Tassonomia integrata morfologica e molecolare di leptocefali del mare Adriatico.

La campagna scientifica di pesca profonda svolta dalla m/n Andrea dell\u2019Universit\ue0 di Bologna nel 2010 nell\u2019Adriatico meridionale ha permesso di collezionare un elevato numero di leptocefali di Anguilliformi. Il \u201cleptocefalo\u201d \ue8 una peculiare forma larvale dei Teleostei del superordine Elopomorpha, la cui caratterizzazione bio-ecologica risulta ancora oggi incompleta (Miller 2009). In particolare, la mancanza di chiavi dicotomiche esaustive limita la conoscenza tassonomica specifica dei leptocefali del Mediterraneo. Questo lavoro ha l\u2019obiettivo di aggiornare la descrizione tassonomica di leptocefali pescati in Adriatico integrando la tradizionale analisi morfologica con quella genetica eseguita mediante DNA barcoding. Il DNA barcoding \ue8 una metodica molecolare basata sull\u2019analisi della variabilit\ue0 di un marcatore di sequenza universale (COI) che consente di ottenere una identificazione tassonomica a livello specifico in modo univoco (Hebert 2003 a,b; Ward 2005)

Complementing morphological classification of Anguilliform leptocephali with DNA barcoding

DNA barcoding is a molecular tool that enables rapid and accurate identification of biological species by sequencing a short, standardized region of the mitochondrial gene cytochrome c oxidase I ( COI) as internal species tag. The aim of the present study was to use DNA barcoding in addition to classical taxonomy in order to obtain a more reliable species-identification of leptocephali, the characteristic larval forms of the superorder Elopomorpha, often difficult to identify and to match with their adult stage. Based on the examination of external morphology, meristics, and pigmentation, 2785 leptocephalus larvae collected in the Adriatic Sea between 2010 and 2012, were ascribed to 7 morphotypes, belonging to Anguilliform order (Ariosoma balearicum, Conger conger, Gnathophis mystax, Nettastoma melanurum, Dalophis imberbis, Chlopsis bicolor, Facciolella sp.) and 69 specimens were sequenced for a 655 bp region of the mitochondrial cytochrome oxidase subunit I gene (COI) to confirm the previous morphological analysis. The highly consistent results obtained revealed a good performance of COI barcoding as a diagnostic method for the identification of these larvae, but the limited number of leptocephali species annotated in the reference databases for barcode (Barcode of Life Data Systems and GenBank) allowed to validate only partially the morphological analysis. Moreover two species, Gnathophis mystax and Facciolella sp., showed unexpected outcomes. The data obtained in this work represent the first results of a wider project aimed at the creation of a new barcode database for the assessment of leptocephali diversity in the Mediterranean Sea (Barcoding of the Adriatic Leptocephali [BAL]), contributing to the knowledge of these unusual larvae and of their adult forms

First Record Of Leptocephali Of The False Moray, Chlopsis Bicolor Rafinesque 1810 Actinopterygii, Anguilliformes, Chlopsidae) From The Adriatic Sea: Morphological And Molecular Identification.

Leptocephali are the characteristic larval forms of the superorder Elopomorpha, very difficult to identify at the species level and sharing few or no characters with the respective adult forms. In this study we used DNA barcoding coupled to classical taxonomy for the species identification of Chlopsis bicolor (Rafinesque, 1810) leptocephali. In the Mediterranean some individuals of C. bicolor in developmental stage, collected along the Sicilian shore of the Messina Strait, were already identified and their detailed morphological descriptions were provided. However, very few specimens of C. bicolor from the Adriatic Sea have been reported and this work represents the first record of its larval form. Three leptocephali of the false moray C.bicolor were morphologically identified among other eel larvae collected during scientific cruises performed in South Adriatic between 2010 and 2012. Moreover we sequenced a 655bp region of COI (cytochrome c oxidase I) in addition to classical taxonomy to provide a larval barcode voucher for species identification. COI barcoding has proven to be successful in larval fish identification, revealing also a good performance as a diagnostic method for the identification of leptocephali. Anyway, the limited number of anguilliform species annotated in the reference databases (Barcode of Life Data Systems and GenBank) allowed us to validate our barcode only at the family level (Chlopsidae). Nevertheless, the morphological description provided in this work together with the barcode sequence published in the project \u2018Barcoding of the Adriatic Leptocephali\u2019 (BAL) on the Barcode of Life Data System (BOLD, http://www.barcodinglife.org), represent a useful contribution to the knowledge of this species

Morphology and Species Composition of Southern Adriatic Sea Leptocephali Evaluated Using DNA Barcoding - Fig 5

<p>A <i>Chlopsis bicolor</i> leptocephalus: (a) lateral view of the body, (b) anterior region, (c) details of the lateral pigmentation and (d) the hypurals.</p

Sampling locations.

<p>Top panel: the sampling area in the Mediterranean Sea is highlighted in grey. Bottom panel: the sampling area is magnified and the sampling sites included in the three cruises are shown.</p

<i>Nettastoma melanurum</i> leptocephali.

<p>Individual 1: (a) lateral view of the body, (b) pigments on the gut thickening, (c) lateral pigment; and Individual 2: (d) head region.</p

<i>Facciolella</i> sp. leptocephali.

<p>Individual 1: (a) lateral view of the body, (b) head region, (c) details of the front teeth, (d) pigmentation of the palate, (e) cross-shaped pigment along the notochord, (f) a lateral view of the pigments at the end of the gut; and Individual 2: (g) anterior region of the body, and (h) the hypurals.</p

Summary of the morphological and molecular genetic identification of the southern Adriatic leptocephali assemblage.

<p>The morphometric and meristic counts measurements were taken from a subsample of each species: total length (TL), total number of myomeres (TM) and not assigned (NA).</p

<i>Dalophis imberbis</i> leptocephali.

<p>Individual 1: (a) lateral view of the body, (b) gallbladder thickening, (c) pigments grouped on the gut, (d) pigmentation after the anus, (e) head region, (f) the hypurals; and Individual 2: (g) lateral pigmentation in the caudal region.</p

<i>Conger conger</i> leptocephali.

<p>Individual 1: (a) lateral view of the body, (b) head region, (c) abdominal pigmentation from a lateral perspective and (d) details of the hypurals. Individual 2: (e) posterior region of the body.</p