DeTox: a pipeline for the detection of toxins in venomous organisms.

Venomous organisms have independently evolved the ability to produce toxins 101 times during their evolutionary history, resulting in over 200 000 venomous species. Collectively, these species produce millions of toxins, making them a valuable resource for bioprospecting and understanding the evolutionary mechanisms underlying genetic diversification. RNA-seq is the preferred method for characterizing toxin repertoires, but the analysis of the resulting data remains challenging. While early approaches relied on similarity-based mapping to known toxin databases, recent studies have highlighted the importance of structural features for toxin detection. The few existing pipelines lack an integration between these complementary approaches, and tend to be difficult to run for non-experienced users. To address these issues, we developed DeTox, a comprehensive and user-friendly tool for toxin research. It combines fast execution, parallelization and customization of parameters. DeTox was tested on published transcriptomes from gastropod mollusks, cnidarians and snakes, retrieving most putative toxins from the original articles and identifying additional peptides as potential toxins to be confirmed through manual annotation and eventually proteomic analysis. By integrating a structure-based search with similarity-based approaches, DeTox allows the comprehensive characterization of toxin repertoire in poorly-known taxa. The effect of the taxonomic bias in existing databases is minimized in DeTox, as mirrored in the detection of unique and divergent toxins that would have been overlooked by similarity-based methods. DeTox streamlines toxin annotation, providing a valuable tool for efficient identification of venom components that will enhance venom research in neglected taxa.

Prey Shifts Drive Venom Evolution in Cone Snails.

Venom systems are complex traits that have independently emerged multiple times in diverse plant and animal phyla. Within each venomous lineage there typically exists interspecific variation in venom composition where several factors have been proposed as drivers of variation, including phylogeny and diet. Understanding these factors is of broad biological interest and has implications for the development of antivenom therapies and venom-based drug discovery. Because of their high species richness and the presence of several major evolutionary prey shifts, venomous marine cone snails (genus Conus) provide an ideal system to investigate drivers of interspecific venom variation. Here, by analyzing the venom gland expression profiles of ∼3,000 toxin genes from 42 species of cone snail, we elucidate the role of prey-specific selection pressures in shaping venom variation. By analyzing overall venom composition and individual toxin structures, we demonstrate that the shifts from vermivory to piscivory in Conus are complemented by distinct changes in venom composition independent of phylogeny. In vivo injections of venom from piscivorous cone snails in fish further showed a higher potency compared with venom of nonpiscivores demonstrating a selective advantage. Together, our findings provide compelling evidence for the role of prey shifts in directing the venom composition of cone snails and expand our understanding of the mechanisms of venom variation and diversification.

Phylogenomics of Neogastropoda: The Backbone Hidden in the Bush.

The molluskan order Neogastropoda encompasses over 15,000 almost exclusively marine species playing important roles in benthic communities and in the economies of coastal countries. Neogastropoda underwent intensive cladogenesis in the early stages of diversification, generating a "bush" at the base of their evolutionary tree, which has been hard to resolve even with high throughput molecular data. In the present study to resolve the bush, we use a variety of phylogenetic inference methods and a comprehensive exon capture dataset of 1817 loci (79.6% data occupancy) comprising 112 taxa of 48 out of 60 Neogastropoda families. Our results show consistent topologies and high support in all analyses at (super)family level, supporting monophyly of Muricoidea, Mitroidea, Conoidea, and, with some reservations, Olivoidea and Buccinoidea. Volutoidea and Turbinelloidea as currently circumscribed are clearly paraphyletic. Despite our analyses consistently resolving most backbone nodes, 3 prove problematic: First, the uncertain placement of Cancellariidae, as the sister group to either a Ficoidea-Tonnoidea clade or to the rest of Neogastropoda, leaves monophyly of Neogastropoda unresolved. Second, relationships are contradictory at the base of the major "core Neogastropoda" grouping. Third, coalescence-based analyses reject monophyly of the Buccinoidea in relation to Vasidae. We analyzed phylogenetic signal of targeted loci in relation to potential biases, and we propose the most probable resolutions in the latter 2 recalcitrant nodes. The uncertain placement of Cancellariidae may be explained by orthology violations due to differential paralog loss shortly after the whole genome duplication, which should be resolved with a curated set of longer loci.

Coupling DNA barcodes and exon-capture to resolve the phylogeny of Turridae (Gastropoda, Conoidea).

Taxon sampling in most phylogenomic studies is often based on known taxa and/or morphospecies, thus ignoring undescribed diversity and/or cryptic lineages. The family Turridae is a group of venomous snails within the hyperdiverse superfamily Conoidea that includes many undescribed and cryptic species. Therefore 'traditional' taxon sampling could constitute a strong risk of undersampling or oversampling Turridae lineages. To minimize potential biases, we establish a robust sampling strategy, from species delimitation to phylogenomics. More than 3,000 cox-1 "barcode" sequences were used to propose 201 primary species hypotheses, nearly half of them corresponding to species potentially new to science, including several cryptic species. A 110-taxa exon-capture tree, including species representatives of the diversity uncovered with the cox-1 dataset, was build using up to 4,178 loci. Our results show the polyphyly of the genus Gemmula, that is split into up to 10 separate lineages, of which half would not have been detected if the sampling strategy was based only on described species. Our results strongly suggest that the use of blind, exploratory and intensive barcode sampling is necessary to avoid sampling biases in phylogenomic studies.

Collaborative Expression: Transcriptomics of Conus virgo Suggests Contribution of Multiple Secretory Glands to Venom Production.

Venomous marine gastropods of the family Conidae are among the most diversified predators in marine realm-in large due to their complex venoms. Besides being a valuable source of bioactive neuropeptides conotoxins, cone-snails venoms are an excellent model for molecular evolution studies, addressing origin of key innovations. However, these studies are handicapped by scarce current knowledge on the tissues involved in venom production, as it is generally assumed the sole prerogative of the venom gland (VG). The role of other secretory glands that are present in all Conus species (salivary gland, SG) or only in some species (accessory salivary gland, ASG) remains poorly understood. Here, for the first time, we carry out a detailed analysis of the VG, SG, and ASG transcriptomes in the vermivorous Conus virgo. We detect multiple transcripts clusters in both the SG and ASG, whose annotations imply venom-related functions. Despite the subsets of transcripts highly-expressed in the VG, SG, and ASG being very distinct, SG expresses an L-, and ASG-Cerm08-, and MEFRR- superfamily conotoxins, all previously considered specific for VG. We corroborate our results with the analysis of published SG and VG transcriptomes from unrelated fish-hunting C. geographus, and C. striatus, possibly fish-hunting C. rolani, and worm-hunting Conus quercinus. In spite of low expression levels of conotoxins, some other specific clusters of putative venom-related peptides are present and may be highly expressed in the SG of these species. Further functional studies are necessary to determine the role that these peptides play in envenomation. In the meantime, our results show importance of routine multi-tissue sampling both for accurate interpretation of tissue-specific venom composition in cone-snails, and for better understanding origin and evolution of venom peptides genes.

Vexitoxins: conotoxin-like venom peptides from predatory gastropods of the genus Vexillum.

Venoms of predatory marine cone snails are intensely studied because of the biomedical applications of the neuropeptides that they contain, termed conotoxins. Meanwhile some gastropod lineages have independently acquired secretory glands strikingly similar to the venom gland of cone snails, suggesting that they possess similar venoms. Here we focus on the most diversified of these clades, the genus Vexillum. Based on the analysis of a multi-species proteo-transcriptomic dataset, we show that Vexillum species indeed produce complex venoms dominated by highly diversified short cysteine-rich peptides, vexitoxins. Vexitoxins possess the same precursor organization, display overlapping cysteine frameworks and share several common post-translational modifications with conotoxins. Some vexitoxins show sequence similarity to conotoxins and adopt similar domain conformations, including a pharmacologically relevant inhibitory cysteine knot motif. The Vexillum envenomation gland (gL) is a notably more recent evolutionary novelty than the conoidean venom gland. Thus, we hypothesize lower divergence between vexitoxin genes, and their ancestral 'somatic' counterparts compared to that in conotoxins, and we find support for this hypothesis in the evolution of the vexitoxin cluster V027. We use this example to discuss how future studies on vexitoxins can inform the origin of conotoxins, and how they may help to address outstanding questions in venom evolution.

A phylogeny-aware approach reveals unexpected venom components in divergent lineages of cone snails.

Marine gastropods of the genus Conus are renowned for their remarkable diversity and deadly venoms. While Conus venoms are increasingly well studied for their biomedical applications, we know surprisingly little about venom composition in other lineages of Conidae. We performed comprehensive venom transcriptomic profiling for Conasprella coriolisi and Pygmaeconus traillii, first time for both respective genera. We complemented reference-based transcriptome annotation by a de novo toxin prediction guided by phylogeny, which involved transcriptomic data on two additional 'divergent' cone snail lineages, Profundiconus, and Californiconus. We identified toxin clusters (SSCs) shared among all or some of the four analysed genera based on the identity of the signal region-a molecular tag present in toxins. In total, 116 and 98 putative toxins represent 29 and 28 toxin gene superfamilies in Conasprella and Pygmaeconus, respectively; about quarter of these only found by semi-manual annotation of the SSCs. Two rare gene superfamilies, originally identified from fish-hunting cone snails, were detected outside Conus rather unexpectedly, so we further investigated their distribution across Conidae radiation. We demonstrate that both these, in fact, are ubiquitous in Conidae, sometimes with extremely high expression. Our findings demonstrate how a phylogeny-aware approach circumvents methodological caveats of similarity-based transcriptome annotation.

Macroevolutionary Analyses Suggest That Environmental Factors, Not Venom Apparatus, Play Key Role in Terebridae Marine Snail Diversification.

How species diversification occurs remains an unanswered question in predatory marine invertebrates, such as sea snails of the family Terebridae. However, the anatomical disparity found throughput the Terebridae provides a unique perspective for investigating diversification patterns in venomous predators. In this study, a new dated molecular phylogeny of the Terebridae is used as a framework for investigating diversification of the family through time, and for testing the putative role of intrinsic and extrinsic traits, such as shell size, larval ecology, bathymetric distribution, and anatomical features of the venom apparatus, as drivers of terebrid species diversification. Macroevolutionary analysis revealed that when diversification rates do not vary across Terebridae clades, the whole family has been increasing its global diversification rate since 25 Ma. We recovered evidence for a concurrent increase in diversification of depth ranges, while shell size appeared to have undergone a fast divergence early in terebrid evolutionary history. Our data also confirm that planktotrophy is the ancestral larval ecology in terebrids, and evolutionary modeling highlighted that shell size is linked to larval ecology of the Terebridae, with species with long-living pelagic larvae tending to be larger and have a broader size range than lecithotrophic species. Although we recovered patterns of size and depth trait diversification through time and across clades, the presence or absence of a venom gland (VG) did not appear to have impacted Terebridae diversification. Terebrids have lost their venom apparatus several times and we confirm that the loss of a VG happened in phylogenetically clustered terminal taxa and that reversal is extremely unlikely. Our findings suggest that environmental factors, and not venom, have had more influence on terebrid evolution.

Data, time and money: evaluating the best compromise for inferring molecular phylogenies of non-model animal taxa.

For over a decade now, High Throughput sequencing (HTS) approaches have revolutionized phylogenetics, both in terms of data production and methodology. While transcriptomes and (reduced) genomes are increasingly used, generating and analyzing HTS datasets remain expensive, time consuming and complex for most non-model taxa. Indeed, a literature survey revealed that 74% of the molecular phylogenetics trees published in 2018 are based on data obtained through Sanger sequencing. In this context, our goal was to identify the strategy that would represent the best compromise among costs, time and robustness of the resulting tree. We sequenced and assembled 32 transcriptomes of the marine mollusk family Turridae, considered as a typical non-model animal taxon. From these data, we extracted the loci most commonly used in gastropod phylogenies (cox1, 12S, 16S, 28S, h3 and 18S), full mitogenomes, and a reduced nuclear transcriptome representation. With each dataset, we reconstructed phylogenies and compared their robustness and accuracy. We discuss the impact of missing data and the use of statistical tests, tree metrics, and supertree and supermatrix methods to further improve phylogenetic data acquisition pipelines. We evaluated the overall costs (time and money) in order to identify the best compromise for phylogenetic data sampling in non-model animal taxa. Although sequencing full mitogenomes seems to constitute the best compromise both in terms of costs and node support, they are known to induce biases in phylogenetic reconstructions. Rather, we recommend to systematically include loci commonly used for phylogenetics and taxonomy (i.e. DNA barcodes, rRNA genes, full mitogenomes, etc.) among the other loci when designing baits for capture.

Exon-Capture-Based Phylogeny and Diversification of the Venomous Gastropods (Neogastropoda, Conoidea).

Transcriptome-based exon capture methods provide an approach to recover several hundred markers from genomic DNA, allowing for robust phylogenetic estimation at deep timescales. We applied this method to a highly diverse group of venomous marine snails, Conoidea, for which published phylogenetic trees remain mostly unresolved for the deeper nodes. We targeted 850 protein coding genes (678,322 bp) in ca. 120 samples, spanning all (except one) known families of Conoidea and a broad selection of non-Conoidea neogastropods. The capture was successful for most samples, although capture efficiency decreased when DNA libraries were of insufficient quality and/or quantity (dried samples or low starting DNA concentration) and when targeting the most divergent lineages. An average of 75.4% of proteins was recovered, and the resulting tree, reconstructed using both supermatrix (IQ-tree) and supertree (Astral-II, combined with the Weighted Statistical Binning method) approaches, are almost fully supported. A reconstructed fossil-calibrated tree dates the origin of Conoidea to the Lower Cretaceous. We provide descriptions for two new families. The phylogeny revealed in this study provides a robust framework to reinterpret changes in Conoidea anatomy through time. Finally, we used the phylogeny to test the impact of the venom gland and radular type on diversification rates. Our analyses revealed that repeated losses of the venom gland had no effect on diversification rates, while families with a breadth of radula types showed increases in diversification rates, thus suggesting that trophic ecology may have an impact on the evolution of Conoidea.

Delimiting species of marine gastropods (Turridae, Conoidea) using RAD sequencing in an integrative taxonomy framework.

Species delimitation in poorly known and diverse taxa is usually performed based on monolocus, DNA-barcoding-like approaches, while multilocus data are often used to test alternative species hypotheses in well-studied groups. We combined both approaches to delimit species in the Xenuroturris/Iotyrris complex, a group of venomous marine gastropods from the Indo-Pacific. First, COI sequences were analysed using three methods of species delimitation to propose primary species hypotheses. Second, RAD sequencing data were also obtained and a maximum-likelihood phylogenetic tree produced. We tested the impact of the level of missing data on the robustness of the phylogenetic tree obtained with the RAD-seq data. Alternative species partitions revealed with the COI data set were also tested using the RAD-seq data and the Bayes factor species delimitation method. The congruence between the species hypotheses proposed with the mitochondrial nuclear data sets, together with the morphological variability of the shell and the radula and the distribution pattern, was used to turn the primary species hypotheses into secondary species hypotheses. Allopatric primary species hypotheses defined with the COI gene were interpreted to correspond to intraspecific structure. Most of the species are found sympatrically in the Philippines, and only one is confidently identified as a new species and described as Iotyrris conotaxis n. sp. The results obtained demonstrate the efficiency of the combined monolocus/multilocus approach to delimit species.

Venom Insulins of Cone Snails Diversify Rapidly and Track Prey Taxa.

A specialized insulin was recently found in the venom of a fish-hunting cone snail, Conus geographus Here we show that many worm-hunting and snail-hunting cones also express venom insulins, and that this novel gene family has diversified explosively. Cone snails express a highly conserved insulin in their nerve ring; presumably this conventional signaling insulin is finely tuned to the Conus insulin receptor, which also evolves very slowly. By contrast, the venom insulins diverge rapidly, apparently in response to biotic interactions with prey and also possibly the cones' own predators and competitors. Thus, the inwardly directed signaling insulins appear to experience predominantly purifying sele\ction to target an internal receptor that seldom changes, while the outwardly directed venom insulins frequently experience directional selection to target heterospecific insulin receptors in a changing mix of prey, predators and competitors. Prey insulin receptors may often be constrained in ways that prevent their evolutionary escape from targeted venom insulins, if amino-acid substitutions that result in escape also degrade the receptor's signaling functions.

Prey-Capture Strategies of Fish-Hunting Cone Snails: Behavior, Neurobiology and Evolution.

The venomous fish-hunting cone snails (Conus) comprise eight distinct lineages evolved from ancestors that preyed on worms. In this article, we attempt to reconstruct events resulting in this shift in food resource by closely examining patterns of behavior, biochemical agents (toxins) that facilitate prey capture and the combinations of toxins present in extant species. The first sections introduce three different hunting behaviors associated with piscivory: 'taser-and-tether', 'net-engulfment' and 'strike-and-stalk'. The first two fish-hunting behaviors are clearly associated with distinct groups of venom components, called cabals, which act in concert to modify the behavior of prey in a specific manner. Derived fish-hunting behavior clearly also correlates with physical features of the radular tooth, the device that injects these biochemical components. Mapping behavior, biochemical components and radular tooth features onto phylogenetic trees shows that fish-hunting behavior emerged at least twice during evolution. The system presented here may be one of the best examples where diversity in structure, physiology and molecular features were initially driven by particular pathways selected through behavior.

iTaxoTools 1.0: Improved DNA Barcode Exploration with TaxI2.

The overall availability of user-friendly software tools tailored to the analysis of DNA barcodes is limited. Several obvious functions such as detecting and visualizing the DNA barcode gap, the calculation of matrices of pairwise distances at the level of species, or the filtering and decontaminating of sets of sequences based on comparisons with reference databases can typically be carried out only by complex procedures that involve various programs and/or a substantial manual work of formatting. The iTaxoTools project aims at contributing user-friendly software solutions to improve the speed and quality of the workflow of alpha-taxonomy. In this chapter, we provide detailed protocols for the use of a substantially improved version of the tool TaxI2 for distance-based exploration of DNA barcodes. The program calculates genetic distances from prealigned data sets, or based on pairwise alignments, or with an alignment-free approach. Sequence and metadata input can be formatted as tab-delimited files and TaxI2 then computes tables, matrices and graphs of distances, and distance summary statistics within and between species and genera. TaxI2 also includes modes to compare a set of sequences against one or two reference data sets and output lists of best matches or filter data according to thresholds or reciprocal matches. Here, detailed step-by-step protocols are provided for the use of TaxI2, as well as for the interpretation of the program's output.

DNA Barcode-Based Species Diagnosis with MolD.

Rapid biodiversity loss sets new requirements for taxonomic research, prompting updating some long-established practices to maximize timely documentation of species before they have gone extinct. One of the crucial procedures associated with the description of new taxa in Linnean taxonomy is assigning them a diagnosis, which is an account of the specific features of the taxon, differentiating it from already described species. Traditionally, diagnostic characters have been morphological, but especially in the case of morphologically cryptic species, molecular diagnoses become increasingly important. In this chapter, we provide detailed protocols for molecular taxon diagnosis with the bioinformatic tool MolD which is available as open-source Python code, command-line driven binary, GUI-driven executable for Windows and Mac, and Galaxy implementation. MolD identifies diagnostic combinations of nucleotides (DNCs) in addition to single (pure) diagnostic sites, enabling users to base DNA diagnoses on a minimal number of diagnostic sites necessary for reliable differentiation of taxa.

Species Delimitation and Exploration of Species Partitions with ASAP and LIMES.

DNA barcoding plays an important role in exploring undescribed biodiversity and is increasingly used to delimit lineages at the species level (see Chap. 4 by Miralles et al.). Although several approaches and programs have been developed to perform species delimitation from datasets of single-locus DNA sequences, such as DNA barcodes, most of these were not initially provided as user-friendly GUI-driven executables. In spite of their differences, most of these tools share the same goal, i.e., inferring de novo a partition of subsets, potentially each representing a distinct species. More recently, a proposed common exchange format for the resulting species partitions (SPART) has been implemented by several of these tools, paving the way toward developing an interoperable digital environment entirely dedicated to integrative and comparative species delimitation. In this chapter, we provide detailed protocols for the use of two bioinformatic tools, one for single locus molecular species delimitation (ASAP) and one for statistical comparison of species partitions resulting from any kind of species delimitation analyses (LIMES).

mold, a novel software to compile accurate and reliable DNA diagnoses for taxonomic descriptions.

DNA data are increasingly being used for phylogenetic inference, and taxon delimitation and identification, but scarcely for the formal description of taxa, despite their undisputable merits in taxonomy. The uncertainty regarding the robustness of DNA diagnoses, however, remains a major impediment to their use. We have developed a new program, mold, that identifies diagnostic nucleotide combinations (DNCs) in DNA sequence alignments for selected taxa, which can be used to provide formal diagnoses of these taxa. To test the robustness of DNA diagnoses, we carry out iterated haplotype subsampling for selected query species in published DNA data sets of varying complexity. We quantify the reliability of diagnosis by diagnosing each query subsample and then checking if this diagnosis remains valid against the entire data set. We demonstrate that widely used types of diagnostic DNA characters are often absent for a query taxon or are not sufficiently reliable. We thus propose a new type of DNA diagnosis, termed "redundant DNC" (or rDNC), which takes into account unsampled genetic diversity, and constitutes a much more reliable descriptor of a taxon. mold successfully retrieves rDNCs for all but two species in the analysed data sets, even in those comprising hundreds of species. mold shows unparalleled efficiency in large DNA data sets and is the only available software capable of compiling DNA diagnoses that suit predefined criteria of reliability.

Neogastropod (Mollusca, Gastropoda) phylogeny: A step forward with mitogenomes.

The Neogastropoda (Mollusca, Gastropoda) encompass more than 15,000 described species of marine predators, including several model organisms in toxinology, embryology and physiology. However, their phylogenetic relationships remain mostly unresolved and their classification unstable. We took advantage of the many mitogenomes published in GenBank to produce a new molecular phylogeny of the neogastropods. We completed the taxon sampling by using an in-house bioinformatic pipeline to retrieve mitochondrial genes from 13 transcriptomes, corresponding to five families not represented in GenBank, for a final dataset of 113 taxa. Because mitogenomic data are prone to reconstruction artefacts, eight different evolutionary models were applied to reconstruct phylogenetic trees with IQTREE, RAxML and MrBayes. If the over-parametrization of some models produced trees with aberrant internal long branches, the global topology of the trees remained stable over models and softwares, and several relationships were revealed or found supported here for the first time. However, even if our dataset encompasses 60% of the valid families of neogastropods, some key taxa are missing and should be added in the future before proposing a revision of the classification of the neogastropods. Our study also demonstrates that even complex models struggle to satisfactorily handle the evolutionary history of mitogenomes, still leading to long-branch attractions in phylogenetic trees. Other approaches, such as reduced-genome strategies, must be envisaged to fully resolve the neogastropod phylogeny.

Maxillo-mandibular Defect Reconstruction with Bilateral Free Fibula Flaps with Dental Implant Placement and Immediate Loading: A Case Report of the Three-team Approach.

Patients with advanced malignant tumors, including both jaws, is a challenging task for a head and neck surgeon. Current treatment landscape demonstrates good functional, anatomical, and aesthetic results in patients who could previously receive only palliative care. The extensive tissue defects resulting from oncological resections in the head and neck region require immediate reconstruction due to the exposure of vital structures and their contact with the external environment. A patient was operated using a three-team multidisciplinary approach involving simultaneous work of three specialized teams of maxillofacial and reconstructive microsurgeons, as well as an implantologist and a prosthodontist. This approach allowed simultaneous tumor resection with subsequent reconstruction of the intraoperative defect involving bilateral harvesting of two revascularized free fibular osteomusculocutaneous flaps with dental implantation and simultaneous rehabilitation of dentition with crowns.

Phylogeny of the genus Turris: correlating molecular data with radular anatomy and shell morphology.

There are over 10,000 species of venomous marine molluscs, the vast majority of these, which are generally referred to as "turrids", are traditionally assigned to a single family, Turridae (Powell 1966). Here, we provide an initial molecular analysis of the type genus of the family, Turris Röding, 1798, thought to be among the most well characterized groups in the family. We show that the type genus is not monophyletic. We analyzed specimens conventionally assigned to 9 different Turris species using molecular markers, combined with the shell morphology and radular anatomy whenever feasible. The results suggest that species assigned to the genus Turris, provisionally assigned to two different subgenera are not monophyletic. Five previously described species belong to the subgenus Turris (s.s.) Röding 1798: Turris babylonia, (Linne, 1758), Turris grandis, (J. E. Gray, 1834), Turris dollyae, (Olivera, 1999), Turris normandavidsoni (Olivera, 1999) and Turris spectabilis (Reeve, 1843). With a change in species designation, Turris assyria (formerly T. babylonia1010) is added to a well-defined clade, which is in turn more closely related to Lophiotoma and Gemmula species than to the other five Turris species. We show that these five species conventionally assigned to Turris do not belong in the same subgenus, and form a clade provisionally designated as AnnulaturrisPowell, 1966: Turris annulata, (Reeve, 1843), Turris undosa, (Lamarck, 1816), Turris cristata, (Vera-Peláez, Vega-Luz, and Lozano-Francisco 2000) Turris cryptorrhaphe (G. B. Sowerby, 1825) and Turris nadaensis (Azuma, 1973). Implications of the molecular phylogenetic results and its correlation with radular morphology are discussed.