Divergence of the Venom Exogene Repertoire in Two Sister Species of Turriconus

Abstract

The genus Conus comprises approximately 700 species of venomous marine cone snails that are highly efficient predators of worms, snails, and fish. In evolutionary terms, cone snails are relatively young with the earliest fossil records occurring in the Lower Eocene, 55 Ma. The rapid radiation of cone snail species has been accompanied by remarkably high rates of toxin diversification. To shed light on the molecular mechanisms that accompany speciation, we investigated the toxin repertoire of two sister species, Conus andremenezi and Conus praecellens, that were until recently considered a single variable species. A total of 196 and 250 toxin sequences were identified in the venom gland transcriptomes of C. andremenezi and C. praecellens belonging to 25 and 29 putative toxin gene superfamilies, respectively. Comparative analysis with closely (Conus tribblei and Conus lenavati) and more distantly related species (Conus geographus) suggests that speciation is associated with significant diversification of individual toxin genes (exogenes) whereas the expression pattern of toxin gene superfamilies within lineages remains largely conserved. Thus, changes within individual toxin sequences can serve as a sensitive indicator for recent speciation whereas changes in the expression pattern of gene superfamilies are likely to reflect more dramatic differences in a species' interaction with its prey, predators, and competitors.

Keywords: conotoxins; exogenes; speciation; venom evolution.

Fig. 1.

—(A) Selected species of the subgenus Turriconus. (i) Conus andremenezi, (ii) Conus praecellens, (iii) Conus excelsus, a famous rarity known from only a few specimens for many centuries, and still a desirable shell collectors treasure, (iv) Conus miniexcelsus, and (v) Conus acutangulus. The shell of C. andremenezi can be distinguished from that of similar closely related species by a generally broader shape, characteristic purplish-brown maculations, undulating spiral ribs, widely spaced ribs on the spire, and a distinct protoconch. (B) Aquarium images of living C. andremenezi (top) and C. praecellens (bottom). Note color difference in siphon (blue arrows) and foot (red arrows). Conus andremenezi has a darker foot but whiter siphon than C. praecellens.

Fig. 2.

—Bayesian phylogenetic tree of some Turriconus and Splinoconus species based on the analysis of the COI fragment. Support values at each node correspond to RaxML bootstrap (B)/Bayesian posterior probability (PP). The specimens for which venom gland transcriptomes were generated are labeled as C. andremenezi Spm1, C. andremenezi Spm2, C. praecellens Spm1, and C. praecellens Spm2. Shells of analyzed Turriconus and Splinoconus species and of C. geographus are shown right to the respective species clade.

Fig. 3.

—Comparison of relative toxin gene superfamily expression in C. andremenezi and C. praecellens with two members of the Splinoconus clades (C. tribblei and C. lenavati) and one member of the Gastridium clade (C. geographus). (A) Donut charts of expression levels. (B) Heat map showing a select number of superfamilies, including all that were highly expressed in the five species compared here. Plotted numbers represented normalized superfamily read counts. Amz1, C. andremenezi specimen 1; Amz2, C. andremenezi specimen 2; Ps1, C. praecellens specimen 1; Ps2, C. praecellens specimen 2; Trb, C. tribblei (pooled data from three individuals); Len, C. lenavati (pooled data from three individuals); Geo, C. geographus.

Fig. 4.

—PCA of expression of individual superfamilies. Relative expression levels of conotoxin superfamilies were determined by mapping reads back to contigs with Bowtie2 (Langmead and Salzberg 2012). Expression levels were normalized to total read counts after trimming. Principal components were calculated using the prcomp package in R (R Core Team 2013). O1∂: ∂-like O1 superfamily; *Superfamilies that did not contribute to separation on PC1 and PC2.

Fig. 5.

—Inter- and intraspecific variation in Conus venom composition. (A) Sequence alignments of a selection of highly expressed conotoxins, where putative orthologs were identified in all four Turriconus specimens (predicted mature peptides and their names are shown); cysteines are highlighted in yellow and differences in amino acids are shown in red. *Predicted C-terminal amidation. (B) Histograms of rbh pair counts versus percent sequence identity for all species analyzed in this study. The total number of rbh pairs is depicted on graphs.

Fig. 6.

—The A2-superfamily sequences identified in the venom gland transcriptomes of C. praecellens and C. andremenezi. For comparison, another A2-superfamily sequence identified in the salivary gland of C. pulicarius PuSG1.1 (Uniprot: P0C8U6), and two A-superfamily sequences, GID and (Uniprot: P60274) GI (Uniprot: P01519) from C. geographus are shown. Signal peptides are underlined in purple, predicted mature peptides are underlined in black.