Maria Vittoria Modica

Large-scale species delimitation method for hyperdiverse groups.

Accelerating the description of biodiversity is a major challenge as extinction rates increase. Integrative taxonomy combining molecular, morphological, ecological and geographical data is seen as the best route to reliably identify species. Classic molluscan taxonomic methodology proposes primary species hypotheses (PSHs) based on shell morphology. However, in hyperdiverse groups, such as the molluscan family Turridae, where most of the species remain unknown and for which homoplasy and plasticity of morphological characters is common, shell‐based PSHs can be arduous. A four‐pronged approach was employed to generate robust species hypotheses of a 1000 specimen South‐West Pacific Turridae data set in which: (i) analysis of COI DNA Barcode gene is coupled with (ii) species delimitation tools GMYC (General Mixed Yule Coalescence Method) and ABGD (Automatic Barcode Gap Discovery) to propose PSHs that are then (iii) visualized using Klee diagrams and (iv) evaluated with additional evidence, such as nuclear gene rRNA 28S, morphological characters, geographical and bathymetrical distribution to determine conclusive secondary species hypotheses (SSHs). The integrative taxonomy approach applied identified 87 Turridae species, more than doubling the amount previously known in the Gemmula genus. In contrast to a predominantly shell‐based morphological approach, which over the last 30 years proposed only 13 new species names for the Turridae genus Gemmula, the integrative approach described here identified 27 novel species hypotheses not linked to available species names in the literature. The formalized strategy applied here outlines an effective and reproducible protocol for large‐scale species delimitation of hyperdiverse groups.

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.

The Neogastropoda: Evolutionary Innovations of Predatory Marine Snails with Remarkable Pharmacological Potential

The Neogastropoda include many familiar molluscs, such as cone snails (Conidae), purple dye snails (Muricidae), mud snails (Nassariidae), olive snails (Olividae), oyster drills (Muricidae), tulip shells (Fasciolariidae), and whelks (Buccinidae). Due to their amazing predatory specializations, neogastropods are often dominant members of the benthic community at the top of the food chain. In a dazzling display that ranges from boring holes to darting harpoons, neogastropods have developed several prey hunting innovations with specialized compounds pharmaceutical companies could only dream about. It has been hypothesized that evolutionary innovations related to feeding were the main drivers of the rapid neogastropod radiation in the late Cretaceous. The anatomical, behavioral, and biochemical specializations of neogastropod families that are promising targets in drug discovery and development are addressed within an evolutionary framework in this chapter.

The venomous cocktail of the vampire snail Colubraria reticulata (Mollusca, Gastropoda)

BackgroundHematophagy arose independently multiple times during metazoan evolution, with several lineages of vampire animals particularly diversified in invertebrates. However, the biochemistry of hematophagy has been studied in a few species of direct medical interest and is still underdeveloped in most invertebrates, as in general is the study of venom toxins. In cone snails, leeches, arthropods and snakes, the strong target specificity of venom toxins uniquely aligns them to industrial and academic pursuits (pharmacological applications, pest control etc.) and provides a biochemical tool for studying biological activities including cell signalling and immunological response. Neogastropod snails (cones, oyster drills etc.) are carnivorous and include active predators, scavengers, grazers on sessile invertebrates and hematophagous parasites; most of them use venoms to efficiently feed. It has been hypothesized that trophic innovations were the main drivers of rapid radiation of Neogastropoda in the late Cretaceous.We present here the first molecular characterization of the alimentary secretion of a non-conoidean neogastropod, Colubraria reticulata. Colubrariids successfully feed on the blood of fishes, throughout the secretion into the host of a complex mixture of anaesthetics and anticoagulants. We used a NGS RNA-Seq approach, integrated with differential expression analyses and custom searches for putative secreted feeding-related proteins, to describe in detail the salivary and mid-oesophageal transcriptomes of this Mediterranean vampire snail, with functional and evolutionary insights on major families of bioactive molecules.ResultsA remarkably low level of overlap was observed between the gene expression in the two target tissues, which also contained a high percentage of putatively secreted proteins when compared to the whole body. At least 12 families of feeding-related proteins were identified, including: 1) anaesthetics, such as ShK Toxin-containing proteins and turripeptides (ion-channel blockers), Cysteine-rich secretory proteins (CRISPs), Adenosine Deaminase (ADA); 2) inhibitors of primary haemostasis, such as novel vWFA domain-containing proteins, the Ectonucleotide pyrophosphatase/phosphodiesterase family member 5 (ENPP5) and the wasp Antigen-5; 3) anticoagulants, such as TFPI-like multiple Kunitz-type protease inhibitors, Peptidases S1 (PS1), CAP/ShKT domain-containing proteins, Astacin metalloproteases and Astacin/ShKT domain-containing proteins; 4) additional proteins, such the Angiotensin-Converting Enzyme (ACE: vasopressive) and the cytolytic Porins.ConclusionsColubraria feeding physiology seems to involve inhibitors of both primary and secondary haemostasis, anaesthetics, a vasoconstrictive enzyme to reduce feeding time and tissue-degrading proteins such as Porins and Astacins. The complexity of Colubraria venomous cocktail and the divergence from the arsenal of the few neogastropods studied to date (mostly conoideans) suggest that biochemical diversification of neogastropods might be largely underestimated and worth of extensive investigation.

Ecological barcoding of corallivory by second internal transcribed spacer sequences: hosts of coralliophiline gastropods detected by the cnidarian DNA in their stomach

The second internal transcribed spacer (ITS2) of the nuclear ribosomal RNA cluster (rDNA) is significantly smaller in the Cnidaria (120–260 bp) than in the rest of the Metazoa. ITS2 is one of the fastest evolving DNA regions among those commonly used in molecular systematics and has been proposed as a possible barcoding gene for Cnidaria to replace the currently problematic mitochondrial sequences used. We have reviewed the intraspecific and interspecific variation of ITS2 rRNA sequences in the Anthozoa. We have observed that the lower limits of the interspecific DNA divergence ranges very often overlap with intraspecific ranges, and identical sequences from individuals of different species are not rare. This finding can result in problems similar to those encountered with the mitochondrial COI, and we conclude that ITS2 does not prove significantly better than COI for standard taxonomic DNA barcoding in Anthozoa.

Correlating Molecular Phylogeny with Venom Apparatus Occurrence in Panamic Auger Snails (Terebridae)

Central to the discovery of neuroactive compounds produced by predatory marine snails of the superfamily Conoidea (cone snails, terebrids, and turrids) is identifying those species with a venom apparatus. Previous analyses of western Pacific terebrid specimens has shown that some Terebridae groups have secondarily lost their venom apparatus. In order to efficiently characterize terebrid toxins, it is essential to devise a key for identifying which species have a venom apparatus. The findings presented here integrate molecular phylogeny and the evolution of character traits to infer the presence or absence of the venom apparatus in the Terebridae. Using a combined dataset of 156 western and 33 eastern Pacific terebrid samples, a phylogenetic tree was constructed based on analyses of 16S, COI and 12S mitochondrial genes. The 33 eastern Pacific specimens analyzed represent four different species: Acus strigatus, Terebra argyosia, T. ornata, and T. cf. formosa. Anatomical analysis was congruent with molecular characters, confirming that species included in the clade Acus do not have a venom apparatus, while those in the clade Terebra do. Discovery of the association between terebrid molecular phylogeny and the occurrence of a venom apparatus provides a useful tool for effectively identifying the terebrid lineages that may be investigated for novel pharmacological active neurotoxins, enhancing conservation of this important resource, while providing supplementary information towards understanding terebrid evolutionary diversification.

Cellular, biochemical, and molecular characterization of nitric oxide synthase expressed in the nervous system of the prosobranch Stramonita haemastoma (Gastropoda, Neogastropoda)

Nitric oxide synthase (NOS) has been characterized in several opistobranchs and pulmonates but it was much less investigated in prosobranchs, which include more than 20,000 species and account for most of the gastropod diversity. Therefore, new data from this large group are needed for a better knowledge of the molecular evolution of NOS enzymes in molluscs. This study focused on NOS expressed in the nervous system of the prosobranch neogastropod Stramonita haemastoma. In this study we report compelling evidence on the expression of a constitutive Ca2+/CaM‐dependent neuronal NOS in the central and peripheral nervous system. The prevailing neuronal localization of NADPHd activity was demonstrated by NADPHd histochemistry in both central and peripheral nervous system structures. L‐arginine/citrulline assays suggested that Stramonita NOS is a constitutive enzyme which is both cytosolic and membrane‐bound. Molecular cloning of the full‐length Stramonita NOS (Sh‐NOS) by reverse‐transcription polymerase chain reaction (RT‐PCR) followed by 5′ and 3′ RACE showed that Sh‐NOS is a protein of 1,517 amino acids, containing a PDZ domain at the N‐terminus and sharing similar regulatory domains to the mammalian neuronal NOS (nNOS). Regional expression of the Sh‐NOS gene was evaluated by RT‐PCR. This analysis showed different expression levels in the nerve ring, the osphradium, the cephalic tentacles, the buccal tissues, and the foot, whereas NOS expression was not found in the salivary glands and the gland of Leiblein. The present data provide a solid background for further studies addressing the specific functions of NO in neogastropods. J. Comp. Neurol. 520:364–383, 2012.

Molecular phylogeny of the nutmeg shells (Neogastropoda, Cancellariidae)

Cancellariidae, or nutmeg shells, is a family of marine gastropods that feed on the body fluids and the egg cases of marine animals. The 300 or so living species are distributed worldwide, mostly on soft bottoms, from intertidal to depths of about 1000 m. Although they are a key group for the understanding of neogastropod evolution, they are still poorly known in terms of anatomy, ecology and systematics. This paper reports the first mitochondrial multi-gene phylogenetic hypothesis for the group. Data were collected for 50 morphospecies, representative of 22 genera belonging to the three currently recognized subfamilies. Sequences from three genes (12S, 16S and COI) were analyzed with Maximum Likelihood analysis and Bayesian Inference, both as single gene datasets and in two partitioned concatenated alignment. Largely consistent topologies were obtained and discussed with respect to the traditional subfamilial arrangements. The obtained phylogenetic trees were also used to produce Robinson-Foulds supertrees. Our results confirmed the monophyly of the subfamily Plesiotritoninae, while Admetinae and Cancellariinae, as currently conceived, were retrieved as polyphyletic. Based on our findings we propose changes to the systematic arrangement of these subfamilies. At a lower taxonomic rank, our results highlighted the rampant homoplasy of many characters traditionally used to segregate genera, and thus the need of a critical re-evaluation of the contents of many genera (e.g. Nipponaphera, Merica, Sydaphera, Bivetia), the monophyly of which was not recovered.

A DNA-barcoding approach to the phenotypic diversity of Mediterranean species of Felimare Ev. Marcus & Er. Marcus, 1967 (Mollusca: Gastropoda), with a preliminary phylogenetic analysis

Abstract The chromodorid nudibranchs Felimare tricolor (Cantraine, 1835), F. fontandraui (Pruvot-Fol, 1951) and F. picta (Shultz in Philippi, 1836) typically show highly variable colour patterns that are misleading for species identification. Their taxonomy has long been confused, and their identification can be difficult if based only on morphological characters. We carried out molecular genetic analyses using the mitochondrial cytochrome c oxidase subunit I (COI) and 16S rDNA markers on Mediterranean samples of these three taxa representing a large part of their range of chromatic variation. Results proved the usefulness of the DNA-barcoding approach in assessing species delimitation in this group, and in identification of specimens with body colours not clear for species recognition. We inferred preliminary phylogenetic relationships among one third of the currently recognised species of Felimare: they indicated with good support sister species relationship among Felimare tricolor and F. fontandraui, and suggested (with low support) potential relationships among F. picta, F. verdensis, F. californiensis and F. agassizi.

Do larval types affect genetic connectivity at sea? Testing hypothesis in two sibling marine gastropods with contrasting larval development

In marine environments, connectivity among populations of benthic invertebrates is provided primarily by dispersion of larvae, with the duration of pelagic larval phase (PLD) supposed to represent one of the major factor affecting connectivity. In marine gastropods, PLD is linked to specific larval development types, which may be entirely intracapsular (thus lacking a pelagic dispersal), or include a short pelagic lecithotrophic or a long planktotrophic phase. In the present study, we investigated two sibling species of the cosmopolitan neogastropod genus Columbella (commonly known as dove shells): Columbella adansoni Menke, 1853, from the Macaronesian Atlantic archipelagos, with planktotrophic development, and Columbella rustica Linnaeus, 1758, from the Mediterranean Sea, with intracapsular development. We expected to find differences between these two sister species, in terms of phylogeographic structure, levels of genetic diversification and spatial distribution of genetic diversity, if PLD was actually a relevant factor affecting connectivity. By analysing the sequence variation at the cytochrome c oxidase subunit I (COI) in 167 specimens of the two species, collected over a comparable geographic range, we found that Columbella adansoni, the species with planktotrophic development, and thus longer PLD, showed no phylogeographic structure, lower levels of genetic diversity, interpopulational variance lower than the intrapopulational one and no spatial structure in the distribution of the genetic diversity; Columbella rustica, the species with intracapsular development, thus with evidently lower dispersal abilities, showed a clear phylogeographic structure, higher levels of genetic diversity, high interpopulational and low intrapopulational variance, and a clear signature of global spatial structure in the distribution of the genetic diversity. Thus, in this study, two sibling species differing almost only in their larval ecology (and PLD), when compared for their genetic variation showed patterns supporting the hypothesis that PLD is a major factor affecting genetic connectivity. Therefore, it seems reasonable to expect that the ecological attributes of the marine communities - also in terms of the variation in larval ecology of the species involved - are taken into the due consideration in conservation actions, like the design of marine protected areas networks.