Marco Oliverio

Phylogeny and evolutionary history of the blister beetles (Coleoptera, Meloidae).

Meloid beetles are well characterised by both morphological and biological features. Previous phylogenetic hypotheses based on morphological characters assumed the repeated parallel evolution of complex biological novelties. In this work relationships among several taxa of the four subfamilies and almost all tribes representing meloid diversity are examined by using mitochondrial (16S) and nuclear (ITS2) DNA sequences, in 25 genera (using Anthicidae as outgroup). Secondary structure of 16S and ITS2 rRNAs were modelled. ITS2 structure represents a synapomorphic condition for the family and informative characters at the tribal level. Phylogenetic hypotheses based on separate and combined analysis of the 16S and ITS2 rDNA sequences, and morpho-biological characters were tested, and compared with previous morphological classifications. Molecular dating allowed an outline of the main steps of the evolutionary history of Meloidae, which evolved during Early Cretaceous and then radiated considerably with the adoption of hypermetaboly and parasitic behaviour, and with repeated, parallel evolution of larval phoresy on its hosts.

A molecular phylogenetic framework for the Muricidae, a diverse family of carnivorous gastropods.

With over 1600 extant described species, the Muricidae are one of the most species-rich and morphologically diverse families of molluscs. As predators of molluscs, polychaetes, anthozoans barnacles and other invertebrates, they form an important component of many benthic communities. Traditionally, the classification of muricids at specific and generic levels has been based primarily on shells, while subfamilies have been defined largely by radular morphology, although the composition and relationships of suprageneric groups have never been studied exhaustively. Here we present the phylogenetic relationships of 77 muricid species belonging to nine of the ten currently recognized subfamilies, based on Bayesian inference and Maximum Likelihood analyses of partial sequences of three mitochondrial (12S, 16S and COI) and one nuclear (28S) genes. The resulting topologies are discussed with respect to traditional subfamilial arrangements, and previous anatomical and molecular findings. We confirm monophyly of each of the subfamilies Ergalataxinae, Rapaninae, Coralliophilinae, Haustrinae, Ocenebrinae and Typhinae as previously defined, but earlier concepts of Muricinae, Trophoninae and Muricopsinae are shown to be polyphyletic. Based on our phylogenetic hypothesis, a new arrangement of these subfamilies is proposed.

Molecular phylogenetics of the Italian Podarcis lizards (Reptilia, Lacertidae)

Abstract Phylogenetic relationships within the Italian species of the lacertid genus Podarcis were examined by parsimony analysis of mitochondrial DNA sequences from the genes encoding the small ribosomal RNA and the phenyl transfer RNA. Lacerta viridis was used as outgroup and Teira dugesii was also included in the ingroup. The 80 phylogenetically informative positions produced four most parsimonious trees, with the Italian Podarcis split into three groups: the first comprised P. filfolensis, P. melisellensis. P. wagleriaria, P. muralis, and P. raffonei, the second P. sicula with its various subspecies. The third lineage was that of P. tiliguerta, whose relationships were resolved as more closely related to the first clade, when weighting transversion three times transitions. Bootstrap analyses on a subset of sequences representing all species herein studied, supported the results from the larger dataset. The present results are only partly in agreement with previous hypotheses based on morphology, immun...

Conus ventricosus venom peptides profiling by HPLC-MS: a new insight in the intraspecific variation.

Conus is a genus of predatory marine gastropods that poison the prey with a complex mixture of compounds active on muscle and nerve cells. An individual cone snails venom contains a mixture of pharmacological agents, mostly short, structurally constrained peptides. This study is focused on the composition of the venom employed by Conus ventricosus Gmelin, 1791, a worm-hunting cone snail living in the Mediterranean Sea. For this purpose, LC coupled to MS techniques has been successfully used to establish qualitative and quantitative differences in conopeptides from minute amounts of venom ducts. We were able to prove variability in the venom conopeptide complement, possibly related to different trophic habits of the species in the Mediterranean Sea. Moreover, the information-rich MS techniques enabled us to identify two novel C. ventricosus peptides, here named Conotoxin-Vn and -Conotoxin-Vn. On the basis of the structural data collected so far, we suggest that Conotoxin-Vn is a conopeptide belonging to the -family that recognizes calcium channels through a specific pharmacophore. Similarly, molecular modeling data suggest that -Conotoxin-Vn should represent a competitive antagonist of neuronal nicotinic acetylcholine receptors (nAChRs).

Shells and heart: Are human laterality and chirality of snails controlled by the same maternal genes?†

The body of most animals display left–right asymmetry of internal organs. Alteration of such asymmetry results in severe congenital defects particularly affecting the cardiovascular system. The earliest known genes involved in asymmetry, the Nodal signalling cascade, are expressed asymmetrically during embryonic development. Nodal was discovered in the mouse, but orthologs (also involved in left‐right specification) were reported in ascidians, sea‐urchins, and snails. Mutations in Nodal‐pathway genes cause alteration of several aspects of chirality, but not entirely mirror phenotypes of the body. Other factors upstream of nodal must be involved in the generation of left–right asymmetry. In snails, breeding experiments have demonstrated that chirality is controlled by a nuclear gene with maternal effect. Given the available evidence, we propose that an evolutionarily conserved genetic basis of chirality (the same that controls left–right asymmetry in snails) is a major synapomorphy of the Bilateria. This hypothesis fits with the observation that: (a) the proportion of patients with heterotaxy and a detected mutation in a gene of the Nodal cascade is actually low, and (b) horizontal recurrence of laterality defects is remarkably more frequent than vertical recurrence, and includes a notable number of affected sibs and/or repeated abortions from unaffected mothers. Identification of the maternal gene(s) involved will allow for the identification of homozygous females at risk of having affected children and spontaneous abortions, and would provide a general medical framework for understanding the genetics of most alterations of chirality.

Surviving the Messinian Salinity Crisis? Divergence patterns in the genus Dendropoma (Gastropoda: Vermetidae) in the Mediterranean Sea.

Four genetically distinct clades were recently described under the name Dendropoma petraeum, a Mediterranean endemic vermetid gastropod. The aim of this work is to date the processes that drove to the diversification within this taxon and to relate them to the corresponding historical events occurred in the Mediterranean Sea. Sequences from mitochondrial and nuclear markers were obtained from specimens collected in 29 localities spanning over 4000km across the entire distribution range of D. petraeum species complex. The phylogenetic and coalescent-based analyses confirmed the four well-supported and largely differentiated lineages of D. petraeum, clearly delimited geographically along a west-east axis within the Mediterranean Sea: Western, Tyrrhenian-Sicilian, Ionian-Aegean and Levantine lineages. Divergence time estimates, obtained using a range of known substitution rates for other marine gastropods, indicated two main stages of diversification. In the first period (between 9.5 and 4.5mya), the ancestral D. petraeum diverged into the current four lineages. The most recent period occurred between 3.72 and 0.66mya in the late Pliocene-early Pleistocene, and included the main within-lineage diversification events. Therefore, if the divergence time between the major lineages of Dendropoma in the Mediterranean actually predated or coincided with the Messinian Salinity Crisis, then they should have survived to this dramatic period within the Mediterranean, as supported by Bayes Factors model comparison. Conversely, if the divergence started after the crisis, congruent with the idea that no true marine organism survived the Messinian Salinity Crisis, then our results indicate substitution rates of Dendropoma much higher than usual (5.16% per million years for COI, 3.04% for 16S). More recent climate changes seem to have conditioned the demographic history of each lineage differently. While Western and Tyrrhenian-Sicilian lineages both underwent an increase in their effective population sizes from 1.5 to 0.6mya coinciding with a long interglacial period, the Ionian-Aegean and Levantine lineages showed constant effective population sizes since 2-2.5mya, suggesting that these eastern lineages might represent small and relict populations surviving the subsequent Quaternary glaciations in isolated refugia.

Accessing novel conoidean venoms: Biodiverse lumun-lumun marine communities, an untapped biological and toxinological resource

Cone snail venoms have yielded pharmacologically active natural products of exceptional scientific interest. However, cone snails are a small minority of venomous molluscan biodiversity, the vast majority being tiny venomous morphospecies in the family Turridae. A novel method called lumun-lumun opens access to these micromolluscs and their venoms. Old fishing nets are anchored to the sea bottom for a period of 1-6months and marine biotas rich in small molluscs are established. In a single lumun-lumun community, we found a remarkable gastropod biodiversity (155 morphospecies). Venomous predators belonging to the superfamily Conoidea (36 morphospecies) were the largest group, the majority being micromolluscs in the family Turridae. We carried out an initial analysis of the most abundant of the turrid morphospecies recovered, Clathurella (Lienardia) cincta (Dunker, 1871). In contrast to all cDNA clones characterized from cone snail venom ducts, one of the C. cincta clones identified encoded two different peptide precursors presumably translated from a single mRNA. The prospect of easily accessing so many different morphospecies of venomous marine snails raises intriguing toxinological possibilities: the 36 conoidean morphospecies in this one net alone have the potential to yield thousands of novel pharmacologically active compounds.

Molecular characterization of native (Italy) and introduced (USA) Podarcis sicula populations (Reptilia, Lacertidae)

Abstract The wall lizard Podarcis sicula has been introduced in many locations outside its native range. Partial sequences from the 12S rDNA, that had proved diagnostic for the three main subspecies, were used to test specimens from marginal native and from intro duced American populations. The mitochondrial haplotype of a specimen from Apulia, traditionally ascribed to P. s. campestris, belongs to the P. s. sicula gene‐pool. Specimens from a P. s. sicu la ‐ P. s. campestris boundary area (southern Latium), with transi tional morphology but P. s. sicula‐like ecology, show mitochondrial haplotypes clearly of the campestris type. The mitochondrial hap‐lotypes of the American specimens (from Kansas and New York), that originated from captive stocks, were included in a P. s. campestris clade. A definitive assessment of the status of both na tive and introduced populations will require the use of nuclear markers.

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.

Cenozoic evolution of Muricidae (Mollusca, Neogastropoda) in the Southern Ocean, with the description of a new subfamily

Barco, A., Schiaparelli, S., Houart, R. & Oliverio, M. (2012). Cenozoic evolution of Muricidae (Mollusca, Neogastropoda) in the Southern Ocean, with the description of a new subfamily. —Zoologica Scripta, 41, 596–616.