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.

Phylogenomics of Neogastropoda: the backbone hidden in the bush.

The molluscan 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 early stages of diversification, generating a 'bush' at the base of their evolutionary tree, that 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 1,817 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, three prove problematic: First, 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 analysed phylogenetic signal of targeted loci in relation to potential biases, and we propose most probable resolutions in the latter two 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.

Whole Genome Duplication and Gene Evolution in the Hyperdiverse Venomous Gastropods.

The diversity of venomous organisms and the toxins they produce have been increasingly investigated, but taxonomic bias remains important. Neogastropods, a group of marine predators representing almost 22% of the known gastropod diversity, evolved a wide range of feeding strategies, including the production of toxins to subdue their preys. However, whether the diversity of these compounds is at the origin of the hyperdiversification of the group and how genome evolution may correlate with both the compounds and species diversities remain understudied. Among the available gastropods genomes, only eight, with uneven quality assemblies, belong to neogastropods. Here, we generated chromosome-level assemblies of two species belonging to the Tonnoidea and Muricoidea superfamilies (Monoplex corrugatus and Stramonita haemastoma). The two obtained high-quality genomes had 3 and 2.2 Gb, respectively, and 92-89% of the total assembly conformed 35 pseudochromosomes in each species. Through the analysis of syntenic blocks, Hox gene cluster duplication, and synonymous substitutions distribution pattern, we inferred the occurrence of a whole genome duplication event in both genomes. As these species are known to release venom, toxins were annotated in both genomes, but few of them were found in homologous chromosomes. A comparison of the expression of ohnolog genes (using transcriptomes from osphradium and salivary glands in S. haemastoma), where both copies were differentially expressed, showed that most of them had similar expression profiles. The high quality of these genomes makes them valuable reference in their respective taxa, facilitating the identification of genome-level processes at the origin of their evolutionary success.

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.

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.

Raising names from the dead: A time-calibrated phylogeny of frog shells (Bursidae, Tonnoidea, Gastropoda) using mitogenomic data.

With 59 Recent species, Bursidae, known as «frog shells¼, are a small but widely distributed group of tropical and subtropical gastropods that are most diverse in the Indo-West Pacific. The present study is aimed at reconstructing phylogenetic relationships of bursid gastropods based on extensive and representative taxon sampling. Five genetic markers (cytochrome c oxidase subunit I (cox1), 16 s and 12 s rRNA mitochondrial genes, 28 s rRNA and Histone H3 nuclear gene) were sequenced for over 30 species in every known genus but Crossata. Furthermore, we sequenced the complete mt-genome of 9 species (10 specimens) (Aspa marginata, Marsupina bufo, Korrigania quirihorai, Korrigania fijiensis, Tutufa rubeta, Bursa lamarckii, Lampasopsis rhodostoma (twice), Bufonaria perelegans and Bursa aff. tuberosissima). Our analysis recovered Bursidae as a monophyletic group, whereas the genus Bursa was found to be polyphyletic. The genera Talisman and Dulcerana are resurrected and the genera Alanbeuella gen. nov. and Korrigania gen. nov. are described. Dating analysis using 21 extinct taxa for node and simplified tip calibrations was performed, showing a diversification of the group in two phases. Diversification may be linked to tectonic events leading to biodiversity relocation from the western Tethys toward the Indo-Pacific.

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.

Lack of Signal for the Impact of Conotoxin Gene Diversity on Speciation Rates in Cone Snails.

Understanding why some groups of organisms are more diverse than others is a central goal in macroevolution. Evolvability, or the intrinsic capacity of lineages for evolutionary change, is thought to influence disparities in species diversity across taxa. Over macroevolutionary time scales, clades that exhibit high evolvability are expected to have higher speciation rates. Cone snails (family: Conidae, $>$900 spp.) provide a unique opportunity to test this prediction because their toxin genes can be used to characterize differences in evolvability between clades. Cone snails are carnivorous, use prey-specific venom (conotoxins) to capture prey, and the genes that encode venom are known and diversify through gene duplication. Theory predicts that higher gene diversity confers a greater potential to generate novel phenotypes for specialization and adaptation. Therefore, if conotoxin gene diversity gives rise to varying levels of evolvability, conotoxin gene diversity should be coupled with macroevolutionary speciation rates. We applied exon capture techniques to recover phylogenetic markers and conotoxin loci across 314 species, the largest venom discovery effort in a single study. We paired a reconstructed timetree using 12 fossil calibrations with species-specific estimates of conotoxin gene diversity and used trait-dependent diversification methods to test the impact of evolvability on diversification patterns. Surprisingly, we did not detect any signal for the relationship between conotoxin gene diversity and speciation rates, suggesting that venom evolution may not be the rate-limiting factor controlling diversification dynamics in Conidae. Comparative analyses showed some signal for the impact of diet and larval dispersal strategy on diversification patterns, though detection of a signal depended on the dataset and the method. If our results remain true with increased taxonomic sampling in future studies, they suggest that the rapid evolution of conid venom may cause other factors to become more critical to diversification, such as ecological opportunity or traits that promote isolation among lineages.

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.

Frogs and tuns and tritons - A molecular phylogeny and revised family classification of the predatory gastropod superfamily Tonnoidea (Caenogastropoda).

The Tonnoidea is a moderately diverse group of large, predatory gastropods with ∼360 valid species. Known for their ability to secrete sulfuric acid, they use it to prey on a diversity of invertebrates, primarily echinoderms. Tonnoideans currently are classified in seven accepted families: the comparatively well known, shallow water Bursidae, Cassidae, Personidae, Ranellidae, and Tonnidae, and the lesser-known, deep water Laubierinidae and Pisanianuridae. We assembled a mitochondrial and nuclear gene (COI, 16S, 12S, 28S) dataset for ∼80 species and 38 genera currently recognized as valid. Bayesian analysis of the concatenated dataset recovered a monophyletic Tonnoidea, with Ficus as its sister group. Unexpectedly, Thalassocyon, currently classified in the Ficidae, was nested within the ingroup as the sister group to Distorsionella. Among currently recognized families, Tonnidae, Cassidae, Bursidae and Personidae were supported as monophyletic but the Ranellidae and Ranellinae were not, with Cymatiinae, Ranella and Charonia supported as three unrelated clades. The Laubierinidae and Pisanianuridae together form a monophyletic group. Although not all currently accepted genera have been included in the analysis, the new phylogeny is sufficiently robust and stable to the inclusion/exclusion of nonconserved regions to establish a revised family-level classification with nine families: Bursidae, Cassidae, Charoniidae, Cymatiidae, Laubierinidae, Personidae, Ranellidae, Thalassocyonidae and Tonnidae. The results reveal that many genera as presently circumscribed are para- or polyphyletic and, in some cases support the rescue of several genus-group names from synonymy (Austrosassia, Austrotriton, Laminilabrum, Lampadopsis, Personella, Proxicharonia, Tritonoranella) or conversely, support their synonymization (Biplex with Gyrineum). Several species complexes are also revealed that merit further investigation (e.g., Personidae: Distorsio decipiens, D. reticularis; Bursidae: Bursa tuberosissima; Cassidae: Echinophoria wyvillei, Galeodea bituminata, and Semicassis bisulcata). Consequently, despite their teleplanic larvae, the apparently circumglobal distribution of some tonnoidean species is the result of excessive synonymy. The superfamily is estimated to have diverged during the early Jurassic (∼186 Ma), with most families originating during a narrow ∼20 My window in Albian-Aptian times as part of the Mesozoic Marine Revolution.

Incorporation of deep-sea and small-sized species provides new insights into gastropods phylogeny.

The use of phylogeny with uneven or limited taxon sampling may bias our interpretation of organismal evolution, for instance, the origin(s) of the deep-sea animals. The Mollusca is the second most speciose phylum, in which the Gastropoda forms the largest group. However, the currently proposed hypotheses of gastropod phylogeny are mainly based on part of their taxonomic diversity, notably on the large-sized and shallow-water species. In this study, we aimed at correcting this bias by reconstructing the phylogeny with new mitogenomes of deep-sea gastropods including Anatoma sp., Bathysciadiidae sp., Bayerotrochus teramachii, Calliotropis micraulax, Coccocrater sp., Cocculina subcompressa, Lepetodrilus guaymasensis, Peltospira smaragdina, Perotrochus caledonicus, Pseudococculinidae sp., and Shinkailepas briandi. This dataset provided the first reports of the mitogenomes for the Cocculiniformia, three vetigastropod superfamilies: Pleurotomarioidea, Lepetelloidea, and Scissurelloidea, and the neritimorph family Phenacolepadidae. The addition of deep-sea representatives also allowed us to evaluate the evolution of habitat use in gastropods. Our results showed a strongly supported sister-group relationship between the deep-sea lineages Cocculiniformia and Neomphalina. Within the Vetigastropoda, the Pleurotomarioidea was revealed as the sister-group of the remaining vetigastropods. Although this clade was presently restricted to the deep sea, fossil records showed that it has only recently invaded this habitat, thus suggesting that shallow waters was the ancestral habitat for the Vetigastropoda. The deep-sea Lepetelloidea and Lepetodriloidea formed a well-supported clade, with the Scissurelloidea sister to it, suggesting an early transition from shallow water to deep sea in this lineage. In addition, the switch between different chemosynthetic habitats was also observed in deep-sea gastropod lineages, notably in Neomphalina and Lepetelloidea. In both cases, the biogenic substrates appeared as the putative ancestral habitat, confirming the previously proposed hypothesis of a wooden-step to deep-sea vents scenario of evolution of habitat use for these taxa.

Quantification of complex modular architecture in plants.

Morphometrics, the assignment of quantities to biological shapes, is a powerful tool to address taxonomic, evolutionary, functional and developmental questions. We propose a novel method for shape quantification of complex modular architecture in thalloid plants, whose extremely reduced morphologies, combined with the lack of a formal framework for thallus description, have long rendered taxonomic and evolutionary studies extremely challenging. Using graph theory, thalli are described as hierarchical series of nodes and edges, allowing for accurate, homologous and repeatable measurements of widths, lengths and angles. The computer program MorphoSnake was developed to extract the skeleton and contours of a thallus and automatically acquire, at each level of organization, width, length, angle and sinuosity measurements. Through the quantification of leaf architecture in Hymenophyllum ferns (Polypodiopsida) and a fully worked example of integrative taxonomy in the taxonomically challenging thalloid liverwort genus Riccardia, we show that MorphoSnake is applicable to all ramified plants. This new possibility of acquiring large numbers of quantitative traits in plants with complex modular architectures opens new perspectives of applications, from the development of rapid species identification tools to evolutionary analyses of adaptive plasticity.

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.

Phylogenetic relationships of the conoidean snails (Gastropoda: Caenogastropoda) based on mitochondrial genomes.

With more than 5,000 species, Conoidea is one of the most diversified superfamilies of Gastropoda. Recently, the family-level classification of these venomous predator snails has undergone substantial changes, on the basis of a phylogenetic tree reconstructed combining partial mitochondrial and nuclear gene sequences, and up to 16 families are now recognized. However, phylogenetic relationships among these families remain largely unresolved. Here, we sequenced 20 complete or nearly complete mitochondrial (mt) genomes, which were combined with mt genomes available in GenBank to construct a dataset that included representatives of 80% of the known families, although for some we had only one species or genus as representative. Most of the sequenced conoidean mt genomes shared a constant genome organization, and observed rearrangements were limited exclusively to tRNA genes in a few lineages. Phylogenetic trees were reconstructed using probabilistic methods. Two main monophyletic groups, termed "Clade A" and "Clade B", were recovered with strong support within a monophyletic Conoidea. Clade A (including families Clavatulidae, Horaiclavidae, Turridae s.s., Terebridae, Drilliidae, Pseudomelatomidae, and Cochlespiridae) was composed of four main lineages, one of which was additionally supported by a rearrangement in the gene order. Clade B (including families Conidae, Borsoniidae, Clathurellidae, Mangeliidae, Raphitomidae, and Mitromorphidae) was composed of five main lineages. The reconstructed phylogeny rejected the monophyly of Clavatulidae, Horaiclavidae, Turridae, Pseudomelatomidae, and Conidae, indicating that several of the currently accepted families may be ill-defined. The reconstructed tree also revealed new phylogenetic positions for genera characterized as tentative (Gemmuloborsonia, Lucerapex, and Leucosyrinx), enigmatic (Marshallena) or challenging to place (Fusiturris), which will potentially impact the classification of the Conoidea.

Exploring the Udoteaceae diversity (Bryopsidales, Chlorophyta) in the Caribbean region based on molecular and morphological data.

The Udoteaceae family (Bryopsidales, Chlorophyta) is known to be highly diverse morphologically in the Caribbean region, but only few studies have studied its genetic diversity. Using an integrative taxonomic approach, this study aimed at (1) exploring the Udoteaceae species diversity using a combination of five DNA-based species delimitation methods and morpho-anatomical data for confirmation; (2) estimating the discriminatory power of traditional diagnostic characters using a morphology-based clustering method and statistical analyses focused on the genus Udotea; and (3) reconstructing the phylogeny of the family based on a multilocus analysis (tufA, rbcL, 18S rDNA). Our results revealed strong congruence between species hypotheses across delimitation methods and markers. Morpho-anatomical characters proved essential to validate these hypotheses, to assign species names and to unveil new species. Morphological analyses led to relevant results for accurately discriminating Udotea morphospecies. Siphon features and cortication were key characters to define supra-specific groups and to revise the taxonomy of the genus Udotea. Phylogenetic analyses confirmed the polyphyly of Udotea, Rhipocephalus and Penicillus, which led us to propose a revised definition of Udotea sensu stricto based on both genetic and morphological data. Finally, our study emphasizes the importance of combining genetic and morphological data for the taxonomic revision of the Udoteaceae, but stresses the need of including more taxa from other geographical regions to better resolve taxonomic issues.

Beyond Conus: Phylogenetic relationships of Conidae based on complete mitochondrial genomes.

Understanding how the extraordinary taxonomic and ecological diversity of cone snails (Caenogastropoda: Conidae) evolved requires a statistically robust phylogenetic framework, which thus far is not available. While recent molecular phylogenies have been able to distinguish several deep lineages within the family Conidae, including the genera Profundiconus, Californiconus, Conasprella, and Conus (and within this one, several subgenera), phylogenetic relationships among these genera remain elusive. Moreover, the possibility that additional deep lineages may exist within the family is open. Here, we reconstructed with probabilistic methods a molecular phylogeny of Conidae using the newly sequenced complete or nearly complete mitochondrial (mt) genomes of the following nine species that represent all main Conidae lineages and potentially new ones: Profundiconus teramachii, Californiconus californicus, Conasprella wakayamaensis, Lilliconus sagei, Pseudolilliconus traillii, Conus (Kalloconus) venulatus, Conus (Lautoconus) ventricosus, Conus (Lautoconus) hybridus, and Conus (Eugeniconus) nobilis. To test the monophyly of the family, we also sequenced the nearly complete mt genomes of the following three species representing closely related conoidean families: Benthomangelia sp. (Mangeliidae), Tomopleura sp. (Borsoniidae), and Glyphostoma sp. (Clathurellidae). All newly sequenced conoidean mt genomes shared a relatively constant gene order with rearrangements limited to tRNA genes. The reconstructed phylogeny recovered with high statistical support the monophyly of Conidae and phylogenetic relationships within the family. The genus Profundiconus was placed as sister to the remaining genera. Within these, a clade including Californiconus and Lilliconus+Pseudolilliconus was the sister group of Conasprella to the exclusion of Conus. The phylogeny included a new lineage whose relative phylogenetic position was unknown (Lilliconus) and uncovered thus far hidden diversity within the family (Pseudolilliconus). Moreover, reconstructed phylogenetic relationships allowed inferring that the peculiar diet of Californiconus based on worms, mollusks, crustaceans and fish is derived, and reinforce the hypothesis that the ancestor of Conidae was a worm hunter. A chronogram was reconstructed under an uncorrelated relaxed molecular clock, which dated the origin of the family shortly after the Cretaceous-Tertiary boundary (about 59million years ago) and the divergence among main lineages during the Paleocene and the Eocene (56-30million years ago).

The phylogeny and systematics of the Nassariidae revisited (Gastropoda, Buccinoidea).

Nassariidae are a group of scavenging, predominantly marine, snails that are diversified on soft bottoms as well as on rocky shores, and are the subject of numerous research papers in ecology, ecotoxicology or paleontology. A weak and/or apparently continuous variation in shell characters has resulted in an intimidating taxonomy, with complex synonymy lists. Over 1320 extant nominal species have been described, of which 442 are currently regarded as valid. Above species level, the state of the art is equally hazy, with four subfamilies and twelve genera currently accepted, and many other names in the graveyard of synonymy. A molecular analysis based on three mitochondrial (COI, 16S, 12S) and two nuclear (28S, H3) markers was conducted. Our dataset includes 218 putative nassariid species, comprising 9 of the 12 valid genera, and 25 nominal genera represented by their type species. The monophyly of the Nassariidae as classically construed is not confirmed. Species of Antillophos, Engoniophos, Phos, Nassaria, Tomlinia and Anentome (formerly considered Buccinidae) are included inside the Nassariidae clade. Within the Nassariinae, the tree unexpectedly demonstrates that species from the Atlantic and the Indo-Pacific form different clades which represent several independent diversification events. Through an integrative approach, the reconstruction of ancestral states was addressed for eight characters supposedly informative for taxonomy. Using numerous fossil calibration points, Nassariidae appear to have originated 120 MYA ago in Atlantic temperate waters during the Lower Cretaceous. Our results have a profound impact on nassariid taxonomy, especially with regard to the validity of subfamily- and genus-level names.

Insights into the molecular phylogeny and historical biogeography of the white-clawed crayfish (Decapoda, Astacidae).

In this study, the evolutionary history of the white-clawed crayfish (WCC) was evaluated using large-scale datasets comprising >1350 specimens from the entire distribution range. Using species delimitation methods on mitochondrial DNA (mtDNA) sequences, we propose four primary species hypotheses for WCC. Sequences for several nuclear regions were screened but none showed significant variation within WCC. This result favours a single secondary species hypothesis and indicates the existence of a mito-nuclear discordance in WCC. Therefore, mtDNA groups were considered only as genetic units that carry information about ancient divergences within WCC and not as taxonomic units. The reconstruction of ancestral ranges and divergence time estimates were used to link the current genetic structure with paleogeographic processes. These results showed that the emergence of mtDNA groups in WCC could be related to the Messinian Salinity Crisis, the climate cooling during the Pliocene and Pleistocene, and (paleo)shifting of the Adriatic Sea coastline in the Padanovenezian Plain. The most recent common ancestor of the mtDNA groups most likely originated from Dalmatia (eastern Adriatic coast) as indicated by the reconstruction of ancestral ranges. This ecoregion, along with the Gulf of Venice Drainages, harbours a high genetic diversity and should be emphasised as an area of the highest conservation priority.