Improving the conservation of Mediterranean Chondrichthyans : the ELASMOMED DNA barcode reference library

Cartilaginous fish are particularly vulnerable to anthropogenic stressors and environmental change because of their K-selected reproductive strategy. Accurate data from scientific surveys and landings are essential to assess conservation status and to develop robust protection and management plans. Currently available data are often incomplete or incorrect as a result of inaccurate species identifications, due to a high level of morphological stasis, especially among closely related taxa. Moreover, several diagnostic characters clearly visible in adult specimens are less evident in juveniles. Here we present results generated by the ELASMOMED Consortium, a regional network aiming to sample and DNA-barcode the Mediterranean Chondrichthyans with the ultimate goal to provide a comprehensive DNA barcode reference library. This library will support and improve the molecular taxonomy of this group and the effectiveness of management and conservation measures. We successfully barcoded 882 individuals belonging to 42 species (17 sharks, 24 batoids and one chimaera), including four endemic and several threatened ones. Morphological misidentifications were found across most orders, further confirming the need for a comprehensive DNA barcoding library as a valuable tool for the reliable identification of specimens in support of taxonomist who are reviewing current identification keys. Despite low intraspecific variation among their barcode sequences and reduced samples size, five species showed preliminary evidence of phylogeographic structure. Overall, the ELASMOMED initiative further emphasizes the key role accurate DNA barcoding libraries play in establishing reliable diagnostic species specific features in otherwise taxonomically problematic groups for biodiversity management and conservation actions.peer-reviewe

Molecular phylogeny and historical zoogeography of four genera of Eastern Atlantic skates (Rajiformes, Rajidae)

The aim of this study was to reconstruct a solid phylogeny of four genera of the Rajidae family (Chondrichthyans: Batoidea) using a concatenated alignment of mtDNA genes. Then use the resultant tree to estimate divergence time between taxa based on molecular clock and fossil calibration and conduct biogeographic analysis. The intent was to prove that the actual distribution of species of Eastern Atlantic and Mediterranean skates is due to a series of vicariant events. The species considered belongs to two different tribe: Rajini (Raja and Dipturus) and Amblyrajini (Leucoraja and Rajella). The choice of this genera is due to their high presence in the area of interest and to the richness of endemic species. The results show that despite the ancient origin of Rajidae (97 MYA), the Eastern Atlantic and Mediterranean faunas originated more recently, during Middle Miocene-Late Pliocene, after the closure of connection between these areas and the Indo-Pacific ocean (15 MYA). The endemic species of the Mediterranean (Raja asterias, R. radula, R. polystigma and Leucoraja melitensis) originated after the Messinian salinity crisis (7-5 MYA), when the recolonization of the basin occurred, and are still maintained in allopatric distribution by the presence of biogeographic barriers. Moreover from 4 to 2.6 MYA we can observe the formation of sister species for Raja, Leucoraja and Rajella, one of which has a Northern distribution, and the other has a Southern distribution (R. clavata vs R. straeleni, L. wallacei vs L. naevus, R. fyllae vs R. caudaspinosa and R. kukujevi vs R. leopardus + R. barnardi). The Quaternary and present oceanographic discontinuities that occur along the western African continental shelf (e.g., Cape Blanc and the Angola–Benguela Front) might contribute to the maintenance of low or null levels of gene flow between these closely related siblings species. Also sympatric speciation must be invoked to explain the evolution of skates, for example for the division between R. leopardus and R. barnardi. The speciation processes followed a south-to-north pathways for Dipturus and a north-to-south pathways for Raja, Leucoraja and Rajella underling that the evolution of the genera occurred independently. In the end, it is conceivable that the evolutionary pathways of the tribes followed the costal line during the gondwana fragmentation. The results demonstrate that the evolution of this family is characterized by a series of parallel and independent speciation events, strictly correlated to the tectonic movement of continental masses and paleogeographic and paleoclimatic events and so can be explained by a panbiogeographical (vicariance) model

Step by step: the unprecedented evolutionary history of family Rajidae

Among skates, Rajidae represents one of the most enigmatic family of cartilaginous fish whose bio- ecological traits contributed to an extraordinary evolutionary success in terms of species richness and endemism. Past and present taxonomic conflicts and species misidentifications are linked to their extraordinary level of morphological stasis. In recent years, these issues have been overcome by wide- scale molecular taxonomy analyses, but also raised questions about their evolutionary history. Concerted actions as the ELASMOMED and ELASMOATL initiatives encouraged and improved large-scale sampling efforts in the Mediterranean and Eastern Atlantic Ocean over years, building unique voucher repositories of thousands of specimens and enabling the exploration of skates\u2019 biogeography. Here, the maximum taxonomic (51 OTUs) and molecular (47 OTUs) diversity of tribes Rajini and Amblyrajini were measured using concatenated mitochondrial genes. We also estimated their evolutionary divergence using the molecular clock approach. Evidences produced so far showed that, despite the ancient origin of Rajidae (97 MYA), the Eastern Atlantic and Mediterranean faunas originated more recently, after the closure of connection between these areas and the Indo-Pacific Ocean (15 MYA). The endemic Mediterranean species originated with the recolonization of the Basin, after the Messinian salinity crisis (7-5 MYA). At least five independent vicariant events contributed from 7.4 to 3.15 MYA to the formation of allopatric or parapatric sister species, each distributed in the N-E Atlantic and S-E Atlantic respectively. On the whole, the Quaternary tectonic movement of continental masses, paleoclimatic events and present oceanographic discontinuities occurring along the western African continental shelf might explain this series of parallel and independent speciation events related to the maintenance of low or null levels of gene flow between closely related sibling and cryptic species

Peer review of the pesticide risk assessment of the active substance pyriproxyfen

Abstract The conclusions of EFSA following the peer review of the initial risk assessments carried out by the competent authorities of the rapporteur Member State the Netherlands and co‐rapporteur Member State Spain for the pesticide active substance pyriproxyfen and the assessment of applications for maximum residue levels (MRLs) are reported. The context of the peer review was that required by Commission Implementing Regulation (EU) No 844/2012. The conclusions were reached on the basis of the evaluation of the representative uses of pyriproxyfen as an insecticide on citrus fruit, pome fruit (apple, pears), tomatoes, ornamentals (field use) and tomatoes, ornamentals (greenhouse application). MRLs were assessed in citrus fruits. The reliable end points, appropriate for use in regulatory risk assessment and the proposed MRLs, are presented. Missing information identified as being required by the regulatory framework is listed. Concerns are identified

Cases of specimens’ misidentification by FAO fishing division.

<p>The number of misidentified individuals over the total number of barcoded individuals is given in parenthesis.</p

Haplotype parsimony networks of <i>Raja miraletus</i>, <i>Raja asterias</i>, <i>Raja polystigma</i>, <i>Raja radula</i> and <i>Torpedo marmorata</i> reconstructed using ELASMOMED COI barcode sequences.

<p>Each circle represents one haplotype and its size is proportional to frequency. Colours indicate the origin of samples according to FAO fishing divisions. Mutations and positions characterizing each haplotype, as well as the size (bp), and the number of the sequences included in each species network, are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170244#pone.0170244.s004" target="_blank">S4 Table</a>.</p

Mean and maximum intraspecific distances and Nearest-Neighbour distance (p-distance values) of the 41 chondrichthyan species barcoded for the ELASMOMED library.

<p>The <i>Dasyatis pastinaca</i> cluster is not separated.</p

Neighbour-Joining tree based on genetic p-distances of COI barcode sequences of 17 shark species.

<p>Numbers near nodes indicate bootstrap values (>50%). The distance scale bar is given. Each species' sample size and geographic origin are detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170244#pone.0170244.s002" target="_blank">S2 Table</a>.</p

Neighbour-Joining tree based on genetic p-distances of COI barcode sequences of 23 skate species.

<p>Numbers near nodes indicate bootstrap values (>50%). The distance scale bar is given. Each species' sample size and geographic origin are detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170244#pone.0170244.s002" target="_blank">S2 Table</a>.</p

Species with discordant BINs found by the ‘BIN Discordance Report’ sequence analysis tool on BOLD.

<p>Taxonomic ranks of conflict are reported including species of the same genus up to taxa belonging to different orders. The column BIN Taxon Variation shows the number of records for each taxon in parenthesis.</p