Nicolas Puillandre

Molecular phylogeny and evolution of the cone snails (Gastropoda, Conoidea)

We present a large-scale molecular phylogeny that includes 320 of the 761 recognized valid species of the cone snails (Conus), one of the most diverse groups of marine molluscs, based on three mitochondrial genes (COI, 16S rDNA and 12S rDNA). This is the first phylogeny of the taxon to employ concatenated sequences of several genes, and it includes more than twice as many species as the last published molecular phylogeny of the entire group nearly a decade ago. Most of the numerous molecular phylogenies published during the last 15years are limited to rather small fractions of its species diversity. Bayesian and maximum likelihood analyses are mostly congruent and confirm the presence of three previously reported highly divergent lineages among cone snails, and one identified here using molecular data. About 85% of the species cluster in the single Large Major Clade; the others are divided between the Small Major Clade (∼12%), the Conus californicus lineage (one species), and a newly defined clade (∼3%). We also define several subclades within the Large and Small major clades, but most of their relationships remain poorly supported. To illustrate the usefulness of molecular phylogenies in addressing specific evolutionary questions, we analyse the evolution of the diet, the biogeography and the toxins of cone snails. All cone snails whose feeding biology is known inject venom into large prey animals and swallow them whole. Predation on polychaete worms is inferred as the ancestral state, and diet shifts to molluscs and fishes occurred rarely. The ancestor of cone snails probably originated from the Indo-Pacific; rather few colonisations of other biogeographic provinces have probably occurred. A new classification of the Conidae, based on the molecular phylogeny, is published in an accompanying paper.

Molecular phylogeny, classification and evolution of conopeptides.

Conopeptides are toxins expressed in the venom duct of cone snails (Conoidea, Conus). These are mostly well-structured peptides and mini-proteins with high potency and selectivity for a broad range of cellular targets. In view of these properties, they are widely used as pharmacological tools and many are candidates for innovative drugs. The conopeptides are primarily classified into superfamilies according to their peptide signal sequence, a classification that is thought to reflect the evolution of the multigenic system. However, this hypothesis has never been thoroughly tested. Here we present a phylogenetic analysis of 1,364 conopeptide signal sequences extracted from GenBank. The results validate the current conopeptide superfamily classification, but also reveal several important new features. The so-called “cysteine-poor” conopeptides are revealed to be closely related to “cysteine-rich” conopeptides; with some of them sharing very similar signal sequences, suggesting that a distinction based on cysteine content and configuration is not phylogenetically relevant and does not reflect the evolutionary history of conopeptides. A given cysteine pattern or pharmacological activity can be found across different superfamilies. Furthermore, a few conopeptides from GenBank do not cluster in any of the known superfamilies, and could represent yet-undefined superfamilies. A clear phylogenetically based classification should help to disentangle the diversity of conopeptides, and could also serve as a rationale to understand the evolution of the toxins in the numerous other species of conoideans and venomous animals at large.

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

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.

The contrasted evolutionary fates of deep-sea chemosynthetic mussels (Bivalvia, Bathymodiolinae)

Bathymodiolinae are giant mussels that were discovered at hydrothermal vents and harboring chemosynthetic symbionts. Due to their close phylogenetic relationship with seep species and tiny mussels from organic substrates, it was hypothesized that they gradually evolved from shallow to deeper environments, and specialized in decaying organic remains, then in seeps, and finally colonized deep-sea vents. Here, we present a multigene phylogeny that reveals that most of the genera are polyphyletic and/or paraphyletic. The robustness of the phylogeny allows us to revise the genus-level classification. Organic remains are robustly supported as the ancestral habitat for Bathymodiolinae. However, rather than a single step toward colonization of vents and seeps, recurrent habitat shifts from organic substrates to vents and seeps occurred during evolution, and never the reverse. This new phylogenetic framework challenges the gradualist scenarios “from shallow to deep.” Mussels from organic remains tolerate a large range of ecological conditions and display a spectacular species diversity contrary to vent mussels, although such habitats are yet underexplored compared to vents and seeps. Overall, our data suggest that for deep-sea mussels, the high specialization to vent habitats provides ecological success in this harsh habitat but also brings the lineage to a kind of evolutionary dead end.

Evolution of Conus Peptide Genes: Duplication and Positive Selection in the A-Superfamily

A remarkable diversity of venom peptides is expressed in the genus Conus (known as “conotoxins” or “conopeptides”). Between 50 and 200 different venom peptides can be found in a single Conus species, each having its own complement of peptides. Conopeptides are encoded by a few gene superfamilies; here we analyze the evolution of the A-superfamily in a fish-hunting species clade, Pionoconus. More than 90 conopeptide sequences from 11 different Conus species were used to build a phylogenetic tree. Comparison with a species tree based on standard genes reveals multiple gene duplication events, some of which took place before the Pionoconus radiation. By analysing several A-conopeptides from other Conus species recorded in GenBank, we date the major duplication events after the divergence between fish-hunting and non-fish-hunting species. Furthermore, likelihood approaches revealed strong positive selection; the magnitude depends on which A-conopeptide lineage and amino-acid locus is analyzed. The four major A-conopeptide clades defined are consistent with the current division of the superfamily into families and subfamilies based on the Cys pattern. The function of three of these clades (the κA-family, the α4/7-subfamily, and α3/5-subfamily) has previously been characterized. The function of the remaining clade, corresponding to the α4/4-subfamily, has not been elucidated. This subfamily is also found in several other fish-hunting species clades within Conus. The analysis revealed a surprisingly diverse origin of α4/4 conopeptides from a single species, Conus bullatus. This phylogenetic approach that defines different genetic lineages of Conus venom peptides provides a guidepost for identifying conopeptides with potentially novel functions.

The Terebridae and teretoxins: Combining phylogeny and anatomy for concerted discovery of bioactive compounds.

The Conoidea superfamily, comprised of cone snails, terebrids, and turrids, is an exceptionally promising group for the discovery of natural peptide toxins. The potential of conoidean toxins has been realized with the distribution of the first Conus (cone snail) drug, Prialt (ziconotide), an analgesic used to alleviate chronic pain in HIV and cancer patients. Cone snail toxins (conotoxins) are highly variable, a consequence of a high mutation rate associated to duplication events and positive selection. As Conus and terebrids diverged in the early Paleocene, the toxins from terebrids (teretoxins) may demonstrate highly divergent and unique functionalities. Recent analyses of the Terebridae, a largely distributed family with more than 300 described species, indicate they have evolutionary and pharmacological potential. Based on a three gene (COI, 12S and 16S) molecular phylogeny, including ~50 species from the West-Pacific, five main terebrid lineages were discriminated: two of these lineages independently lost their venom apparatus, and one venomous lineage was previously unknown. Knowing the phylogenetic relationships within the Terebridae aids in effectively targeting divergent lineages with novel peptide toxins. Preliminary results indicate that teretoxins are similar in structure and composition to conotoxins, suggesting teretoxins are an attractive line of research to discover and develop new therapeutics that target ion channels and receptors. Using conotoxins as a guideline, and innovative natural products discovery strategies, such as the Concerted Discovery Strategy, the potential of the Terebridae and their toxins are explored as a pioneering pharmacological resource.

Evolution of the Toxoglossa Venom Apparatus as Inferred by Molecular Phylogeny of the Terebridae

Toxoglossate marine gastropods, traditionally assigned to the families Conidae, Terebridae, and Turridae, are one of the most populous animal groups that use venom to capture their prey. These marine animals are generally characterized by a venom apparatus that consists of a muscular venom bulb and a tubular venom gland. The toxoglossan radula, often compared with a hypodermic needle for its use as a conduit to inject toxins into prey, is considered a major anatomical breakthrough that assisted in the successful initial radiation of these animals in the Cretaceous and early Tertiary. The pharmacological success of toxins from cone snails has made this group a star among biochemists and neuroscientists, but very little is known about toxins from the other Toxoglossa, and the phylogeny of these families is largely in doubt. Here we report the first molecular phylogeny for the Terebridae and use the results to infer the evolution of the venom apparatus for this group. Our findings indicate that most of the genera of terebrids are polyphyletic, and one species (“Terebra” (s.l.) jungi) is the sister group to all other terebrids. Molecular analyses combined with mapping of venom apparatus morphology indicate that the Terebridae have lost the venom apparatus at least twice during their evolution. Species in the genera Terebra and Hastula have the typical venom apparatus found in most toxoglossate gastropods, but all other terebrid species do not. For venomous organisms, the dual analysis of molecular phylogeny and toxin function is an instructive combination for unraveling the larger questions of phylogeny and speciation. The results presented here suggest a paradigm shift in the current understanding of terebrid evolution, while presenting a road map for discovering novel terebrid toxins, a largely unexplored resource for biomedical research and potential therapeutic drug development.

Morphological proxies for taxonomic decision in turrids (Mollusca, Neogastropoda): a test of the value of shell and radula characters using molecular data.

Abstract The state of the art of turrid (=Turridae s. l.) systematics is that shells — when they include the protoconch — are reliable species-level identifiers, but inadequate proxies for allocation to genera or subfamilies. Generally, the radula is used for allocation to a (sub)family, but the hypothesis that the radula is a more adequate proxy than the shell for relationships has not yet been tested by molecular data. Species of Xenuroturris may have drastically different radulae, with either “semi-enrolled” or “duplex” marginal teeth, although their shells are very similar or even almost indistinguishable. Molecular data confirm that specimens with different types of radulae constitute different species, but two species of a pair with respectively semi-enrolled and duplex teeth end up being not closely related. However, it is still unresolved whether species with semi-enrolled (=Iotyrris) and duplex teeth (=Xenuroturris) form two supported monophyletic clades. Iotyrris devoizei n.sp. and I. musivum n.sp. are described from Vanuatu, where they occur sympatrically with I. cingulifera and Xenuroturris legitima.

Loss of planktotrophy and speciation: geographical fragmentation in the deep-water gastropod genus Bathytoma (Gastropoda, Conoidea) in the western Pacific

Dispersal capabilities are crucial in how speciation patterns are determined in marine invertebrates. Species possessing a long-living planktonic larva apparently have a dispersal advantage over those with non-planktotrophic development, and their distant populations may exchange genetic material, maintaining a broad geographical range for the species. Recent species of the gastropod genus Bathytoma (Conoidea) are all characterized by non-planktotrophic development, having most probably lost a free-swimming larva in the pre-Pliocene, as Miocene fossils have protoconchs indicating planktotrophic larval development. All have a bathyal distribution (100–1500 m), which implies that their capability for direct expansion on the bottom is restricted by both deep-sea basins and shallow-water areas, especially in insular West and South-West Indo-Pacific. Therefore, it can be hypothesized that Bathytoma populations should represent numerous, mostly allopatric taxa restricted to a single or contiguous island groups. We tested this hypothesis using molecular and morphological characters independently. One hundred and thirty-eight specimens from the Philippines, Solomons, Vanuatu, and the Coral Sea were sequenced for one mitochondrial (COI) and one nuclear (ITS2) gene, and 14 operational molecular units were recognized. When these molecular units are overlaid over shell characters, 13 species (11 unnamed) and one form of uncertain status are recognized: three occur in the Philippines, six in the Solomons and one in New Caledonia. Broad distributions (inter-archipelagic) are uncommon (three species). On the whole, the phylogeographic pattern of the diversity in the genus is rather complex and probably also reflects processes of sympatric and fine-scale allopatric speciation, and local extinctions. The eleven new species are described and named.

A good compromise: rapid and robust species proxies for inventorying biodiversity hotspots using the Terebridae (Gastropoda: Conoidea).

Devising a reproducible approach for species delimitation of hyperdiverse groups is an ongoing challenge in evolutionary biology. Speciation processes combine modes of passive and adaptive trait divergence requiring an integrative taxonomy approach to accurately generate robust species hypotheses. However, in light of the rapid decline of diversity on Earth, complete integrative approaches may not be practical in certain species-rich environments. As an alternative, we applied a two-step strategy combining ABGD (Automated Barcode Gap Discovery) and Klee diagrams, to balance speed and accuracy in producing primary species hypotheses (PSHs). Specifically, an ABGD/Klee approach was used for species delimitation in the Terebridae, a neurotoxin-producing marine snail family included in the Conoidea. Delimitation of species boundaries is problematic in the Conoidea, as traditional taxonomic approaches are hampered by the high levels of variation, convergence and morphological plasticity of shell characters. We used ABGD to analyze gaps in the distribution of pairwise distances of 454 COI sequences attributed to 87 morphospecies and obtained 98 to 125 Primary Species Hypotheses (PSHs). The PSH partitions were subsequently visualized as a Klee diagram color map, allowing easy detection of the incongruences that were further evaluated individually with two other species delimitation models, General Mixed Yule Coalescent (GMYC) and Poisson Tree Processes (PTP). GMYC and PTP results confirmed the presence of 17 putative cryptic terebrid species in our dataset. The consensus of GMYC, PTP, and ABGD/Klee findings suggest the combination of ABGD and Klee diagrams is an effective approach for rapidly proposing primary species proxies in hyperdiverse groups and a reliable first step for macroscopic biodiversity assessment.