BioInvasions Records (2020) Volume 9, Issue 3: 496–503

CORRECTED PROOF

Rapid Communication First record of the Red Sea gastropod sanguinolenta Menke, 1829 (: : ) from the Israeli Mediterranean coast

Chen Rabi1,2,*, Gil Rilov1,2, Arseniy R. Morov1 and Tamar Guy-Haim1,* 1Israel Oceanographic and Limnological Research, National Institute of Oceanography, P.O. Box 8030, Haifa 31080, Israel 2Marine Biology Department, The Leon H. Charney School of Marine Sciences, University of Haifa, Mt. Carmel, Haifa 31905, Israel *Corresponding authors E-mails: [email protected] (CR), [email protected] (TGH)

Citation: Rabi C, Rilov G, Morov AR, Guy-Haim T (2020) First record of the Red Abstract Sea gastropod Menke, 1829 (Gastropoda: Cycloneritida: The Red Sea intertidal gastropod Nerita sanguinolenta is reported here for the first Neritidae) from the Israeli Mediterranean time from the Northern Mediterranean coast of Israel, where it was found at the coast. BioInvasions Records 9(3): 496–503, landward face of a vermetid reef on 11 November 2019, and identified morphologically https://doi.org/10.3391/bir.2020.9.3.06 and by using molecular barcoding. Possible vectors for its introduction primarily Received: 8 March 2020 include ship ballast or larval transport via the Suez Canal in multiple migration Accepted: 12 April 2020 events. Investigation of the thermal affinities of N. sanguinolenta is required for Published: 12 June 2020 understanding the potential for its proliferation in the Mediterranean Sea. Handling editor: Fred Wells Thematic editor: Andrew David Key words: , Red Sea endemic, Lessepsian migration, intertidal, molecular barcoding, COI, introduction vector Copyright: © Rabi et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International - CC BY 4.0). Introduction OPEN ACCESS. Nerita is a of marine and freshwater snails that is found mainly on tropical and subtropical intertidal coasts, with few exceptions that occur in temperate waters (Frey and Vermeij 2008). Contrary to most Nerita that inhabit the Red Sea and have an Indo-Pacific distribution, the species Nerita sanguinolenta (Menke, 1829) is endemic to the Red Sea. Nonetheless, despite having a narrow distribution in its native range, N. sanguinolenta is abundant where it occurs (Dekker 2000) and considered as a key-species, indicating the lower mid-littoral zone (Ayal and Safriel 1980). It was first recorded in the Mediterranean Sea in 1969 in Karpathos, Greece (Nordsieck 1973) by the synonym Nerita kinzelbachi (Nordsieck, 1973) as a new species, and was later recognized as a synonym of Nerita sanguinolenta Menke (Dekker 2000; Mienis, 1974). To date, living specimens of N. sanguinolenta were recorded in three other Mediterranean sites (Figure 1; Supplementary material Table S1): in 1994 in Tobrouk, Libya (Giannuzzi-Savelli 1994), in 2004 in Antalya Bay, Turkey (Mutaf et al. 2008) and in 2012 in Chebba, Gulf of Gabès, Tunisia (Zaouali and Ben Souissi 2013). Additionally, two dead shells of N. sanguinolenta were recorded in 2013 in Port Said, Egypt (Mytilineou et al. 2016).

Rabi et al. (2020), BioInvasions Records 9(3): 496–503, https://doi.org/10.3391/bir.2020.9.3.06 496 First record of Nerita sanguinolenta from the Israeli Mediterranean coast

Figure 1. The distribution map of Nerita sanguinolenta in the Mediterranean Sea presenting recoded sites and years (in brackets), including the molecular reference population area at the Red Sea in Port Safaga, Egypt (The list of geo-referenced sites is detailed in Supplementary material Table S1).

Due to the high variety of colors, patterns and details of shell morphology, Nerita species have given rise to a plethora of subjective synonyms (e.g., Spencer et al. 2007), which highlights the need for a thorough phylogenetic and molecular analysis and the establishment of a taxonomically- substantiated reference library, to validate the identity of specimens of this genus found outside of their native ranges (i.e., alien species). Here we report a new and the easternmost Mediterranean record of Nerita sanguinolenta from the Israeli Mediterranean intertidal, and the first molecular verification of the Red Sea origin of this Lessepsian invader.

Materials and methods Specimen collection A single, live, Nerita sanguinolenta specimen was collected at Shikmona, Haifa (32.82592°N; 34.95569°E), in the Northern Mediterranean coast of Israel (Figure 1) on 11 November 2019 (NPA permit no. 42282). The specimen was located on horizontal rocks at the back reef area in the high intertidal zone of a vermetid reef, during a spring-low tide. The gastropod was collected alive, and then was administered to anesthesia with menthol solution to get easily a part of the foot tissue. Within 24 hours, the specimen was preserved in 95% ethanol. Mean and maximum tide amplitudes in the region are 0.25 m and 0.45 m respectively. Water temperature at the time of collection was 25 °C and is annually extending between 17.0 and 31.5 °C.

Rabi et al. (2020), BioInvasions Records 9(3): 496–503, https://doi.org/10.3391/bir.2020.9.3.06 497 First record of Nerita sanguinolenta from the Israeli Mediterranean coast

Molecular identification Mitochondrial DNA was extracted from 5 mg of foot tissue using DNA Blood and Tissue Kit (Qiagen, Germany), according to the manufacturer specifications. Following the DNA extraction, COI gene was amplified using PCR with universal primers LCO1490 and HCO2198 (Folmer et al. 1994). Reaction conditions were as follows: 95 °C for 5 min, followed by 35 cycles of 95 °C for 30 s, 50 °C for 45 s, and 72 °C for 1 min, and an additional elongation step of 72 °C for 5 min. The PCR products were purified and sequenced by Hylabs (Israel). The obtained forward and reverse sequences were edited, corrected and assembled (as a consensus) using Geneious Prime (Kearse et al. 2012). To identify the specimen to species, we compared the specimen sequence to: (1) NCBI database by performing nucleotide blast search (blastn, https://blast.ncbi.nlm.nih.gov/), and (2) BOLD (Barcode of Life Data Systems) identification system for COI (http://www.boldsystems.org/). We further added the obtained sequence to the constructed COI-based Nerita phylogeny including 49 Nerita species, following Frey and Vermeij (2008). Fifty COI sequences of Nerita and a COI sequence of Thalassonerita naticoidea (formerly naticoidea Clarke, 1989) that was used as an outgroup (following Postaire et al. 2014) were downloaded from NCBI GenBank (Table S2), added to the sequence of the collected specimen and aligned using ClustalW in MEGA7 software (Kumar et al. 2016). Evolutionary models and parameter estimates were selected using the lowest AICc score obtained with ModelTest in MEGA7. A maximum likelihood tree was constructed in MEGA7 using GTR+G+I model of evolution with 1000 bootstrapping replicates. Upon molecular analysis, the specimen was deposited in the Steinhardt Collections of Natural History, Tel Aviv University, Israel (SMNH MO 83499).

Results Systematic results One specimen (Figure 2) was recorded from the intertidal zone of Shikmona beach, Haifa, Israel.

Order ARCHEOGASTROPODA Thiele, 1925 Family NERITIDAE Rafinesque, 1815 Genus Nerita Linnaeus, 1758 Nerita sanguinolenta Menke, 1829 Synonyms: Nerita (Theliostyla) sanguinolenta Menke, 1829 (alternate representation); Nerita forskali Récluz, 1841; Nerita erythrea Récluz, 1850; Nerita marginata var. coccinea Mörch, 1852; Nerita crassilabra Smith, E.A. 1885; kinzelbachi F. Nordsieck, 1973 (original description). Type locality: Karpathos, Greece. Description: Solid semi oval shell, with expanded-swollen body . Very small and flat . Large columellar callus, few notches in its middle.

Rabi et al. (2020), BioInvasions Records 9(3): 496–503, https://doi.org/10.3391/bir.2020.9.3.06 498 First record of Nerita sanguinolenta from the Israeli Mediterranean coast

Figure 2. Dorsal, side and ventral view of Nerita sanguinolenta specimen that was found in November 2019 at Shikmona, Haifa, Israel. Photo: Oz Rittner, The Steinhardt Museum of Natural History, Tel-Aviv University.

Inconspicuous denticles in the inner surface of the outer . Flat mouth. Color black-and-white (common type). Rough thick granulated flat operculum. Measurements of the specimen: width 22.5 mm, height 21.1 mm, weight 4.60 g.

Molecular results We analyzed 58 COI sequences of Nerita including one sequence of the specimen obtained from Shikmona, Israel. The COI sequence was uploaded to NCBI GenBank (accession number MN783108) and to www.boldsystems.org with BIN BOLD:AAH3049. This is the first sequence of Nerita sanguinolenta from the Mediterranean deposited in Genbank or BOLD. Blastn analysis revealed 99.04–99.36% identity (97% query cover) to two sequences of Nerita sanguinolenta specimens collected in Port Safaga, Egypt (EU732305.1 and EU732306.1). The phylogenetic position is visualized using the maximum likelihood tree (Figure 3). The ML tree shows that the collected specimen clusters with the two specimens of N. sanguinolenta from Egypt, within the Red Sea “albicilla” clade in the Theliostyla subgenus (Frey and Vermeij 2008).

Discussion The establishment status of Nerita sanguinolenta in the Mediterranean Sea was previously defined as casual (Pancucci-Papadopoulou et al. 2005; Zenetos et al. 2005) and as established in the Eastern Mediterranean basin by the European Union’s Marine Strategy Framework Directive (MSFD) based on its multiple findings in different locations (Zenetos et al. 2010). Yet, until now, this species has not been recorded in the easternmost part of the Mediterranean Sea and there is no evidence for self-perpetuating populations in the sites where it was previously recorded. In a recent comprehensive review of the marine gastropod fauna of Lebanon (Crocetta et al. 2020), N. sanguinolenta was not listed. The spatially and temporally disjunct pattern of its Mediterranean distribution may suggest multiple migration events via the Suez Canal but with no successful proliferation in

Rabi et al. (2020), BioInvasions Records 9(3): 496–503, https://doi.org/10.3391/bir.2020.9.3.06 499 First record of Nerita sanguinolenta from the Israeli Mediterranean coast

Figure 3. Molecular Phylogenetic analysis by Maximum Likelihood method. The evolutionary history was inferred by using the Maximum Likelihood method based on the General Time Reversible model (GTR+G+I). The percentage of trees in which the associated taxa clustered together is shown next to the branches (bootstrapping value). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 51 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 270 positions in the final dataset. Evolutionary analyses were conducted in MEGA7.

these locations. According to Zenetos et al. (2005), N. sanguinolenta is a suspected Lessepsian migrant rather than a true Lessepsian migrant because the species has never been documented in the Suez Canal itself. Since that report, two empty shells of N. sanguinolenta were found in Port

Rabi et al. (2020), BioInvasions Records 9(3): 496–503, https://doi.org/10.3391/bir.2020.9.3.06 500 First record of Nerita sanguinolenta from the Israeli Mediterranean coast

Said, Egypt in 2013 (Mytilineou et al. 2016), implying the possibility of Suez Canal passage. In our analysis, we molecularly confirmed the identity and Red Sea origin of a Mediterranean N. sanguinolenta. Therefore, two plausible vectors of N. sanguinolenta to the Eastern Mediterranean basin can be considered: (1) ballast water of ships traveling through Suez Canal, or (2) larval migration through the Canal, facilitated by the long pelagic larval duration (PLD) of 5–6 months characterizing this species (Waters et al. 2005). The combination of these vectors, i.e., veligers or trochophores traveling in the ballast, is also possible (Murphy et al. 2002). Guy‐Haim et al. (2017) found living gastropods among other benthic fauna in the guts and feces of Indo-Pacific invasive rabbitfish, Siganus luridus (Rüppell, 1829) and Siganus rivulatus (Forsskål & Niebuhr, 1775), and suggested that ichthyochory, live passage via fish gut, as an introduction and dispersal vector for other non-indigenous species in the Mediterranean Sea. This vector seems less probable as an explanation for the introduction of N. sanguinolenta, which inhabits the mid-high intertidal and is thus less exposed to incidental ingestion by fish. Ounifi Ben Amor et al. (2016) suggested marine agriculture as another plausible vector of N. sanguinolenta to the central Mediterranean Sea – mainly fish farms, that are abundant in sheltered areas like ports and gulfs in Tunisia. N. sanguinolenta was found attached to an artificial marine agriculture cage with another invasive gastropod, Morula aspera, an Indo-Pacific invasive species. Along the Israeli coast, fish farms are not common; nonetheless, other artificial structures prevalent in marinas and ports may have provided a shelter and facilitated the survival of this species. Nerita are gregarious herbivores that graze microalgae from rock surfaces and inhabit cracks and hidden niches in the high rocky intertidal zone. In the Red Sea, N. sanguinolenta inhabit the lower midlittoral and shallow tide pools, often forming a narrow belt on the lee of wide platforms (Ayal and Safriel 1980). In the Eastern Mediterranean, the native gregarious gastropod herbivores are two abundant topshells, turbinatus (Born, 1778) and Phorcus articulatus (Lamarck, 1822). These topshells graze on microalgae and inhabit the rocky intertidal, therefore sharing the same habitat and trophic niche with N. sanguinolenta. Evidence for competitive displacement of native biota by Lessepsian invaders have already been recorded in algae, mollusks, starfish and fish (reviewed in Galil 2007). Whether this single specimen is evidence for an upcoming invasion is unclear, given its past sporadic occurrences in the Mediterranean over the past 50 years, but should such establishment of N. sanguinolenta does occur in the intertidal rocky coast of the Mediterranean Sea it may lead to competition with the native grazers. N. sanguinolenta individuals from their native range at Ras Sudr region (Gulf of Suez, Egypt) were found at a colorful rocky surrounding that matched the color pattern of their shells (Hamdi 2011). For example, red shell individuals were found on red

Rabi et al. (2020), BioInvasions Records 9(3): 496–503, https://doi.org/10.3391/bir.2020.9.3.06 501 First record of Nerita sanguinolenta from the Israeli Mediterranean coast

granite rocks, black and white shell individuals were found on black and white rocks, and greenish individuals were found on greenish rocks. According to Rusmore-Villaume (2008), this may indicate a color-sensitive predator that can detect individuals, which differ in their color from the surrounding. Similarly, the shell color of the N. sanguinolenta that was found at Haifa beach was grey and beige, which is similar to the local rock colors in the intertidal coast, allowing an efficient camouflage. This phenotypic plasticity may provide a competitive advantage to N. sanguinolenta over the native topshells. The rapid warming of the Mediterranean Sea and especially of the Israeli coast (Rilov 2016) over the past few decades has already facilitated the introduction and propagation of non-indigenous, mostly thermophilic species (Zenetos 2019; Katsanevakis et al. 2014) and possibly the collapse of many native molluscan species (Rilov 2016). A further increase in seawater temperatures might increase the fitness of N. sanguinolenta populations and their westward spread. An investigation of the thermal performance of this species under present and predicted temperatures, in comparison to the thermal performances of native grazers, and using models to predict future distributions, can shed light on its potential establishment throughout the Mediterranean Sea.

Acknowledgements

We thank Henk Mienis for the taxonomic authentication and Oz Rittner for the specimen photographs. We thank the anonymous reviewers.

Funding Declaration

This research was partly supported by BIOMOD project funding (to T.G.H. and G.R.) from the Ministry of Science & Technology of the State of Israel and Federal Ministry of Education and Research (BMBF), Germany.

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Supplementary material The following supplementary material is available for this article: Table S1. Geo-referenced Nerita sanguinolenta records. Table S2. Nerita species and outgroup included in the phylogenetic analysis. This material is available as part of online article from: http://www.reabic.net/journals/bir/2020/Supplements/BIR_2020_Rabi_etal_SupplementaryMaterial.xlsx

Rabi et al. (2020), BioInvasions Records 9(3): 496–503, https://doi.org/10.3391/bir.2020.9.3.06 503