Hidden species diversity and mito-nuclear discordance within the Mediterranean cone snail, Lautoconus ventricosus.

The Mediterranean cone snail, Lautoconus ventricosus, is currently considered a single species inhabiting the whole Mediterranean basin and the adjacent Atlantic coasts. Yet, no population genetic study has assessed its taxonomic status. Here, we collected 245 individuals from 75 localities throughout the Mediterranean Sea and used cox1 barcodes, complete mitochondrial genomes, and genome skims to test whether L. ventricosus represents a complex of cryptic species. The maximum likelihood phylogeny based on complete mitochondrial genomes recovered six main clades (hereby named blue, brown, green, orange, red, and violet) with sufficient sequence divergence to be considered putative species. On the other hand, phylogenomic analyses based on 437 nuclear genes only recovered four out of the six clades: blue and orange clades were thoroughly mixed and the brown one was not recovered. This mito-nuclear discordance revealed instances of incomplete lineage sorting and introgression, and may have caused important differences in the dating of main cladogenetic events. Species delimitation tests proposed the existence of at least three species: green, violet, and red + blue + orange (i.e., cyan). Green plus cyan (with sympatric distributions) and violet, had West and East Mediterranean distributions, respectively, mostly separated by the Siculo-Tunisian biogeographical barrier. Morphometric analyses of the shell using species hypotheses as factor and shell length as covariate showed that the discrimination power of the studied parameters was only 70.2%, reinforcing the cryptic nature of the uncovered species, and the importance of integrative taxonomic approaches considering morphology, ecology, biogeography, and mitochondrial and nuclear population genetic variation.

Comparative Venomics of the Cryptic Cone Snail Species Virroconus ebraeus and Virroconus judaeus.

The venom duct transcriptomes and proteomes of the cryptic cone snail species Virroconus ebraeus and Virroconus judaeus were obtained and compared. The most abundant and shared conotoxin precursor superfamilies in both species were M, O1, and O2. Additionally, three new putative conotoxin precursor superfamilies (Virro01-03) with cysteine pattern types VI/VII and XVI were identified. The most expressed conotoxin precursor superfamilies were SF-mi2 and M in V. ebraeus, and Cerm03 and M in V. judaeus. Up to 16 conotoxin precursor superfamilies and hormones were differentially expressed between both species, and clustered into two distinct sets, which could represent adaptations of each species to different diets. Finally, we predicted, with machine learning algorithms, the 3D structure model of selected venom proteins including the differentially expressed Cerm03 and SF-mi2, an insulin type 3, a Gastridium geographus GVIA-like conotoxin, and an ortholog to the Pionoconus magus ω-conotoxin MVIIA (Ziconotide).

Chromosome-level genome of the venomous snail Kalloconus canariensis: a valuable model for venomics and comparative genomics.

BACKGROUND: Genomes are powerful resources to understand the evolutionary mechanisms underpinning the origin and diversification of the venoms of cone snails (Conidae: Caenogastropoda) and could aid in the development of novel drugs. FINDINGS: Here, we used PacBio continuous long reads and Omni-C data to assemble the chromosome-level genome of Kalloconus canariensis, a vermivorous cone endemic to the Canary Islands. The final genome size was 2.87 Gb, with a N50 of 79.75 Mb and 91% of the reads located into the 35 largest scaffolds. Up to 55.80% of the genome was annotated as repetitive regions, being class I of transposable elements (16.65%) predominant. The annotation estimated 34,287 gene models. Comparative analysis of this genome with the 2 cone snail genomes released to date (Dendroconus betulinus and Lautoconus ventricosus) revealed similar genome sizes and organization, although chromosome sizes tended to be shorter in K. canariensis. Phylogenetic relationships within subclass Caenogastropoda were recovered with strong statistical support. The family Conidae was recovered as a clade, with K. canariensis plus L. ventricosus sister to D. betulinus. CONCLUSIONS: Despite the great diversity of cone snails (>900 species) and their venoms (hundreds of peptides per species), only 2 recently reported genomes are available for the group. The high-quality chromosome-level assembly of K. canariensis will be a valuable reference for studying the origin and evolution of conotoxin genes as well as whole-genome duplication events during gastropod evolution.

The genome of the venomous snail Lautoconus ventricosus sheds light on the origin of conotoxin diversity.

BACKGROUND: Venoms are deadly weapons to subdue prey or deter predators that have evolved independently in many animal lineages. The genomes of venomous animals are essential to understand the evolutionary mechanisms involved in the origin and diversification of venoms. RESULTS: Here, we report the chromosome-level genome of the venomous Mediterranean cone snail, Lautoconus ventricosus (Caenogastropoda: Conidae). The total size of the assembly is 3.59 Gb; it has high contiguity (N50 = 93.53 Mb) and 86.6 Mb of the genome assembled into the 35 largest scaffolds or pseudochromosomes. On the basis of venom gland transcriptomes, we annotated 262 complete genes encoding conotoxin precursors, hormones, and other venom-related proteins. These genes were scattered in the different pseudochromosomes and located within repetitive regions. The genes encoding conotoxin precursors were normally structured into 3 exons, which did not necessarily coincide with the 3 structural domains of the corresponding proteins. Additionally, we found evidence in the L. ventricosus genome for a past whole-genome duplication event by means of conserved gene synteny with the Pomacea canaliculata genome, the only one available at the chromosome level within Caenogastropoda. The whole-genome duplication event was further confirmed by the presence of a duplicated hox gene cluster. Key genes for gastropod biology including those encoding proteins related to development, shell formation, and sex were located in the genome. CONCLUSIONS: The new high-quality L. ventricosus genome should become a reference for assembling and analyzing new gastropod genomes and will contribute to future evolutionary genomic studies among venomous animals.

Comparative transcriptomics of the venoms of continental and insular radiations of West African cones.

The transcriptomes of the venom glands of 13 closely related species of vermivorous cones endemic to West Africa from genera Africonus and Varioconus were sequenced and venom repertoires compared within a phylogenetic framework using one Kalloconus species as outgroup. The total number of conotoxin precursors per species varied between 108 and 221. Individuals of the same species shared about one-fourth of the total conotoxin precursors. The number of common sequences was drastically reduced in the pairwise comparisons between closely related species, and the phylogenetical signal was totally eroded at the inter-generic level (no sequence was identified as shared derived), due to the intrinsic high variability of these secreted peptides. A common set of four conotoxin precursor superfamilies (T, O1, O2 and M) was expanded in all studied cone species, and thus, they are considered the basic venom toolkit for hunting and defense in the West African vermivorous cone snails. Maximum-likelihood ancestral character reconstructions inferred shared conotoxin precursors preferentially at internal nodes close to the tips of the phylogeny (between individuals and between closely related species) as well as in the common ancestor of Varioconus. Besides the common toolkit, the two genera showed significantly distinct catalogues of conotoxin precursors in terms of type of superfamilies present and the abundance of members per superfamily, but had similar relative expression levels indicating functional convergence. Differential expression comparisons between vermivorous and piscivorous cones highlighted the importance of the A and S superfamilies for fish hunting and defense.

Conotoxin Diversity in the Venom Gland Transcriptome of the Magician's Cone, Pionoconus magus.

The transcriptomes of the venom glands of two individuals of the magician's cone, Pionoconus magus, from Okinawa (Japan) were sequenced, assembled, and annotated. In addition, RNA-seq raw reads available at the SRA database from one additional specimen of P. magus from the Philippines were also assembled and annotated. The total numbers of identified conotoxin precursors and hormones per specimen were 118, 112, and 93. The three individuals shared only five identical sequences whereas the two specimens from Okinawa had 30 sequences in common. The total number of distinct conotoxin precursors and hormones for P. magus was 275, and were assigned to 53 conotoxin precursor and hormone superfamilies, two of which were new based on their divergent signal region. The superfamilies that had the highest number of precursors were M (42), O1 (34), T (27), A (18), O2 (17), and F (13), accounting for 55% of the total diversity. The D superfamily, previously thought to be exclusive of vermivorous cones was found in P. magus and contained a highly divergent mature region. Similarly, the A superfamily alpha 4/3 was found in P. magus despite the fact that it was previously postulated to be almost exclusive of the genus Rhombiconus. Differential expression analyses of P. magus compared to Chelyconus ermineus, the only fish-hunting cone from the Atlantic Ocean revealed that M and A2 superfamilies appeared to be more expressed in the former whereas the O2 superfamily was more expressed in the latter.

Conotoxin Diversity in Chelyconus ermineus (Born, 1778) and the Convergent Origin of Piscivory in the Atlantic and Indo-Pacific Cones.

The transcriptome of the venom duct of the Atlantic piscivorous cone species Chelyconus ermineus (Born, 1778) was determined. The venom repertoire of this species includes at least 378 conotoxin precursors, which could be ascribed to 33 known and 22 new (unassigned) protein superfamilies, respectively. Most abundant superfamilies were T, W, O1, M, O2, and Z, accounting for 57% of all detected diversity. A total of three individuals were sequenced showing considerable intraspecific variation: each individual had many exclusive conotoxin precursors, and only 20% of all inferred mature peptides were common to all individuals. Three different regions (distal, medium, and proximal with respect to the venom bulb) of the venom duct were analyzed independently. Diversity (in terms of number of distinct members) of conotoxin precursor superfamilies increased toward the distal region whereas transcripts detected toward the proximal region showed higher expression levels. Only the superfamilies A and I3 showed statistically significant differential expression across regions of the venom duct. Sequences belonging to the alpha (motor cabal) and kappa (lightning-strike cabal) subfamilies of the superfamily A were mainly detected in the proximal region of the venom duct. The mature peptides of the alpha subfamily had the α4/4 cysteine spacing pattern, which has been shown to selectively target muscle nicotinic-acetylcholine receptors, ultimately producing paralysis. This function is performed by mature peptides having a α3/5 cysteine spacing pattern in piscivorous cone species from the Indo-Pacific region, thereby supporting a convergent evolution of piscivory in cones.

Phylogenetic relationships of cone snails endemic to Cabo Verde based on mitochondrial genomes.

BACKGROUND: Due to their great species and ecological diversity as well as their capacity to produce hundreds of different toxins, cone snails are of interest to evolutionary biologists, pharmacologists and amateur naturalists alike. Taxonomic identification of cone snails still relies mostly on the shape, color, and banding patterns of the shell. However, these phenotypic traits are prone to homoplasy. Therefore, the consistent use of genetic data for species delimitation and phylogenetic inference in this apparently hyperdiverse group is largely wanting. Here, we reconstruct the phylogeny of the cones endemic to Cabo Verde archipelago, a well-known radiation of the group, using mitochondrial (mt) genomes. RESULTS: The reconstructed phylogeny grouped the analyzed species into two main clades, one including Kalloconus from West Africa sister to Trovaoconus from Cabo Verde and the other with a paraphyletic Lautoconus due to the sister group relationship of Africonus from Cabo Verde and Lautoconus ventricosus from Mediterranean Sea and neighboring Atlantic Ocean to the exclusion of Lautoconus endemic to Senegal (plus Lautoconus guanche from Mauritania, Morocco, and Canary Islands). Within Trovaoconus, up to three main lineages could be distinguished. The clade of Africonus included four main lineages (named I to IV), each further subdivided into two monophyletic groups. The reconstructed phylogeny allowed inferring the evolution of the radula in the studied lineages as well as biogeographic patterns. The number of cone species endemic to Cabo Verde was revised under the light of sequence divergence data and the inferred phylogenetic relationships. CONCLUSIONS: The sequence divergence between continental members of the genus Kalloconus and island endemics ascribed to the genus Trovaoconus is low, prompting for synonymization of the latter. The genus Lautoconus is paraphyletic. Lautoconus ventricosus is the closest living sister group of genus Africonus. Diversification of Africonus was in allopatry due to the direct development nature of their larvae and mainly triggered by eustatic sea level changes during the Miocene-Pliocene. Our study confirms the diversity of cone endemic to Cabo Verde but significantly reduces the number of valid species. Applying a sequence divergence threshold, the number of valid species within the sampled Africonus is reduced to half.

Mitogenomic phylogeny of cone snails endemic to Senegal.

Cone snails attain in Senegal one of their highest peaks of species diversity throughout the continental coast of Western Africa. A total of 15 endemic species have been described, all placed in the genus Lautoconus. While there is ample data regarding the morphology of the shell and the radular tooth of these species, virtually nothing is known regarding the genetic diversity and phylogenetic relationships of one of the most endangered groups of cones. In this work, we determined the complete or near-complete (only lacking the control region) mitochondrial (mt) genomes of 17 specimens representing 11 endemic species (Lautoconus belairensis, Lautoconus bruguieresi, Lautoconus cacao, Lautoconus cloveri, Lautoconus cf. echinophilus, Lautoconus guinaicus, Lautoconus hybridus, Lautoconus senegalensis, Lautoconus mercator, Lautoconus taslei, and Lautoconus unifasciatus). We also sequenced the complete mt genome of Lautoconus guanche from the Canary Islands, which has been related to the cones endemic to Senegal. All mt genomes share the same gene arrangement, which conforms to the consensus reported for Conidae, Neogastropoda and Caenogastropoda. Phylogenetic analyses using probabilistic methods recovered three major lineages, whose divergence coincided in time with sea level and ocean current changes as well as temperature fluctuations during the Messinian salinity crisis and the Plio-Pleistocene transition. Furthermore, the three lineages corresponded to distinct types of radular tooth (robust, small, and elongated), suggesting that dietary specialization could be an additional evolutionary driver in the diversification of the cones endemic to Senegal. The reconstructed phylogeny showed several cases of phenotypic convergence (cryptic species) and questions the validity of some species (ecotypes or phenotypic plasticity), both results having important taxonomic and conservation consequences.

Evolution at a different pace: distinctive phylogenetic patterns of cone snails from two ancient oceanic archipelagos.

Ancient oceanic archipelagos of similar geological age are expected to accrue comparable numbers of endemic lineages with identical life history strategies, especially if the islands exhibit analogous habitats. We tested this hypothesis using marine snails of the genus Conus from the Atlantic archipelagos of Cape Verde and Canary Islands. Together with Azores and Madeira, these archipelagos comprise the Macaronesia biogeographic region and differ remarkably in the diversity of this group. More than 50 endemic Conus species have been described from Cape Verde, whereas prior to this study, only two nonendemic species, including a putative species complex, were thought to occur in the Canary Islands. We combined molecular phylogenetic data and geometric morphometrics with bathymetric and paleoclimatic reconstructions to understand the contrasting diversification patterns found in these regions. Our results suggest that species diversity is even lower than previously thought in the Canary Islands, with the putative species complex corresponding to a single species, Conus guanche. One explanation for the enormous disparity in Conus diversity is that the amount of available habitat may differ, or may have differed in the past due to eustatic (global) sea level changes. Historical bathymetric data, however, indicated that sea level fluctuations since the Miocene have had a similar impact on the available habitat area in both Cape Verde and Canary archipelagos and therefore do not explain this disparity. We suggest that recurrent gene flow between the Canary Islands and West Africa, habitat losses due to intense volcanic activity in combination with unsuccessful colonization of new Conus species from more diverse regions, were all determinant in shaping diversity patterns within the Canarian archipelago. Worldwide Conus species diversity follows the well-established pattern of latitudinal increase of species richness from the poles towards the tropics. However, the eastern Atlantic revealed a striking pattern with two main peaks of Conus species richness in the subtropical area and decreasing diversities toward the tropical western African coast. A Random Forests model using 12 oceanographic variables suggested that sea surface temperature is the main determinant of Conus diversity either at continental scales (eastern Atlantic coast) or in a broader context (worldwide). Other factors such as availability of suitable habitat and reduced salinity due to the influx of large rivers in the tropical area also play an important role in shaping Conus diversity patterns in the western coast of Africa.

Replaying the tape: recurring biogeographical patterns in Cape Verde Conus after 12 million years.

Isolated oceanic islands are excellent natural laboratories to test the relative role of historical contingency and determinism in evolutionary diversification. Endemics of the marine venomous snail Conus in the Cape Verde archipelago were originated from at least two independent colonizations of 'small' and 'large' shelled species separated by 12 million years. In this study, we have reconstructed phylogenetic relationships within large-shelled Conus (C. ateralbus, C. pseudonivifer, C. trochulus, and C. venulatus) based on mitochondrial cox1 and nad4 haplotype sequences. The reconstructed molecular phylogeny revealed three well-supported and relatively divergent clades (A, B, and C) that do not correspond to current species classification based on shell colour and banding patterns. Clade A grouped specimens assigned either to C. pseudonivifer or C. trochulus, clade B is composed of specimens assigned to C. venulatus, and clade C comprises specimens assigned either to C. venulatus or C. ateralbus. Geometric morphometric analyses found significant differences between the radular teeth shape of C. pseudonivifer/C. trochulus and C. venulatus/C. ateralbus. In clades A and B, northwestern Boavista and Maio specimens cluster together to the exclusion of eastern Boavista samples. In Sal, populations form a monophyletic island assemblage (clade C). The large-shelled Conus have remarkably replicated biogeographical patterns of diversification of small-shelled Conus. Similar selective forces (i.e. nonplanktonic lecithotrophy with limited larval dispersal and allopatric diversification) together with repeated instances of low sea level stands during glacial maxima that allowed connection between islands, have overcome the effect of historical contingency, and explain the observed recurring biogeographical patterns.