gray lines represent the genomic regions of other species compared to the Flag gene, the purple line represents the alignment position of the Flag gene. (C) N-terminal sequence alignment of MaSp1, Flag from 3 spiders of Araneoidea and H. dav spiders. (D) C-terminal sequence alignment of MaSp1, Flag from 3 spiders of Araneoidea and H. dav spiders.