Evolution of Conus peptide toxins: analysis of Conus californicus Reeve, 1844

Abstract

Conus species are characterized by their hyperdiverse toxins, encoded by a few gene superfamilies. Our phylogenies of the genus, based on mitochondrial genes, confirm previous results that C. californicus is highly divergent from all other species. Genetic and biochemical analysis of their venom peptides comprise the fifteen most abundant conopeptides and over 50 mature cDNA transcripts from the venom duct. Although C. californicus venom retains many of the general properties of other Conus species, they share only half of the toxin gene superfamilies found in other Conus species. Thus, in these two lineages, approximately half of the rapidly diversifying gene superfamilies originated after an early Tertiary split. Such results demonstrate that, unlike endogenously acting gene families, these genes are likely to be significantly more restricted in their phylogenetic distribution. In concordance with the evolutionary distance of C. californicus from other species, there are aspects of prey-capture behavior and prey preferences of this species that diverges significantly from all other Conus.

Figure 1

Phylogenetic tree inferred from the concatenation of 12S and 16SrRNAs and COI sequences from 16 Conus species and five outgroup species. Branches are labelled with posterior probabilities from the Bayesian analysis on the left and bootstrap percentages on the right.

Figure 2

Phylogenetic tree inferred from the concatenation of 12S and 16S rRNAs sequences from 57 Conus species and 2 outgroup species. Branches are labelled with Bayesian posterior probabilities (left) and maximum likelihood bootstrap percentages (right or bottom) that are greater than 50% Even with the addition of far more Conus species, the tree shows strong support for placing Conus califonicus on a long branch well outside of and sister to the other Conus speices.

Figure 3

Shells and prey capture behavior of Conus californicus “Hinds, R.B” Reeve, L.A., 1844. Conus californicus, the California cone, inhabits the temperate costal waters off California and Baja California. Live animals (top left) were collected on the Western coast of Baja California roughly 40 km north of Ensenada, Mexico. A photograph of a shell from one of these specimens is shown (top right). An interesting feature of C. californicus feeding behavior is that individual snails cooperate in order to subdue prey items larger than themselves, like a marauding wolf pack converging on larger prey. Such behavior has never been documented for any other Conus species.

Figure 4

Identification of conopeptides from venom fractions of Conus californicus. (a) Fractionation of C. californicus venom extract on a Vydac C₁₈ analytical column with a linear gradient of 1.0% B90/min at a flow rate of 1.0 mL/min. Lettered arrows represent HPLC fractions used to identify primary amino acid sequences depicted in Table 1. (b) Subfractionation of peak c from panel (a), after reduction with 15 mM DDT and alkylation with 4-vinylpyridine using an elution gradient of 15-40% B90 over 30 minutes; three peptides, cl 6a, cl 9a and cl 9b were sequenced from this fractionation (c) Fractionation of peak i from panel (a), leading to the characterization of cl 6b the most abundant peptide within the venom of Conus californicus, using a Vydac C₁₈ analytical column eluded with a gradient of 15-60% buffer B90 over 60 min. The sequences of the four peptides marked in panels (b) and (c) were deduced from an automated Edman degredation and are presented in Table 1.

Figure 5

Reconstruction of conotoxin superfamilies evolution within selected Conoidea. The tree is a collapsed cladogram of Figure 1.The vertical bars on the tree represent the appearance of the corresponding superfamily (unlabeled bars unnamed superfamilies). The apparent absence and presence of each conotoxin superfamily is also reported in the adjacent table (conotoxins in three lineages – T. mirabilis, Lienardia sp. and “minor” clade have not been investigated).