Aquatic Invasions (2016) Volume 11, Issue 3: 247–255 DOI: http://dx.doi.org/10.3391/ai.2016.11.3.03 Open Access © 2016 The Author(s). Journal compilation © 2016 REABIC Research Article The Mediterranean Sea as a gateway for invasion of the Red Sea: the case of the Indo-West Pacific head-shield slug Chelidonura fulvipunctata Baba, 1938 Manuel António E. Malaquias1,*, Andrea Zamora-Silva1, Dyana Vitale2, Andrea Spinelli2, Sergio De Matteo2, Salvatore Giacobbe2, Deneb Ortigosa3 and Juan L. Cervera3 1Phylogenetic Systematics and Evolution Research Group, Section of Taxonomy and Evolution, Department of Natural History, University Museum of Bergen, University of Bergen, PB 7800, 5020-Bergen, Norway 2Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Viale F. Stagno d'Alcontres, 31, 98166 Messina, Italy 3Department of Biology, Faculty of Marine and Environmental Sciences, University of Cádiz, International Campus of Excellence on the Sea (CEIMAR), Polígono del Rio San Pedro s/n, Apdo. 40, 11510 Puerto Real (Cádiz), Spain *Corresponding author E-mail: [email protected] Received: 1 December 2015 / Accepted: 9 March 2016 / Published online: 7 April 2016 Handling editor: Vadim Panov Abstract The number of tropical species established in the Mediterranean Sea has risen at an unprecedented rate in the last 40–50 years, often entering the basin from the Red Sea and to a lesser extent by shipping or mariculture. The taxonomic identity of these alien species can be difficult to determine and DNA barcoding can help to clarify the status of questionable species. One of these cases is the W-shaped mark Aglajidae slug usually identified as being the Indo-West Pacific species Chelidonura fulvipunctata Baba, 1938 but was first described in the Mediterranean Sea as a native species under the name C. mediterranea Swennen, 1961. A Bayesian phylogeny using the barcode marker cytochrome c oxidase subunit I and an ABGD species delimitation analysis unequivocally showed that specimens from the eastern and central Mediterranean Sea are conspecific with specimens from the Indian and Pacific Oceans. In this work, we hypothesize that C. fulvipunctata (presently occurring in the entire Mediterranean Sea and Indo-West Pacific; only once recorded in the Red Sea) has entered the Red Sea through the Mediterranean Sea. Thus the Red Sea can also be a receiver of tropical species that have arrived in the Mediterranean by other routes than Lessepsian immigration, with the Suez Canal acting as a “revolving door” allowing both species of Indo-Pacific origin to enter the Mediterranean and species established in the Mediterranean resilient to tropical/subtropical environmental conditions to move into the Red Sea. Key words: alien species, biodiversity, Cephalaspidea, DNA barcode, Mollusca, species range shifts Introduction on morphological similarities with their tropical counterparts. The influx of tropical and subtropical species into Barcoding methodologies have seldom been applied the Mediterranean Sea has reached an unprecedented but could help to clarify the status of questionable magnitude over the last 40–50 years. There are about species (sensu Gofas and Zenetos 2003). For example 212 non-native molluscan species reported in the the taxonomic identity of the Mediterranean W- basin, 21 being opisthobranchs (reviewed by Gofas shaped mark slug (Aglajidae) is uncertain. Originally and Zenetos 2003; Gosliner et al. 2008a; Zenetos et described as a native species Chelidonura al. 2010). Three main routes are often proposed to mediterranea Swennen, 1961; it was later identified explain the arrival of these exotic elements; the most as the Indo-West Pacific species Chelidonura common is the influx from the Red Sea through the fulvipunctata Baba, 1938. No similar head-shield Suez Canal, followed by introduction via shipping slug occurs in the Atlantic Ocean, and there is a on ships’ hulls or in ballast water, and to a less single record from the Red Sea in the Gulf of Aqaba extent mariculture (Gofas and Zenetos 2003). Most (reported online; Koretz 2005). The Red Sea is a of these exotic species have been recognized based well-studied basin surveyed by a wealth of 247 M.A.E. Malaquias et al. expeditions from as early as the second half of the Sea and the role of this basin as a possible gateway eighteen century, which has resulted in a large for other species of tropical origin to enter the Red number of studies on the opisthobrachs of the region Sea as opposed to the traditional colonization of the (see reviews by Yonow 2008; 2012). It is therefore Mediterranean Sea from the Red Sea. significant that none have reported the occurrence of C. fulvipunctata. Swennen (1961) described the species C. medi- Methods terranea from a single specimen collected in the Bay Sampling, DNA extraction, amplification, and of Antalya, Turkey, in the eastern Mediterranean sequencing Sea. The specimen was 18 mm in length with a whitish foot and greyish-brown background colour Samples from Mozambique (ZMBN 94192.1, ZMBN with orange spots scattered over the dorsum, a 94192.2) and Italy (ZMBN 106843, ZMBN 106844, yellowish-white W-shaped mark on the anterior part ZMBN 106845) were collected by the authors, whereas of the cephalic shield, and a median dorsal stripe of those from Marshall Islands (ZMBN 106813, ZMBN similar colour. The presence of a W-shaped mark led 106814) and Cyprus (MNCN/ADN: 86334) have Swennen (1961) to compare this species with the been donated by colleagues (see Acknowledgements). Indo-West Pacific C. fulvipunctata (type-locality: The DNA sequence of the specimen from Lizard Seto, Kii Peninsula, Japan; Baba 1938) which, despite Island, Australia, was obtained from GenBank. being known for its striking chromatic variability The specimen from Cyprus was collected by divers (Gosliner 1987; Wells and Bryce 1993; Nakano 2004; using SCUBA on 21 October 2015 on a rocky wall Gosliner et al. 2015), was thought to never have a in water 2 m deep, and it was crawling on algae. median dorsal stripe. The dorsal stripe is, however, Specimens from Italy were collected by snorkeling present on specimens from Hawaii (Gosliner 1980) in water 0.9 to 1.5 m deep at Capo Peloro Lagoon, and C. mediterranea has since been considered to be NE Sicily, Mediterranean Sea, between March–June a junior synonym of C. fulvipunctata. In all 2015. This shallow Lagoon is a transitional environ- subsequent reports, the W-shaped mark head-shield ment formed by two interconnected basins, Lago di slug in the Mediterranean Sea has been identified as Faro and Lago di Ganzirri, each of them communi- the Indo-West Pacific species. cating with the Strait of Messina and is characterized It took more than 20 years for additional specimens by bottoms of coarse sand and shell debris with of Chelidonura slug with a W-shape mark be found coralline algae and artificial rocky banks. Three in the Mediterranean Sea: Mienis and Gat (1987) specimens were collected and preserved in 96% reported an occurrence for the shores of Israel and ethanol and an additional 12 specimens were obser- Perrone and Sammut (1997) and Sammut and Perrone ved. All specimens were found crawling actively, (1998) found three additional specimens in the but mating and egg-laying were not witnessed. Maltese islands. All these specimens lacked the DNA was extracted from tissue obtained from median dorsal line and resembled Indo-West Pacific parapodial lobes using the Qiagen DNeasy Blood morphs that occur, for example, in Japan and South and Tissue Kit following the protocol recommended Africa and typically have a black background and by the manufacturer. Partial sequences of the many orange dots scattered over the body (Gosliner mitochondrial gene cytochrome c oxidase subunit I 1987; Perrone and Sammut 1997; Nakano 2004). (COI; ca. 660 bp) were amplified using the universal More recently, Tsiakkiros and Zenetos (2011) cited primers: LCO1490 (F) GGTCAACAAATCATAA- the presence of this slug in Cyprus and illustrate two AGATATTGG and HCO2198 (R) TAAACTT- specimens with different chromatic patterns; one CAGGGTGACCAAAAATCA (Folmer et al. 1994), lighter with a striking cephalic W-mark and a darker following the protocol described by Malaquias et al. one with an almost imperceptible cephalic W-mark. (2009). The quality and quantity of PCR products Horst (2015) reported the first occurrence in the were assessed by gel-electrophoresis following western Mediterranean Sea from the coast of France standards methods (see Eilertsen and Malaquias 2013). and Karachle et al. (2016) report its occurrence in Successful PCR products were purified according to the Balearic Islands off the coast of Spain. the EXO-SAP method described by Eilertsen and In this paper, we review the historical records and Malaquias (2013). Sequence reactions were run on an provide new records of C. fulvipunctata in the ABI 3730XL DNA Analyser (Applied Biosystems). Mediterranean Sea, and we use DNA barcoding to Acronyms: ZMBN, Department of Natural History, test the conspecificity of Mediterranean and Indo- University Museum of Bergen, University of Bergen, West Pacific specimens. We discuss the possible Norway. MNCN/ADN: DNA collection, Museo means of entrance of this species in the Mediterranean Nacional de Ciencias Naturales, Madrid, Spain. 248 Tropical Chelidonura fulvipunctata in the Mediterranean and Red Sea distances of 3.4% between a specimen from Italy Phylogenetic analyses and molecular species and Lizard Island and 3.1% between two specimens delimitation from Italy. Minimum differences ranged between 0– 0.2% and were found between the specimen from The programme Geneious (v. 6.1.4 Biomatters Ltd.) Cyprus and the two specimens from Mozambique was used to inspect, edit, and assemble the chromato- (Table 1). grams of the forward and reverse DNA strands. All The ABGD species delimitation method retrieved sequences were blasted in GenBank to check for seven “initial partitions” and four “recursive partitions” contamination. Single gene sequences were aligned with eleven species each rendering all samples of C.