Figure 1.
Species tree based on multi-locus data showing phylogenetic relationships and approximate dates of divergence for all Sistrurus taxa [22] and Crotalus atrox and Agkistrodon c. contortrix outgroups [57].
Branch lengths for the in group taxa and Agkistrodon are as in Kubatko et al. [22]. Branch lengths for C. atrox were estimated based on mtDNA ATP6–8 gene sequence divergences between C. atrox [30] and S. miliarius and S. catenatus [22].
Figure 2.
The venom proteome of S. m. streckeri.
The proteins from 2 mg of pooled crude venom were fractionated on a C18 column as described in the Experimental section. HPLC fractions were collected manually and analyzed by SDS-PAGE (insert; under non-reduced (upper panel) and reducing (lower paner) conditions), N-terminal sequencing, and molecular mass determination by ESI-MS or SDS-PAGE. Protein bands excised from SDS-polyacrylamide gel were identified by tryptic peptide mass fingerprinting and CID-MS/MS. The results are listed in Table 1.
Figure 3.
The venom proteome of S. m. miliarius.
The proteins from 2 mg of crude venom were fractionated on a C18 column as described in as described in the Experimental section. HPLC fractions were collected manually and analyzed as in Fig. 2. The results are listed in Table 1.
Figure 4.
Comparison of the overall venom proteomes of S. m. streckeri and S. m. miliarius.
Pie charts display the relative occurrence of proteins from different toxin families in the venoms of S. m. streckeri (panel A) and S. m. miliarius (panel B). svNGF, snake venom nerve growth factor; CTL, C-type lectin-like protein; DISI, disintegrin; PEP, peptides (including tripeptide SVMP inhibitors, bradykinin-potentiating peptides (BPP), and C-natriuretic peptide, C-NP); CRISP, cysteine-rich secretory protein; D49-PLA2, D49-phospholipase A2; SP, serine proteinase; PI- and PIII-SVMP, snake venom Zn2+-metalloproteinase of class I and class III, respectively. The percentages of the different toxin families in S. m. streckeri and S. m. miliarius venoms are listed in Table 2.
Table 1.
Assignment of the reverse-phase fractions from the venoms of Sistrurus miliarius streckeri (Sms) and S. m. miliarius (Smm), isolated respectively as in Figs.1 and 2, to protein families by N-terminal Edman sequencing, mass spectrometry, and collision-induced fragmentation by nESI-MS/MS of selected peptide ions from in-gel digested protein bands separated by SDS-PAGE (inserts in Figs. 1 and 2).
Table 2.
Overview of the relative occurrence of proteins of different families (in percentage of the total HPLC-separated proteins) in the venoms of Sistrurus m. streckeri (Sms) and Sistrurus m. miliarius (Smm).
Table 3.
Summary of proteomics and diet information used for comparative evolutionary analysis of rattlesnake venom composition.
Table 4.
Measures of phylogenetic signal (estimated as K values) for venom and diet variation across the 8 taxa shown in Fig. 1.
Table 5.
Phylogenetic General Least Squares analyses for associations between diet and individual and composite venom traits for the 8 taxa shown in Fig. 1.