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Baeolidia moebii Bergh, 1888 (Mollusca: Gastropoda: Nudibranchia) is spreading in the eastern Mediterranean Sea
Regional Studies in Marine Science 32 (2019) 100830
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Baeolidia moebii Bergh, 1888 (Mollusca: Gastropoda: Nudibranchia) is spreading in the eastern Mediterranean Sea ∗
Sofia Paz-Sedano a , , Valentina Tanduo a , Nathalie Yonow b , Mehmet Baki Yokeş c , Demetris Kletou d , Fabio Crocetta a a
Department of Integrative Marine Ecology, Stazione Zoologica Anton Dohrn, Villa Comunale, I-80121 Napoli, Italy Department of Biosciences, Swansea University, Singleton Park, Swansea SA2 8PP, Wales, UK c AMBRD Laboratories, Hanimefendi Sokak 160/6, 34384 Sisli, Istanbul, Turkey d Marine and Environmental Research (MER) Lab Ltd., 4533, Limassol, Cyprus b
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Article history: Received 17 May 2019 Received in revised form 28 July 2019 Accepted 8 September 2019 Available online 12 September 2019 Keywords: Aeolidiidae Alien species Bioinvasions Cyprus Sea slugs Turkey
a b s t r a c t The knowledge of alien species dispersal in new geographic areas is a matter of major concern, since alien species may involve both ecological and faunistic threats in the newly colonized habitats. The geographical range of the genus Baeolidia Bergh, 1888 is mostly in the Indo-Pacific realm, including the Red Sea. However, one of the species belonging to this genus, Baeolidia moebii Bergh, 1888, has been also reported as an ephemeral Mediterranean invader from Turkey. In addition, its identification is highly debatable due to Red Sea records of a sibling species, Baeolidia australis (Rudman, 1982). The presence of two additional Baeolidia in the Mediterranean basin is reported here, based on photographs from Turkey and collected material from Cyprus, thus reinforcing the presence of this alien species in the former country and extending its distribution to the latter. In addition, morphological analyses of a specimen from the Mediterranean Sea were performed for the first time, and it was also barcoded through unpublished species-specific primers designed for the Baeolidia cytochrome c oxidase subunit I gene. Our results confirm the previous identification of the alien taxon and suggest that the presence of B. australis in the Red Sea should be discarded. Finally, the present records suggest that the Mediterranean spreading of B. moebii may be overlooked due to sea slugs’ cryptic behaviour. © 2019 Elsevier B.V. All rights reserved.
1. Introduction The introduction of alien species from one geographic area to another has highly increased in marine environments, mainly due to anthropogenic dispersal through artificial canals and fouling, but also enhanced by ballast transport, mariculture, bait and pet trade, and market discards (Molnar et al., 2008; Hulme, 2009). The Mediterranean Sea has been documented as one of the areas with highest rates of alien species invasions, mainly due to Lessepsian migration, i.e., species originating from the Indo-Pacific entering the Mediterranean basin through the Suez Canal. This attracted the interest of several researchers in the last decades, which resulted in the production of numerous scientific databases, inventories, and reviews (Zenetos et al., 2017; Galil et al., 2018 and references therein). Regarding Mollusca, more than 200 alien species have been recorded so far in the Mediterranean (Sabelli and Taviani, 2014), among which approximately 30 sea slug species are included, recorded mostly in the Levant Basin (e.g., Yokeş and Rudman, 2004; Tsiakkiros and Zenetos, ∗ Corresponding author. E-mail address: [email protected] (S. Paz-Sedano). https://doi.org/10.1016/j.rsma.2019.100830 2352-4855/© 2019 Elsevier B.V. All rights reserved.
2011; Yokeş et al., 2012; Crocetta et al., 2013). Some emblematic examples of well-established species include Bulla arabica (Malaquias & Reid, 2008), Goniobranchus annulatus (Eliot, 1904), Haminoea cyanomarginata (Heller & Thompson, 1983), and Melibe viridis (Kelaart, 1858), two of which have also reached the central and the westernmost parts of the Mediterranean (Crocetta et al., 2013, 2017; Fernández-Vilert et al., 2018; Servello et al., 2019). However, the fate of alien invaders may vary. In fact, apart from the taxa mentioned above, other sea slugs are only known to date from sporadic or even single Mediterranean records — e.g. Baeolidia moebii (Bergh, 1888), Caloria indica (Bergh, 1896), Goniobranchus obsoletus (Rüppell & Leuckart, 1830), and Plocamopherus tilesii (Bergh, 1877) (Gat, 1993; Turk and Furlan, 2011; Yokeş et al., 2012; Kleitou et al., 2019). Among them, B. moebii is known as being a Mediterranean invader since 2007, but it is only known from the area based on a single individual photographed in Kaş, Turkey (Turk and Furlan, 2011 as Spurilla major); this led some authors to consider this sea slug species as an ephemeral introduction in the Mediterranean, and not an established species (Crocetta et al., 2013; Zenetos et al., 2017). Based on the recent sightings of two Baeolidia specimens from the Mediterranean Sea, we took the opportunity to properly investigate the systematics
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of this alien species through an integrative taxonomic approach, and to revise the Red Sea records of this and its sibling species B. australis. 2. Materials and methods 2.1. Source of material Material examined in the present paper was found by SCUBA diving (for amateur purposes) in Turkey and Cyprus. The sample from Turkey was only studied externally by photographs. The specimen from Cyprus was photographed alive, fixed in 96% ethanol, and subsequently studied anatomically. The reproductive system and the SEM (Scanning Electron Microscopy) stubs of the analysed material are currently preserved in the institutional collection of Stazione Zoologica Anton Dohrn (SZN) (Naples, Italy), whilst the remaining tissues were dissolved for sequencing. 2.2. Morphological examination The external morphology of the specimen was studied by both photographs of the living animal and laboratory observations. Internal anatomy was studied under a Leica M60 microscope. Internal organs were removed by a dorsal incision, separated, and photographed using a Leica IC80HD Camera attached. Subsequently, they were drawn using Adobe Photoshop CC. To study the labial cuticle and radula, the buccal mass was dissolved in 10% sodium hydroxide until the surrounding tissue was completely removed, the structures then rinsed in water. The labial cuticle, radula, and penis were mounted on SEM stubs and gold-palladium coated in an SC7640 Sputter coater for SEM examination with a Jeol JSM-6700 F microscope. 2.3. Molecular analyses 2.3.1. DNA extraction, amplification, and sequencing The genomic DNA of the Baeolidia specimen from Cyprus R was extracted from the body by using the NucleoSpin⃝ Tissue (Macherey-Nagel) kit, following the manufacturer’s protocol. As amplifications with universal primers for invertebrates (Folmer et al., 1994) failed, partial sequence of cytochrome c oxidase I (COI) was amplified using the following species-specific primers designed for the present study: forward 5′ -TWTTAATTCGTTTY GAGYTW-3′ and reverse 5′ -AAYCTWGTRTTRAAATTHCG-3′ . PCR was conducted in 25 µl volume reaction, prepared in the following order: 12.75 µl of water, 2.5 µl of Qiagen buffer and magnesium chloride (10X), 2.5 µl of dNTPs (2 mM), 1 µl each of forward (74.4 nM) and reverse (76.8 nM) primers, 0.25 µl of DNA polymerase (5 U/µl), and 5 µl of DNA. Amplification was performed with an initial denaturation for 5 min at 95 ◦ C, followed by 39 cycles of 1 min at 95 ◦ C, 1 min annealing at 40 ◦ C and 45 s at 72 ◦ C, with a final extension of 7 min at 72 ◦ C. The successful PCR product was purified and sequenced at the Molecular Biology and Sequencing Service of SZN. 2.3.2. GenBank data mining and phylogenetic analyses A GenBank search was carried out to check for barcodes of Baeolidia taxa. Sequences of Tritonia challengeriana (Bergh, 1888) and Luisella babai (Schmekel, 1972) were also included as outgroups. The COI sequence obtained was assembled and edited using BioEdit v7.2.5 (Hall, 1999) and a search was subsequently run in GenBank to check for contamination. Alignment was carried out using MEGA7 (Kumar et al., 2016) and confirmed by translating sequences into amino acids using the genetic code of invertebrate mitochondrial DNA. Sequences were trimmed to 486 base pairs. The evolutionary models were determined separately
for the first, second, and third codon positions using jModelTest2,1,7 (Darriba et al., 2012) under Akaike information criteria (Akaike, 1974). Bayesian Inference (BI) and Maximum Likelihood (ML) analyses were conducted. The first was performed using the software package MrBayes v3.1.2b (Ronquist and Huelsenbeck, 2003) for ten million generations, two independent runs, and 1000 sampling frequency. ML analysis was performed using the software package RAxML-NG (Kozlov et al., 2018), with 0.03 automatic boot-stopping cut-off implemented. Only nodes supported by posterior probabilities ≥ 0.96 (Alfaro et al., 2003) and bootstrap values ≥ 75 (Hillis and Bull, 1993) were considered statistically significant. The trees obtained were visualized in FigTree v1.3.1 and edited in Adobe Photoshop CC 2014. 2.3.3. Species delimitation analyses Genetic distances amongst specimens of Baeolidia were compared using pairwise uncorrected p-distances for COI. The analysis was carried out using MEGA7 (Kumar et al., 2016) considering all codon positions. An Automatic Barcode Gab Discovery (ABGD) analysis (Puillandre et al., 2011) was conducted on the COI ingroup sequences through the ABGD web tool (http://wwwabi. snv.jussieu.fr/public/abgd/abgdweb.html) using the Kimura (K80) model. The parameters selected were a relative gap width (X) of 1, a range of prior values for maximum divergence of intraspecific diversity (P) from 0.0001 to 0.1, and Nb bins = 20. 3. Results 3.1. Molecular results Seven different Baeolidia taxa were found in GenBank (Table 1). A fragment of the COI sequence of the specimen from Cyprus was successfully obtained and the evolutionary models for the first, second, and third positions were TIM1+I, F81, and HKY+G, respectively. The phylogenetic tree obtained is shown in Fig. 1. The species of the genus Baeolidia are gathered in a well-supported group (BI = 0.99, ML = 83). The relationships between the different species are not resolved; however, the Baeolidia specimen from Cyprus clearly clustered with other B. moebii from Hawaii, Philippines, and the Marshall Islands, with a high value for both analyses (BI = 1, ML = 100). Species delimitation analyses confirm this result, showing uncorrected p-distances amongst B. moebii specimens of 0–0.2 (Table 2) and grouping together as the same species, B. moebii, in ABGD species delimitation analysis (Fig. 1). 3.2. Systematics Order Nudibranchia Cuvier, 1817 Family Aeolidiidae Gray, 1827 Genus Baeolidia (Bergh, 1888) Baeolidia moebii (Bergh, 1888) (Figs. 2–4) Material examined. Turkey – Limanagzi – Kaş (Antalya) (36.1824◦ N, 29.6438◦ E), one individual photographed by Murat Draman, October 2013, ∼8–10 m depth (Fig. 2a). The individual was not sampled. Cyprus — Nissia Natura 2000 (35.0056◦ N, 34.0691◦ E), one specimen collected by one of us (DK), June 2017, ∼20 m depth (Fig. 2b) (SZN-MOL-0020). Ecology. Both specimens were detected crawling on the leaves of the marine phanerogam Posidonia oceanica (Linnaeus) Delile. No additional data are available for the specimen from Turkey but in Cyprus, the leaves were covered by hydrozoans such as Aglaophenia harpago (Schenck, 1965) and Sertularia sp., and by bryozoans such as Electra posidoniae Gautier, 1954 and Calpensia nobilis (Esper, 1796).
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Table 1 Species used for molecular analyses, including voucher, locality, and COI GenBank accession number. Abbreviations used: CASIZ — California Academy of Science (California, USA); MNCN and MNCN/ADN — Museo Nacional de Ciencias Naturales (Madrid, Spain); SZN — Stazione Zoologica Anton Dohrn (Naples, Italy).
a
Species
Voucher
Locality
GenBank accession number
Tritonia challengeriana (Bergh, 1884) Luisella babai (Schmekel 1972) Baeolidia japonica (Baba, 1933) Baeolidia japonica (Baba, 1933) Baeolidia japonica (Baba, 1933) Baeolidia moebii (Bergh, 1888) Baeolidia moebii (Bergh, 1888) Baeolidia moebii (Bergh, 1888) Baeolidia moebii (Bergh, 1888) Baeolidia moebii (Bergh, 1888) Baeolidia moebii (Bergh, 1888) Baeolidia moebii (Bergh, 1888)a Baeolidia ransoni (Pruvot-Fol, 1956) Baeolidia rieae (Carmona, Pola, Gosliner & Cervera, 2014) Baeolidia salaamica (Rudman, 1982) Baeolidia salaamica (Rudman, 1982) Baeolidia salaamica (Rudman, 1982) Baeolidia scottjohnsoni (Carmona, Pola, Gosliner & Cervera, 2014) Baeolidia variabilis (Carmona, Pola, Gosliner & Cervera, 2014) Baeolidia variabilis (Carmona, Pola, Gosliner & Cervera, 2014) Baeolidia variabilis (Carmona, Pola, Gosliner & Cervera, 2014) Baeolidia variabilis (Carmona, Pola, Gosliner & Cervera, 2014)
CASIZ 171177 MNCN 15.05/53698 CASIZ186795 CASIZ184520 CASIZ 181357 CASIZ186211 CASIZ177602 CASIZ 180327 MNCN 15.05/54987 MNCN/ADN: 51948 MNCN/ADN: 51949 SZN-MOL0020 CASIZ 186209 CASIZ 184525 CASIZ 177599 CASIZ 177397 CASIZ 180330 CASIZ 184503 CASIZ 187741.1 CASIZ 187741.2 CASIZ 186210 CASIZ 177716
Bouvetoya Spain Marshall Island Japan Philippines Philippines Philippines Hawaii Hawaii Marshall Island Marshall Island Cyprus Philippines Japan Philippines Philippines Philippines Marshall Islands Marshall Islands Marshall Islands Philippines Philippines
HM162718 HQ616754 JQ997059 JQ997058 JQ997057 JQ997061 HQ616770 JQ997060 HQ616771 JX087550 JX087551 MK922511 JQ997043 JQ997046 JQ997062 JQ997047 JQ997048 JQ997045 JQ997055 JQ997056 JQ997054 JQ997051
The Baeolidia specimen sequenced during the present study.
Fig. 1. Phylogenetic tree based on COI. Numbers above branches represent posterior probabilities (BI) and bootstrap values (ML). Specimen sequenced from Cyprus in bold. Coloured bands show ABGD species delimitation analysis result. Abbreviations used: CASIZ — California Academy of Science (California, USA); MNCN and MNCN/AND — Museo Nacional de Ciencias Naturales (Madrid, Spain); SZN — Stazione Zoologica Anton Dohrn (Naples, Italy).
External morphology (Fig. 2). Body elongate, broad, narrowing towards posterior end of foot. Small tentaculiform foot corners in anterior part of foot (Fig. 2). Body colour brown. Dorsum with scattered small white spots. Larger white patch behind rhinophores and pericardial path. Head bluish with oval purplish blue spot on anteriormost margin of head (Cyprus) (Fig. 2b). Oral tentacles long, brown with white bands. One white band surrounding the beginning of the oral tentacles, two located in the middle part. Tips of oral tentacles bluish (Turkey) (Fig. 2a)
or brownish (Cyprus) (Fig. 2b). Rhinophores brown, studded with minute whitish knobs, but two bands of darker brown may be present (Cyprus). Cerata flattened. Branches of digestive gland brownish, visible through translucent body wall, with whitish spots of variable sizes. Terminal part of cerata with bluish band, followed by yellowish band, ending with a white spot on the tip. Cerata irregularly arranged on body. Nine or ten arches can be observed, each with up to 11 or 12 cerata counted in the specimen from Cyprus.
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Fig. 2. Baeolidia moebii specimens from the Mediterranean Sea. Photographs of the living animals. A, juvenile individual from Limanagzi-Kaş, Turkey, crawling on Posidonia oceanica leaves, photographed by Murat Draman; B, specimen from Nissia Natura 2000, Cyprus, photographed by Demetris Kletou (∼3 cm). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Table 2 COI gene pairwise uncorrected p-distances (%) amongst species of Baeolidia. Species
COI p-distances (%)
B. B. B. B. B.
0–0.4 23.9–24.3 19.3–19.5 13.2–13.8 20.6–21
moebii vs. B. moebii moebii vs. B. japonica moebii vs. B. salaamica scottjohnsoni vs. B. japonica salaamica vs. B. variabilis
Anatomy (Figs. 3, 4). Radular formula 16 × 0.1.0. Radular teeth of up to 120 elongate, thin, pointed denticles. Denticles larger and longer in central part of radula, progressively smaller and thinner outwards (Fig. 3a, b). Masticatory border of jaws smooth (Fig. 3c, d). Oral and salivary glands absent. Reproductive system diaulic (Fig. 4). Preampullary duct widening into kidney-shaped ampulla. Postampullary duct split into oviduct and vas deferens. Vas deferens wide and elongate, expanding into wider proximal portion of penial sac. Penis short, thin, unarmed, inside the distal part of the penial sac. Receptaculum seminis rounded, with a large stalk connecting to large oviduct. Vagina ventral to penis. Remarks. The genus Baeolidia has had a controversial taxonomic history due to lack of defined morphological characteristics (Bergh, 1888; Miller, 2001; Gosliner, 1985). The main characters used to differentiate species until recently were colouration, ornamentation of rhinophores, and radular morphology. However, all these may vary within a single species, so that the taxonomic status of several taxa is still debated (Carmona et al., 2014). Baeolidia australis (Rudman, 1982), a species very similar to B. moebii, is one of these. In fact, despite both taxa having been widely investigated in the past with regards to several anatomical features (Bergh, 1888; Edmunds, 1969; Rudman, 1982; Miller, 2001; Carmona et al., 2014), putative differences between these species were questioned by several authors (Rudman, 2007; Carmona et al., 2014) and the taxonomic validity of B. australis still remains speculative, as type or topotypical material of this taxon was never analysed molecularly. According to Carmona et al. (2014), the only valid difference between these two sibling species is the colouration of live animals, with an orange band in the cerata of B. australis, yellow in B. moebii, and a reticulate body pattern in B. australis, absent in B. moebii. In addition, the reproductive system of B. australis was only described by Miller (2001), but his description seems to have some omissions, among which the shape and size of the different reproductive organs that were not listed. The presence of a bursa copulatrix was also highlighted, a structure which seems to be absent in all the other species assigned to Baeolidia (see Carmona et al., 2014).
Despite such uncertainties, Red Sea records of both B. moebii and B. australis occur in the recent literature (Yonow, 2000, 2008; Dekker and Orlin, 2000). However, the Mediterranean specimens of B. moebii analysed here are not only clearly conspecific with all the Indo-Pacific B. moebii sequenced, and with the Mediterranean specimen previously identified by Turk and Furlan (2011) as Spurilla major, but also with the Red Sea individuals figured by Yonow (2000, 2008) as B. australis. This suggests that only B. moebii is present in the Red Sea and the species, originally described from Rodrigues Island (Mauritius) and widely distributed into the Indo-Pacific Ocean (Carmona et al., 2013), has been invading the Mediterranean Sea since at least 2007. 4. Discussion To date, the genus Baeolidia was only known from the Mediterranean Sea based on a single photograph. In our study, we report a new photographic record of B. moebii from this Turkish locality where the species was already sighted, and extend its known distribution to Cyprus with a collected specimen. The new records reported here immediately raise the question of the origin of these sea slugs. The fact that the genus Baeolidia has mostly an Indo-Pacific distribution (Yonow, 2008; Carmona et al., 2014; Gosliner et al., 2015), as well as the fact that conspecific specimens were recorded from the nearby Red Sea (Yonow, 2000, 2008), strongly suggest that specimens recorded from the Mediterranean Sea (Turk and Furlan, 2011; present paper) actually belong to a new alien arrival, presumably through Lessepsian migration. In addition, the first Mediterranean record held in 2007 (Turk and Furlan, 2011), coupled with the present sightings, may suggest a recent spread or even an establishment of B. moebii in the Mediterranean Sea. Although on the one hand the three records may simply represent introduced larvae or juveniles which have not established a viable population, sea slugs often possess cryptic behaviour and may require specific and focused surveys and sampling methods (Jörger et al., 2014; Araya and Valdés, 2016), thereby suggesting that its spreading in the Mediterranean Sea may be partially overlooked. This is also in agreement with the fact that this species has not been recorded from additional Levantine countries such as the Mediterranean coasts of Egypt, Israel, Lebanon, and Syria, or even from the Red Sea again after records held by Yonow (2000, 2008). An alternative hypothesis is that this species may have locally arrived through ballast water and that all the records reported here and in the previous literature may be the result of casual but repetitive introductions. However, lack of records from the additional
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Fig. 3. Baeolidia moebii from Cyprus. Scanning Electron Microscope photographs. A–B, radular teeth; C, jaw; D, detailed view of jaw masticatory border. Scale bars: 100 µm.
Fig. 4. Baeolidia moebii from Cyprus. Reproductive system. Scale bar: 1 mm. Abbreviations: am, ampulla; fgm, female gland mass; ps, penial sac; rs, receptaculum seminis; va, vagina; vd, vas deferens.
Levantine countries listed above may easily be an artefact due to
help in confirming whether or not B. moebii is spreading in the
the small size of this nudibranch, sampling bias, and/or taxonomic
Mediterranean Sea.
impediments, and thus we are inclined to discard the latter
Finally, it is worth mentioning that the role of citizen scientists
hypothesis. Further field excursions with laboratory work may
and of scuba divers in assessing the distribution of alien species
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has been previously highlighted in several papers (Katsanevakis et al., 2012; Poursanidis and Zenetos, 2013; Adriaens et al., 2015; Kleitou et al., 2019), and the present records add more support for this. Despite the fact that Cavo Greco and Nissia (Cyprus) areas have been recently monitored for two years using visual census by scientists, there was no record of B. moebii in the different habitats investigated. Our specimen from Cyprus was found in the same area by one of us (DK) during a purely observational recreational scuba diving. The same situation occurs with the Kaş (Turkey) individual reported here, which was found by an amateur scuba photographer. Thus, the development of speciesspecific observational projects is strongly suggested in light of the ongoing changes to which the Mediterranean biota is being subjected. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements Murat Draman (Turkey) provided data of the Turkish specimen. The Electron Microscopy and the Molecular Biology services of Stazione Zoologica Anton Dohrn (Italy) assisted in various stages. We are grateful to all of them. We would also like to thank the various referees who provided useful comments on previous drafts. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. References Adriaens, T., Sutton-Croft, M., Owen, K., Brosens, D., Van Valkenburg, J., Kilbey, D., Groom, Q., Ehmig, C., Thürkow, F., Van Hende, P., Schneider, K., 2015. Trying to engage the crowd in recording invasive alien species in Europe: experiences from two smartphone applications in northwest Europe. Manag. Biol. Invasions 6 (2), 215–225. http://dx.doi.org/10.3391/mbi.2015.6.2.12. Akaike, H., 1974. A new look at the statistical model identification. IEEE. Trans. Automat. Control 19, 716–723. http://dx.doi.org/10.1109/TAC.1974.1100705. Alfaro, M.E., Zoller, S., Lutzoni, F., 2003. Bayes or bootstraps? A simulation study comparing the performance of Bayesian Markov chain Monte Carlo sampling and bootstrapping in assessing phylogenetic confidence. Mol. Biol. Evol. 20, 255–266. http://dx.doi.org/10.1093/molbev/msg028. Araya, J.F., Valdés, A., 2016. Shallow water heterobranch sea slugs (Gastropoda: Heterobranchia) from the Región de Atacama. North. Chil. PeerJ 4 (e1963), http://dx.doi.org/10.7717/peerj.1963. Bergh, L.S.R., 1888. Malacologische untersuchungen. In: Reisen im archipel der philippinen von Dr. Carl gottfried semper. Zweiter Theil. Wiss. Result. 2 (3), 755–814. Carmona, L., Pola, M., Gosliner, T.M., Cervera, J.L., 2013. A tale that morphology fails to tell: a molecular phylogeny of Aeolidiidae (Aeolidida, Nudibranchia, Gastropoda). PloS one 8 (5), e63000. Carmona, L., Pola, M., Gosliner, T.M., Cervera, J.L., 2014. Review of Baeolidia, the largest genus of Aeolidiidae (Mollusca: Nudibranchia), with the description of five new species. Zootaxa 3802 (4), 477–514. http://dx.doi.org/10.11646/ zootaxa.3802.4.5. Crocetta, F., Gofas, S., Salas, C., Tringali, L.P., Zenetos, A., 2017. Local ecological knowledge versus published literature: a review of non-indigenous Mollusca in Greek marine waters. Aquat. Invasions 12 (4), 415–434. http://dx.doi.org/ 10.3391/ai.2017.12.4.01. Crocetta, F., Zibrowius, H., Bitar, G., Templado, J., Oliverio, M., 2013. Biogeographical homogeneity in the eastern Mediterranean Sea - I: the opisthobranchs (Mollusca: Gastropoda) from Lebanon. Mediterr. Mar. Sci. 14 (2), 403–408. http://dx.doi.org/10.12681/mms.404. Darriba, D., Taboada, G.L., Doallo, R., Posada, D., 2012. JModelTest 2: more models, new heuristics and parallel computing. Nature Methods 9 (8), 722. http://dx.doi.org/10.1038/nmeth.2109. Dekker, H., Orlin, Z., 2000. Check-list of red sea Mollusca. Spirula 1, 3–46. Edmunds, M., 1969. Opisthobranchiate Mollusca from Tanzania: I. Eolids (Eubranchidae and Aeolidiidae). J. Mollus. Stud. 38 (5), 451–469.
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Exploring the diversity of mesopsammic gastropods: How to collect, identify, and delimitate small and elusive sea slugs? Am. Malacol. Bull. 32 (2), 290–308. http://dx.doi.org/10.4003/006.032.0205. Katsanevakis, S., Bogucarskis, K., Gatto, F., Vandekerkhove, J., Deriu, I., Cardoso, A.C., 2012. Building the European Alien Species Information Network (EASIN): a novel approach for the exploration of distributed alien species data. BioInvasions Rec. 1 (4), 235–245. http://dx.doi.org/10.3391/bir.2012.1. 4.01. Kleitou, P., Giovos, I., Wolf, W., Crocetta, F., 2019. On the importance of citizenscience: first record of Goniobranchus obsoletus (Rüppell & Leuckart, 1830) (Mollusca: Gastropoda: Nudibranchia) from Cyprus. BioInvasions Rec. 8 (2), 252–257. Kozlov, A.M., Darriba, D., Flouri, T., Morel, B., Stamatakis, A., 2018. RAxML-NG: A fast, scalable, and user-friendly tool for maximum likelihood phylogenetic inference, bioRxiv http://dx.doi.org/10.1101/447110. 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MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572–1574. Rudman, W.B., 1982. The taxonomy and biology of further Aeolidacean and Arminacean nudibranch molluscs with symbiotic zooxanthellae. Zool. J. Linn. Soc. 74 (2), 147–196. http://dx.doi.org/10.1111/j.1096-3642.1982.tb01146.x. Rudman, W.B., 2007. Comment on Spurilla australis from Lembeh Straits, Sulawesi by Matt Oldfield. [Message in] Sea Slug Forum. Australian Museum, Sydney. Available from: http://www.seaslugforum.net/find/19744 (accessed 1.18). Sabelli, B., Taviani, M., 2014. The making of the Mediterranean molluscan biodiversity. In: Goffredo, S., Dubinsky, Z. (Eds.), The Mediterranean Sea. Springer, Dordrecht, pp. 285–306. http://dx.doi.org/10.1007/978-94-007-6704-1_16. Servello, G., Andaloro, F., Azzurro, E., Castriota, L., Catra, M., Chiarore, A., Crocetta, F., D’Alessandro, M., Denitto, F., Froglia, C., Gravili, C., Langer, M., Lo Brutto, S., Mastrototaro, F., Petrocelli, A., Pipitone, C., Piraino, S., Relini, G., Serio, D., Xentidis, N.J., Zenetos, A., 2019. Marine alien species in Italy: a contribution to the implementation of descriptor D2 of the Marine Strategy Framework Directive. Mediterr. Mar. Sci. 20 (1), 1–48. http://dx.doi.org/10. 12681/mms.18711. Tsiakkiros, L., Zenetos, A., 2011. Further additions to the alien mollusc fauna along the Cypriot coast: new opisthobranchia species. Acta Adriat. 52 (1), 115–123.
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Yonow, N., 2000. Red Sea Opisthobranchia 4: the orders Cephalaspidea, Anaspidea, Notaspidea and Nudibranchia: Dendronotacea, and Aeolidacea. Fauna Arab. 18, 87–132. Yonow, N., 2008. Sea Slugs of the Red Sea. Pensoft Publishers, Bulgaria. Zenetos, A., Çinar, M.E., Crocetta, F., Golani, D., Rosso, A., Servello, G., Shenkar, N., Turon, X., Verlaque, M., 2017. Uncertainties and validation of alien species catalogues: The Mediterranean as an example. Estuar. Coast. Shelf. Sci. 191, 171–187. http://dx.doi.org/10.1016/j.ecss.2017.03.031.
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Regional Studies in Marine Science journal homepage: www.elsevier.com/locate/rsma
Baeolidia moebii Bergh, 1888 (Mollusca: Gastropoda: Nudibranchia) is spreading in the eastern Mediterranean Sea ∗
Sofia Paz-Sedano a , , Valentina Tanduo a , Nathalie Yonow b , Mehmet Baki Yokeş c , Demetris Kletou d , Fabio Crocetta a a
Department of Integrative Marine Ecology, Stazione Zoologica Anton Dohrn, Villa Comunale, I-80121 Napoli, Italy Department of Biosciences, Swansea University, Singleton Park, Swansea SA2 8PP, Wales, UK c AMBRD Laboratories, Hanimefendi Sokak 160/6, 34384 Sisli, Istanbul, Turkey d Marine and Environmental Research (MER) Lab Ltd., 4533, Limassol, Cyprus b
article
info
Article history: Received 17 May 2019 Received in revised form 28 July 2019 Accepted 8 September 2019 Available online 12 September 2019 Keywords: Aeolidiidae Alien species Bioinvasions Cyprus Sea slugs Turkey
a b s t r a c t The knowledge of alien species dispersal in new geographic areas is a matter of major concern, since alien species may involve both ecological and faunistic threats in the newly colonized habitats. The geographical range of the genus Baeolidia Bergh, 1888 is mostly in the Indo-Pacific realm, including the Red Sea. However, one of the species belonging to this genus, Baeolidia moebii Bergh, 1888, has been also reported as an ephemeral Mediterranean invader from Turkey. In addition, its identification is highly debatable due to Red Sea records of a sibling species, Baeolidia australis (Rudman, 1982). The presence of two additional Baeolidia in the Mediterranean basin is reported here, based on photographs from Turkey and collected material from Cyprus, thus reinforcing the presence of this alien species in the former country and extending its distribution to the latter. In addition, morphological analyses of a specimen from the Mediterranean Sea were performed for the first time, and it was also barcoded through unpublished species-specific primers designed for the Baeolidia cytochrome c oxidase subunit I gene. Our results confirm the previous identification of the alien taxon and suggest that the presence of B. australis in the Red Sea should be discarded. Finally, the present records suggest that the Mediterranean spreading of B. moebii may be overlooked due to sea slugs’ cryptic behaviour. © 2019 Elsevier B.V. All rights reserved.
1. Introduction The introduction of alien species from one geographic area to another has highly increased in marine environments, mainly due to anthropogenic dispersal through artificial canals and fouling, but also enhanced by ballast transport, mariculture, bait and pet trade, and market discards (Molnar et al., 2008; Hulme, 2009). The Mediterranean Sea has been documented as one of the areas with highest rates of alien species invasions, mainly due to Lessepsian migration, i.e., species originating from the Indo-Pacific entering the Mediterranean basin through the Suez Canal. This attracted the interest of several researchers in the last decades, which resulted in the production of numerous scientific databases, inventories, and reviews (Zenetos et al., 2017; Galil et al., 2018 and references therein). Regarding Mollusca, more than 200 alien species have been recorded so far in the Mediterranean (Sabelli and Taviani, 2014), among which approximately 30 sea slug species are included, recorded mostly in the Levant Basin (e.g., Yokeş and Rudman, 2004; Tsiakkiros and Zenetos, ∗ Corresponding author. E-mail address: [email protected] (S. Paz-Sedano). https://doi.org/10.1016/j.rsma.2019.100830 2352-4855/© 2019 Elsevier B.V. All rights reserved.
2011; Yokeş et al., 2012; Crocetta et al., 2013). Some emblematic examples of well-established species include Bulla arabica (Malaquias & Reid, 2008), Goniobranchus annulatus (Eliot, 1904), Haminoea cyanomarginata (Heller & Thompson, 1983), and Melibe viridis (Kelaart, 1858), two of which have also reached the central and the westernmost parts of the Mediterranean (Crocetta et al., 2013, 2017; Fernández-Vilert et al., 2018; Servello et al., 2019). However, the fate of alien invaders may vary. In fact, apart from the taxa mentioned above, other sea slugs are only known to date from sporadic or even single Mediterranean records — e.g. Baeolidia moebii (Bergh, 1888), Caloria indica (Bergh, 1896), Goniobranchus obsoletus (Rüppell & Leuckart, 1830), and Plocamopherus tilesii (Bergh, 1877) (Gat, 1993; Turk and Furlan, 2011; Yokeş et al., 2012; Kleitou et al., 2019). Among them, B. moebii is known as being a Mediterranean invader since 2007, but it is only known from the area based on a single individual photographed in Kaş, Turkey (Turk and Furlan, 2011 as Spurilla major); this led some authors to consider this sea slug species as an ephemeral introduction in the Mediterranean, and not an established species (Crocetta et al., 2013; Zenetos et al., 2017). Based on the recent sightings of two Baeolidia specimens from the Mediterranean Sea, we took the opportunity to properly investigate the systematics
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S. Paz-Sedano, V. Tanduo, N. Yonow et al. / Regional Studies in Marine Science 32 (2019) 100830
of this alien species through an integrative taxonomic approach, and to revise the Red Sea records of this and its sibling species B. australis. 2. Materials and methods 2.1. Source of material Material examined in the present paper was found by SCUBA diving (for amateur purposes) in Turkey and Cyprus. The sample from Turkey was only studied externally by photographs. The specimen from Cyprus was photographed alive, fixed in 96% ethanol, and subsequently studied anatomically. The reproductive system and the SEM (Scanning Electron Microscopy) stubs of the analysed material are currently preserved in the institutional collection of Stazione Zoologica Anton Dohrn (SZN) (Naples, Italy), whilst the remaining tissues were dissolved for sequencing. 2.2. Morphological examination The external morphology of the specimen was studied by both photographs of the living animal and laboratory observations. Internal anatomy was studied under a Leica M60 microscope. Internal organs were removed by a dorsal incision, separated, and photographed using a Leica IC80HD Camera attached. Subsequently, they were drawn using Adobe Photoshop CC. To study the labial cuticle and radula, the buccal mass was dissolved in 10% sodium hydroxide until the surrounding tissue was completely removed, the structures then rinsed in water. The labial cuticle, radula, and penis were mounted on SEM stubs and gold-palladium coated in an SC7640 Sputter coater for SEM examination with a Jeol JSM-6700 F microscope. 2.3. Molecular analyses 2.3.1. DNA extraction, amplification, and sequencing The genomic DNA of the Baeolidia specimen from Cyprus R was extracted from the body by using the NucleoSpin⃝ Tissue (Macherey-Nagel) kit, following the manufacturer’s protocol. As amplifications with universal primers for invertebrates (Folmer et al., 1994) failed, partial sequence of cytochrome c oxidase I (COI) was amplified using the following species-specific primers designed for the present study: forward 5′ -TWTTAATTCGTTTY GAGYTW-3′ and reverse 5′ -AAYCTWGTRTTRAAATTHCG-3′ . PCR was conducted in 25 µl volume reaction, prepared in the following order: 12.75 µl of water, 2.5 µl of Qiagen buffer and magnesium chloride (10X), 2.5 µl of dNTPs (2 mM), 1 µl each of forward (74.4 nM) and reverse (76.8 nM) primers, 0.25 µl of DNA polymerase (5 U/µl), and 5 µl of DNA. Amplification was performed with an initial denaturation for 5 min at 95 ◦ C, followed by 39 cycles of 1 min at 95 ◦ C, 1 min annealing at 40 ◦ C and 45 s at 72 ◦ C, with a final extension of 7 min at 72 ◦ C. The successful PCR product was purified and sequenced at the Molecular Biology and Sequencing Service of SZN. 2.3.2. GenBank data mining and phylogenetic analyses A GenBank search was carried out to check for barcodes of Baeolidia taxa. Sequences of Tritonia challengeriana (Bergh, 1888) and Luisella babai (Schmekel, 1972) were also included as outgroups. The COI sequence obtained was assembled and edited using BioEdit v7.2.5 (Hall, 1999) and a search was subsequently run in GenBank to check for contamination. Alignment was carried out using MEGA7 (Kumar et al., 2016) and confirmed by translating sequences into amino acids using the genetic code of invertebrate mitochondrial DNA. Sequences were trimmed to 486 base pairs. The evolutionary models were determined separately
for the first, second, and third codon positions using jModelTest2,1,7 (Darriba et al., 2012) under Akaike information criteria (Akaike, 1974). Bayesian Inference (BI) and Maximum Likelihood (ML) analyses were conducted. The first was performed using the software package MrBayes v3.1.2b (Ronquist and Huelsenbeck, 2003) for ten million generations, two independent runs, and 1000 sampling frequency. ML analysis was performed using the software package RAxML-NG (Kozlov et al., 2018), with 0.03 automatic boot-stopping cut-off implemented. Only nodes supported by posterior probabilities ≥ 0.96 (Alfaro et al., 2003) and bootstrap values ≥ 75 (Hillis and Bull, 1993) were considered statistically significant. The trees obtained were visualized in FigTree v1.3.1 and edited in Adobe Photoshop CC 2014. 2.3.3. Species delimitation analyses Genetic distances amongst specimens of Baeolidia were compared using pairwise uncorrected p-distances for COI. The analysis was carried out using MEGA7 (Kumar et al., 2016) considering all codon positions. An Automatic Barcode Gab Discovery (ABGD) analysis (Puillandre et al., 2011) was conducted on the COI ingroup sequences through the ABGD web tool (http://wwwabi. snv.jussieu.fr/public/abgd/abgdweb.html) using the Kimura (K80) model. The parameters selected were a relative gap width (X) of 1, a range of prior values for maximum divergence of intraspecific diversity (P) from 0.0001 to 0.1, and Nb bins = 20. 3. Results 3.1. Molecular results Seven different Baeolidia taxa were found in GenBank (Table 1). A fragment of the COI sequence of the specimen from Cyprus was successfully obtained and the evolutionary models for the first, second, and third positions were TIM1+I, F81, and HKY+G, respectively. The phylogenetic tree obtained is shown in Fig. 1. The species of the genus Baeolidia are gathered in a well-supported group (BI = 0.99, ML = 83). The relationships between the different species are not resolved; however, the Baeolidia specimen from Cyprus clearly clustered with other B. moebii from Hawaii, Philippines, and the Marshall Islands, with a high value for both analyses (BI = 1, ML = 100). Species delimitation analyses confirm this result, showing uncorrected p-distances amongst B. moebii specimens of 0–0.2 (Table 2) and grouping together as the same species, B. moebii, in ABGD species delimitation analysis (Fig. 1). 3.2. Systematics Order Nudibranchia Cuvier, 1817 Family Aeolidiidae Gray, 1827 Genus Baeolidia (Bergh, 1888) Baeolidia moebii (Bergh, 1888) (Figs. 2–4) Material examined. Turkey – Limanagzi – Kaş (Antalya) (36.1824◦ N, 29.6438◦ E), one individual photographed by Murat Draman, October 2013, ∼8–10 m depth (Fig. 2a). The individual was not sampled. Cyprus — Nissia Natura 2000 (35.0056◦ N, 34.0691◦ E), one specimen collected by one of us (DK), June 2017, ∼20 m depth (Fig. 2b) (SZN-MOL-0020). Ecology. Both specimens were detected crawling on the leaves of the marine phanerogam Posidonia oceanica (Linnaeus) Delile. No additional data are available for the specimen from Turkey but in Cyprus, the leaves were covered by hydrozoans such as Aglaophenia harpago (Schenck, 1965) and Sertularia sp., and by bryozoans such as Electra posidoniae Gautier, 1954 and Calpensia nobilis (Esper, 1796).
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Table 1 Species used for molecular analyses, including voucher, locality, and COI GenBank accession number. Abbreviations used: CASIZ — California Academy of Science (California, USA); MNCN and MNCN/ADN — Museo Nacional de Ciencias Naturales (Madrid, Spain); SZN — Stazione Zoologica Anton Dohrn (Naples, Italy).
a
Species
Voucher
Locality
GenBank accession number
Tritonia challengeriana (Bergh, 1884) Luisella babai (Schmekel 1972) Baeolidia japonica (Baba, 1933) Baeolidia japonica (Baba, 1933) Baeolidia japonica (Baba, 1933) Baeolidia moebii (Bergh, 1888) Baeolidia moebii (Bergh, 1888) Baeolidia moebii (Bergh, 1888) Baeolidia moebii (Bergh, 1888) Baeolidia moebii (Bergh, 1888) Baeolidia moebii (Bergh, 1888) Baeolidia moebii (Bergh, 1888)a Baeolidia ransoni (Pruvot-Fol, 1956) Baeolidia rieae (Carmona, Pola, Gosliner & Cervera, 2014) Baeolidia salaamica (Rudman, 1982) Baeolidia salaamica (Rudman, 1982) Baeolidia salaamica (Rudman, 1982) Baeolidia scottjohnsoni (Carmona, Pola, Gosliner & Cervera, 2014) Baeolidia variabilis (Carmona, Pola, Gosliner & Cervera, 2014) Baeolidia variabilis (Carmona, Pola, Gosliner & Cervera, 2014) Baeolidia variabilis (Carmona, Pola, Gosliner & Cervera, 2014) Baeolidia variabilis (Carmona, Pola, Gosliner & Cervera, 2014)
CASIZ 171177 MNCN 15.05/53698 CASIZ186795 CASIZ184520 CASIZ 181357 CASIZ186211 CASIZ177602 CASIZ 180327 MNCN 15.05/54987 MNCN/ADN: 51948 MNCN/ADN: 51949 SZN-MOL0020 CASIZ 186209 CASIZ 184525 CASIZ 177599 CASIZ 177397 CASIZ 180330 CASIZ 184503 CASIZ 187741.1 CASIZ 187741.2 CASIZ 186210 CASIZ 177716
Bouvetoya Spain Marshall Island Japan Philippines Philippines Philippines Hawaii Hawaii Marshall Island Marshall Island Cyprus Philippines Japan Philippines Philippines Philippines Marshall Islands Marshall Islands Marshall Islands Philippines Philippines
HM162718 HQ616754 JQ997059 JQ997058 JQ997057 JQ997061 HQ616770 JQ997060 HQ616771 JX087550 JX087551 MK922511 JQ997043 JQ997046 JQ997062 JQ997047 JQ997048 JQ997045 JQ997055 JQ997056 JQ997054 JQ997051
The Baeolidia specimen sequenced during the present study.
Fig. 1. Phylogenetic tree based on COI. Numbers above branches represent posterior probabilities (BI) and bootstrap values (ML). Specimen sequenced from Cyprus in bold. Coloured bands show ABGD species delimitation analysis result. Abbreviations used: CASIZ — California Academy of Science (California, USA); MNCN and MNCN/AND — Museo Nacional de Ciencias Naturales (Madrid, Spain); SZN — Stazione Zoologica Anton Dohrn (Naples, Italy).
External morphology (Fig. 2). Body elongate, broad, narrowing towards posterior end of foot. Small tentaculiform foot corners in anterior part of foot (Fig. 2). Body colour brown. Dorsum with scattered small white spots. Larger white patch behind rhinophores and pericardial path. Head bluish with oval purplish blue spot on anteriormost margin of head (Cyprus) (Fig. 2b). Oral tentacles long, brown with white bands. One white band surrounding the beginning of the oral tentacles, two located in the middle part. Tips of oral tentacles bluish (Turkey) (Fig. 2a)
or brownish (Cyprus) (Fig. 2b). Rhinophores brown, studded with minute whitish knobs, but two bands of darker brown may be present (Cyprus). Cerata flattened. Branches of digestive gland brownish, visible through translucent body wall, with whitish spots of variable sizes. Terminal part of cerata with bluish band, followed by yellowish band, ending with a white spot on the tip. Cerata irregularly arranged on body. Nine or ten arches can be observed, each with up to 11 or 12 cerata counted in the specimen from Cyprus.
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Fig. 2. Baeolidia moebii specimens from the Mediterranean Sea. Photographs of the living animals. A, juvenile individual from Limanagzi-Kaş, Turkey, crawling on Posidonia oceanica leaves, photographed by Murat Draman; B, specimen from Nissia Natura 2000, Cyprus, photographed by Demetris Kletou (∼3 cm). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Table 2 COI gene pairwise uncorrected p-distances (%) amongst species of Baeolidia. Species
COI p-distances (%)
B. B. B. B. B.
0–0.4 23.9–24.3 19.3–19.5 13.2–13.8 20.6–21
moebii vs. B. moebii moebii vs. B. japonica moebii vs. B. salaamica scottjohnsoni vs. B. japonica salaamica vs. B. variabilis
Anatomy (Figs. 3, 4). Radular formula 16 × 0.1.0. Radular teeth of up to 120 elongate, thin, pointed denticles. Denticles larger and longer in central part of radula, progressively smaller and thinner outwards (Fig. 3a, b). Masticatory border of jaws smooth (Fig. 3c, d). Oral and salivary glands absent. Reproductive system diaulic (Fig. 4). Preampullary duct widening into kidney-shaped ampulla. Postampullary duct split into oviduct and vas deferens. Vas deferens wide and elongate, expanding into wider proximal portion of penial sac. Penis short, thin, unarmed, inside the distal part of the penial sac. Receptaculum seminis rounded, with a large stalk connecting to large oviduct. Vagina ventral to penis. Remarks. The genus Baeolidia has had a controversial taxonomic history due to lack of defined morphological characteristics (Bergh, 1888; Miller, 2001; Gosliner, 1985). The main characters used to differentiate species until recently were colouration, ornamentation of rhinophores, and radular morphology. However, all these may vary within a single species, so that the taxonomic status of several taxa is still debated (Carmona et al., 2014). Baeolidia australis (Rudman, 1982), a species very similar to B. moebii, is one of these. In fact, despite both taxa having been widely investigated in the past with regards to several anatomical features (Bergh, 1888; Edmunds, 1969; Rudman, 1982; Miller, 2001; Carmona et al., 2014), putative differences between these species were questioned by several authors (Rudman, 2007; Carmona et al., 2014) and the taxonomic validity of B. australis still remains speculative, as type or topotypical material of this taxon was never analysed molecularly. According to Carmona et al. (2014), the only valid difference between these two sibling species is the colouration of live animals, with an orange band in the cerata of B. australis, yellow in B. moebii, and a reticulate body pattern in B. australis, absent in B. moebii. In addition, the reproductive system of B. australis was only described by Miller (2001), but his description seems to have some omissions, among which the shape and size of the different reproductive organs that were not listed. The presence of a bursa copulatrix was also highlighted, a structure which seems to be absent in all the other species assigned to Baeolidia (see Carmona et al., 2014).
Despite such uncertainties, Red Sea records of both B. moebii and B. australis occur in the recent literature (Yonow, 2000, 2008; Dekker and Orlin, 2000). However, the Mediterranean specimens of B. moebii analysed here are not only clearly conspecific with all the Indo-Pacific B. moebii sequenced, and with the Mediterranean specimen previously identified by Turk and Furlan (2011) as Spurilla major, but also with the Red Sea individuals figured by Yonow (2000, 2008) as B. australis. This suggests that only B. moebii is present in the Red Sea and the species, originally described from Rodrigues Island (Mauritius) and widely distributed into the Indo-Pacific Ocean (Carmona et al., 2013), has been invading the Mediterranean Sea since at least 2007. 4. Discussion To date, the genus Baeolidia was only known from the Mediterranean Sea based on a single photograph. In our study, we report a new photographic record of B. moebii from this Turkish locality where the species was already sighted, and extend its known distribution to Cyprus with a collected specimen. The new records reported here immediately raise the question of the origin of these sea slugs. The fact that the genus Baeolidia has mostly an Indo-Pacific distribution (Yonow, 2008; Carmona et al., 2014; Gosliner et al., 2015), as well as the fact that conspecific specimens were recorded from the nearby Red Sea (Yonow, 2000, 2008), strongly suggest that specimens recorded from the Mediterranean Sea (Turk and Furlan, 2011; present paper) actually belong to a new alien arrival, presumably through Lessepsian migration. In addition, the first Mediterranean record held in 2007 (Turk and Furlan, 2011), coupled with the present sightings, may suggest a recent spread or even an establishment of B. moebii in the Mediterranean Sea. Although on the one hand the three records may simply represent introduced larvae or juveniles which have not established a viable population, sea slugs often possess cryptic behaviour and may require specific and focused surveys and sampling methods (Jörger et al., 2014; Araya and Valdés, 2016), thereby suggesting that its spreading in the Mediterranean Sea may be partially overlooked. This is also in agreement with the fact that this species has not been recorded from additional Levantine countries such as the Mediterranean coasts of Egypt, Israel, Lebanon, and Syria, or even from the Red Sea again after records held by Yonow (2000, 2008). An alternative hypothesis is that this species may have locally arrived through ballast water and that all the records reported here and in the previous literature may be the result of casual but repetitive introductions. However, lack of records from the additional
S. Paz-Sedano, V. Tanduo, N. Yonow et al. / Regional Studies in Marine Science 32 (2019) 100830
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Fig. 3. Baeolidia moebii from Cyprus. Scanning Electron Microscope photographs. A–B, radular teeth; C, jaw; D, detailed view of jaw masticatory border. Scale bars: 100 µm.
Fig. 4. Baeolidia moebii from Cyprus. Reproductive system. Scale bar: 1 mm. Abbreviations: am, ampulla; fgm, female gland mass; ps, penial sac; rs, receptaculum seminis; va, vagina; vd, vas deferens.
Levantine countries listed above may easily be an artefact due to
help in confirming whether or not B. moebii is spreading in the
the small size of this nudibranch, sampling bias, and/or taxonomic
Mediterranean Sea.
impediments, and thus we are inclined to discard the latter
Finally, it is worth mentioning that the role of citizen scientists
hypothesis. Further field excursions with laboratory work may
and of scuba divers in assessing the distribution of alien species
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has been previously highlighted in several papers (Katsanevakis et al., 2012; Poursanidis and Zenetos, 2013; Adriaens et al., 2015; Kleitou et al., 2019), and the present records add more support for this. Despite the fact that Cavo Greco and Nissia (Cyprus) areas have been recently monitored for two years using visual census by scientists, there was no record of B. moebii in the different habitats investigated. Our specimen from Cyprus was found in the same area by one of us (DK) during a purely observational recreational scuba diving. The same situation occurs with the Kaş (Turkey) individual reported here, which was found by an amateur scuba photographer. Thus, the development of speciesspecific observational projects is strongly suggested in light of the ongoing changes to which the Mediterranean biota is being subjected. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements Murat Draman (Turkey) provided data of the Turkish specimen. The Electron Microscopy and the Molecular Biology services of Stazione Zoologica Anton Dohrn (Italy) assisted in various stages. We are grateful to all of them. We would also like to thank the various referees who provided useful comments on previous drafts. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. References Adriaens, T., Sutton-Croft, M., Owen, K., Brosens, D., Van Valkenburg, J., Kilbey, D., Groom, Q., Ehmig, C., Thürkow, F., Van Hende, P., Schneider, K., 2015. Trying to engage the crowd in recording invasive alien species in Europe: experiences from two smartphone applications in northwest Europe. Manag. Biol. Invasions 6 (2), 215–225. http://dx.doi.org/10.3391/mbi.2015.6.2.12. Akaike, H., 1974. A new look at the statistical model identification. IEEE. Trans. Automat. Control 19, 716–723. http://dx.doi.org/10.1109/TAC.1974.1100705. Alfaro, M.E., Zoller, S., Lutzoni, F., 2003. Bayes or bootstraps? A simulation study comparing the performance of Bayesian Markov chain Monte Carlo sampling and bootstrapping in assessing phylogenetic confidence. Mol. Biol. Evol. 20, 255–266. http://dx.doi.org/10.1093/molbev/msg028. Araya, J.F., Valdés, A., 2016. Shallow water heterobranch sea slugs (Gastropoda: Heterobranchia) from the Región de Atacama. North. Chil. PeerJ 4 (e1963), http://dx.doi.org/10.7717/peerj.1963. Bergh, L.S.R., 1888. Malacologische untersuchungen. In: Reisen im archipel der philippinen von Dr. Carl gottfried semper. Zweiter Theil. Wiss. Result. 2 (3), 755–814. Carmona, L., Pola, M., Gosliner, T.M., Cervera, J.L., 2013. A tale that morphology fails to tell: a molecular phylogeny of Aeolidiidae (Aeolidida, Nudibranchia, Gastropoda). PloS one 8 (5), e63000. Carmona, L., Pola, M., Gosliner, T.M., Cervera, J.L., 2014. Review of Baeolidia, the largest genus of Aeolidiidae (Mollusca: Nudibranchia), with the description of five new species. Zootaxa 3802 (4), 477–514. http://dx.doi.org/10.11646/ zootaxa.3802.4.5. Crocetta, F., Gofas, S., Salas, C., Tringali, L.P., Zenetos, A., 2017. Local ecological knowledge versus published literature: a review of non-indigenous Mollusca in Greek marine waters. Aquat. Invasions 12 (4), 415–434. http://dx.doi.org/ 10.3391/ai.2017.12.4.01. Crocetta, F., Zibrowius, H., Bitar, G., Templado, J., Oliverio, M., 2013. Biogeographical homogeneity in the eastern Mediterranean Sea - I: the opisthobranchs (Mollusca: Gastropoda) from Lebanon. Mediterr. Mar. Sci. 14 (2), 403–408. http://dx.doi.org/10.12681/mms.404. Darriba, D., Taboada, G.L., Doallo, R., Posada, D., 2012. JModelTest 2: more models, new heuristics and parallel computing. Nature Methods 9 (8), 722. http://dx.doi.org/10.1038/nmeth.2109. Dekker, H., Orlin, Z., 2000. Check-list of red sea Mollusca. Spirula 1, 3–46. Edmunds, M., 1969. Opisthobranchiate Mollusca from Tanzania: I. Eolids (Eubranchidae and Aeolidiidae). J. Mollus. Stud. 38 (5), 451–469.
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