BioInvasions Records (2021) Volume 10, Issue 1: 136–148

CORRECTED PROOF

Research Article An outbreak of perna (Linnaeus, 1758) (, , ) in the Eastern Mediterranean

Jacob Douek1, Guy Paz1, Kfir Gayer2, Moti Mendelson3, Baruch Rinkevich1 and Bella S. Galil4,* 1Israel Oceanographic & Limnological Research, National Institute of Oceanography, Tel Shikmona, 3108001, 2School of Zoology, The George S. Wise Faculty of Life Sciences, and the Steinhardt Museum of Natural History, Tel Aviv University, Tel Aviv, 69978, Israel 33/A HaNoter St., Kiryat Hayim 26307, Israel 4Steinhardt Museum of Natural History, Tel Aviv University, Tel Aviv 69978, Israel Author e-mails: [email protected] (JD), [email protected] (GP), [email protected] (KG), [email protected] (MM), [email protected] (BR), [email protected] (BSG) *Corresponding author

Citation: Douek J, Paz G, Gayer K, Abstract Mendelson M, Rinkevich B, Galil BS An outbreak of the widely invasive mytilid is reported off Israel, (2021) An outbreak of Perna perna (Linnaeus, 1758) (Mollusca, Bivalvia, long after its previously documented ephemeral occurrence in the mid-1960s. No Mytilidae) in the Eastern Mediterranean. contemporary populations are known east of the Gulf of Tunis, in the central BioInvasions Records 10(1): 136–148, . Four molecular markers, the mitochondrial cytochrome c oxidase https://doi.org/10.3391/bir.2021.10.1.15 subunit I (COI), ribosomal 18S, 28S subunits (18S rRNA, 28S rRNA) and histone Received: 17 October 2020 H3 have been used to validate samples identities. COI haplotype diversity suggests Accepted: 26 December 2020 that the Israeli population is highly polymorphic, originating from multiple propagules. Published: 31 December 2020 The present extensive beds in Haifa Bay, home to Haifa port, may point to vessel- Thematic editor: Stelios Katsanevakis transported introduction. The dense beds of P. perna may change intertidal habitats and their communities, and cause fouling of coastal infrastructure (e.g. marine Copyright: © Douek et al. outfalls, seawalls, jetties), as well as offshore infrastructure (e.g. offshore terminals, This is an open access article distributed under terms of the Creative Commons Attribution License energy production platforms), and may incur heavy maintenance costs. (Attribution 4.0 International - CC BY 4.0). Key words: invasive alien species, , molecular markers, genetic diversity, OPEN ACCESS. Levant Sea

Introduction The widely invasive mytilid mussel Perna perna (Linnaeus, 1758) forms dense beds intertidally on hard substrates, natural and man-made, providing multilayered habitats for both sessile and mobile epifauna (Berry and Schleyer 1983; Lambert and Steinke 1986; Jacobi 1987; Cole and McQuaid 2011). When present in large numbers the may impose significant ecosystem changes (Hicks and Tunnell 1995). The species is considered indigenous to the Red Sea, eastern and southwestern , and established non-indigenous to the Gulf of Mexico, the Atlantic coast of , southern India, and Sri Lanka (Gardner et al. 2016). Its populations in the western Mediterranean and the adjacent Atlantic coast are considered cryptogenic (Fofonoff et al. 2018).

Douek et al. (2021), BioInvasions Records 10(1): 136–148, https://doi.org/10.3391/bir.2021.10.1.15 136 Outbreak of Perna perna off Israel

Monterosato (1878: 65) listed P. perna (as Mytilus pictus, Born = M. africanus, Chemnitz) from Algeria, southern Spain and . Pérès and Picard (1964) reported the species from the Alboran Sea (Motril, Spain, Morocco and Algeria), and Zaouali (1973) documented its presence in the vicinity of ports in Tunisia (, and the Gulf of Tunis). The pattern of occurrence in the vicinity of ports has been further supported with records from Toulon, France (Mars 1965), between Aspra and Capo Zafferano, near , Sicily, Italy (Buccheri and Palisano 1976), and Malta (live specimens in Valletta’s Grand Harbour, shells in Marsaxlokk Bay) (Cachia et al. 2004), though no live specimens have been collected recently (P. Schembri pers. comm.). In the port of Trieste, Italy, live specimens were collected from an oil-drilling platform arriving from Senegal (De Min and Vio 1998). No living specimens of P. perna have been collected in Israel in the past 55 years. In 1965, live and freshly dead specimens were collected near the Reading power station, Tel Aviv, and a single valve was collected off Akhziv in 1982; listings from Akko and Kiryat Haim, Israel, and Bardawil, Egypt (Barash and Danin 1992) remain unconfirmed (Mienis 2019). The population died off naturally as by the late 1960s no P. perna, living individuals or shells, occurred in the vicinity of the Reading power station and along the beaches of Tel Aviv (BSG unpublished). In the present study, we employed the analyses of four molecular markers, the mitochondrial cytochrome c oxidase subunit I (COI), ribosomal 18S, 28S subunits (18S rRNA, 28S rRNA) and histone H3 to validate the taxonomic identity of a mytilid mussel newly abundant off the Israeli coast, its haplotype diversity, and to assess the population’s possible source of invasion. Our results suggest that the Israeli population of P. perna is highly polymorphic, originating from multiple propagules. We provide preliminary documentation of its recent outbreak (Figure 1) and consider possible vectors and impacts.

Materials and methods Study site and sampling Identification is based on live-collected material (Figure 1). A total of ten bivalves were collected from intertidal rocks on the Mediterranean coast of Israel (Figure 2). Seven bivalves were sampled in Haifa Bay (32°49′27″N; 35°2′12″E, intertidal, 25 August 2020) and tagged as AP 112–118, and three bivalves were sampled from Neurim, (32°21′57″N; 34°51′26″E, intertidal rocks, 5 September 2020 and tagged as AP 121–123. The water temperature in September 2020 was 30.2–31.4 °C (https://isramar.ocean.org.il/isramar 2009/Hadera/default.aspx). The specimens collected in Haifa Bay were preserved in 70% EtOH, and the Neurim specimens were transported freshly frozen to the National Institute of Oceanography, Haifa, kept frozen until sampling, and then fixed

Douek et al. (2021), BioInvasions Records 10(1): 136–148, https://doi.org/10.3391/bir.2021.10.1.15 137 Outbreak of Perna perna off Israel

Figure 1. Subtidal Perna perna bed off Havazelet HaSharon, Israel. Photo: K. Gayer.

Figure 2. Sites along the Israeli Mediterranean coast where Perna perna specimens were collected recently (Haifa Bay, Neurim, Havazelet HaSharon), and sites of earlier (1960s) collections (confirmed: Tel Aviv, Akhziv; unconfirmed: Akko, Kiryat Haim).

Douek et al. (2021), BioInvasions Records 10(1): 136–148, https://doi.org/10.3391/bir.2021.10.1.15 138 Outbreak of Perna perna off Israel

Table 1. Primers sequences for the COI, 18S rRNA, 28S rRNA and histone H3 genes. Genes Primer IDs Primer sequences References HCO1490 5’-GGTCAACAAATCATAAAGATATTGG-3’ COI Folmer et al. 1994 LCO2180 5’-TAAACTTCAGGGTGACCAAAAAATCA-3’ 28S C1 5’-ACCCGCTGAATTTAAGCAT-3’ 28S Lê et al. 1993 28S D2 5’-TCCGTGTTTCAAGACGGG-3’ H3 F1 5’-ATGGCTCGTACCAAGCAGACVGC-3’ Histone H3 Colgan et al. 1998 H3 R1 5’-ATATCCTTRGGCATRATRGTGAC-3’ 18S a 5’-AACCTGGTTGATCCTGCCAGT-3’ 18S Medlin et al. 1988 18S b 5’-GATCCTTCTGCAGGTTCACCTAC-3’

in 70% EtOH. In the laboratory, specimens were tagged, photographed and a tissue sample taken for DNA extraction. The voucher specimens are preserved in 70% EtOH and deposited at the Steinhardt Museum of Natural History, Tel Aviv University. A P. perna bed off Havazelet HaSharon (32°21′39″N; 34°51′17″E, depth 60–100 cm, 7 October 2020) was sampled quantitatively. The water temperature in early October 2020 was 28.4–28.6 °C (https://isramar.ocean.org.il/isramar2009/Hadera_CTD/). All P. perna specimens within six randomly selected quadrats (45 cm2 frame) were counted by snorkeling; 49 individuals were randomly collected, blotted dry and weighed, and their length measured.

DNA extraction DNA isolation was performed using Phenol/chloroform extraction according to Douek et al. (2002). The extracted DNA was resuspended in 50 μl of double-distilled water (DDW) and further diluted 1:20 in DDW for the PCR amplification.

PCR amplification The PCR amplification conditions for the mitochondrial cytochrome c oxidase subunit I (COI) gene, 18S rRNA, 28S rRNA and histone H3 nuclear genes were performed as follows (a different reaction for each gene): One μl of diluted DNA (1:20) from each sample was added to a reaction mixture containing 5 μM of each of the forward and reverse primers (Table 1) and 30 μl of a Taq PCR MasterMix (Tiangen, Beijing, China), for a total solution volume of 60 μl. The thermal profile was 95 °C for 2 min followed with 35 cycles of 95 °C for 60 sec, annealing temperature 45 °C for 60 sec and 72 °C for 60 sec, and ended with one cycle of 72 °C for 10 min. The PCR products were screened on 1.0% TAE agarose gel and 50 μl of the successful products were sent for sequencing (Macrogen Inc, Amsterdam, The Netherlands) employing the same primers used for the PCR.

Sequence analyses Forward and reverse sequences of each sample ware aligned and corrected using BioEdit (Hall 1999) and were compared to the NCBI GenBank sequences using BLASTN as to the BOLD system for species identification,

Douek et al. (2021), BioInvasions Records 10(1): 136–148, https://doi.org/10.3391/bir.2021.10.1.15 139 Outbreak of Perna perna off Israel

followed by downloading all sequences of this species available in the NCBI GenBank. All sequences were aligned and compared using BioEdit and ClustalX version 2.0 (Larkin et al. 2007). Haplotype analysis and haplotype diversity were calculated using DNAsp ver 6.12.03 (Rozas et al. 2017). Estimates of evolutionary divergence between sequences and maximum likelihood phylogenetic trees were inferred by using the Maximum Likelihood method and Tamura 3-parameter model using MEGA version X (Kumar et al. 2018), employing the best-fit substitution models assessed using ModelTest implemented in MEGAX. Haplotype network was drawn using Haplotype Viewer (http://www.cibiv.at/~greg/haploviewer) based on neighbor joining tree (ClustalX) and multiple alignment (BioEdit).

Results The cytochrome c oxidase subunit I sequences (COI) assigned the 10 bivalves collected in Haifa and Neurim as Perna perna with 97–100% identity to P. perna clades 1 and 2 (Figure 3a, c) from the (KY454031.1, García-Souto et al. 2017), Venezuela (DQ343598.1, Blair et al. 2006), South Africa, and Mozambique (KC691990.1, KC692009.1, Cunha et al. 2014) (Figure 3a, b). The P. perna sequences further showed about 94% similarity to P. indica Kuriakose & Nair, 1976 (FJ428753.1, Thankakkon and Edward 2013) (actually misidentified P. perna, see discussion), and lower similarity, 83% and 81% to P. canaliculus and P. viridis, respectively. Further analysis of the 10 Israeli P. perna COI sequences revealed high polymorphism as consisting of 9 haplotypes. The two identical samples are AP 112 Haifa and AP 121 Neurim. The diversity among the Israeli samples ranged up to 0.03 (Figure 3b) and correlated with the diversity between the P. perna clades 1, 2 and 3 (Figure 3c). Then, we employed a COI haplotype analysis on the 10 Israeli samples comparing them to 1048 global P. perna COI sequences available in the NCBI GenBank. Results revealed a total number of 229 distinct COI haplotypes in which two Israeli COI haplotypes from Haifa (AP 114 Haifa and AP 115 Haifa), emerged as unique haplotypes. The other 7 COI haplotypes were distributed between existing COI haplotypes (Figure 4) from locations around the world (Brazil, Morocco, Namibia, New Zealand, Senegal, South Africa, Spain, Tunisia, and Venezuela; Blair et al. 2006; Zardi et al. 2007; Wood et al. 2007; Pochon et al. 2013; Alves et al. 2012; Teske et al. 2012; Cunha et al. 2014; de Oliveira et al. 2017; Micklem et al. 2016; García-Souto et al. 2017; Lourenço et al. 2017; Supplementary material Table S1) and among clades 1 and 2 as defined by Cunha et al. (2014, Figure 3a). The AP 112 Haifa COI sequence and the AP 121 Neurim COI sequence are identical to 119 COI sequences of samples collected from South America, southern and North Africa. The AP 116 Haifa sequence is identical to 441 COI sequences collected primarily from southern Europe and northern Africa. The AP 123 Neurim sequence is identical to 64 COI sequences from

Douek et al. (2021), BioInvasions Records 10(1): 136–148, https://doi.org/10.3391/bir.2021.10.1.15 140 Outbreak of Perna perna off Israel

Figure 3. a. A Dendrogram (Maximum Likelihood and Tamura 3-parameter model in MEGAX) of COI sequences of the Israeli Perna perna. P. perna samples from the Atlantic Ocean (KY454031.1), Venezuela (DQ343598.1) and South Africa, Mozambique (KC691990.1, KC692009.1) and P. indica (FJ428753.1) as an outgroup. The tree with the highest log likelihood (−1102.44) is shown. b. Evolutionary divergences between the sequences in a, revealed by disparity (percentages). c. Estimates of evolutionary divergence over sequence pairs between the clades.

South America, South Africa and New Zealand (Table S1, Figure 4). The 10 sampled Israeli specimens further revealed a high haplotype diversity (H = 0.978; Table 2), a genetic pattern identified in most native and introduced P. perna populations worldwide (Table 2). The 28S ribosomal genes and histone 3 (H3) gene sequences revealed just a single haplotype per gene for the 10 Israeli samples and the 18S revealed 5 haplotypes with a single base change between the sequences (GenBank accession No. in Table S2). These genes were not used for further analyses.

Douek et al. (2021), BioInvasions Records 10(1): 136–148, https://doi.org/10.3391/bir.2021.10.1.15 141 Outbreak of Perna perna off Israel

Figure 4. Worldwide Perna perna COI haplotype network, listing all 229 haplotypes (and considerable numbers of specimen/haplotype) as base differences between haplotypes. The 9 Israeli haplotypes are marked.

Table 2. Perna perna COI. Number of haplotypes and haplotype diversity at different sampling locations (H = haplotype diversity; VarHd = haplotype diversity variance; NA – data not available). Number of Number of GenBank accession No. Location H VarHd Source Sequences haplotypes MW Israel 10 9 0.978 0.00292 This study KY51 Different locations 1 730 81 0.611 0.00038 Lourenço et al. 2017 KC Different locations 2 30 20 0.952 0.00069 Cunha et al. 2014 KY45 Atlantic Ocean 2 2 1.000 0.25 García-Souto et al. 2017 DQ35 South Africa 140* 49 NA NA Zardi et al. 2007 JX South Africa 158 54 0.847 0.00063 Teske et al. 2012 KT South Africa 37** 11 NA NA Micklem et al. 2016 HG New Zealand 1 1 0.000 0 Pochon et al. 2013 DQ34 Venezuela 3 3 1.000 0.07407 Blair et al. 2006 DQ91 Different locations 3 8 5 0.893 0.00736 Wood et al. 2007 JQ Brazil 2 2 1.000 0.25 Alves et al. 2012 KP Brazil 158*** 44 NA NA de Oliveira et al. 2017 KU South India 10 2 0.356 0.02532 Gardner et al. 2016 Total sequences available All locations 1058 229 0.806 0.00014 1 Tunisia, Algeria, Morocco, Spain, Portugal, Western Sahara 2 Tunisia, South Africa, Angola, Namibia, Mozambique, Oman. 3 Venezuela, Brazil *140 sequences, only 49 represented sequences (haplotypes) are available. **37 sequences, only 11 represented sequences (haplotypes) are available. ***158 sequences, only 44 represented sequences (haplotypes) are available.

Taxonomy, measurements, photos, the contiguous COI sequences and their trace files have been uploaded to BIM project at BOLD system database at http://v4.boldsystems.org/ (Table S2). The vouchers have been preserved in 70% EtOH and deposited at the Steinhardt Museum of Natural History, Tel Aviv University with museum voucher IDs (Table S2). The numbers of P. perna in the quadrats sampled off Havazelet HaSharon averaged 66.3 specimens per 0.2025 m2 (45, 53, 62, 67, 80, 97 specimens per quadrat). The average (± SD) shell length was 41.6 ± 7.0 mm, shell weight 5.9 ± 1.5 gr.

Douek et al. (2021), BioInvasions Records 10(1): 136–148, https://doi.org/10.3391/bir.2021.10.1.15 142 Outbreak of Perna perna off Israel

Discussion Policy, regulations, and management of an invasive non-indigenous species are dependent for their success on ascertaining its taxonomic identity and thus non-indigenous status, its transfer mechanisms and its potential effects in the novel environment. Perna perna has had a confused subject to nomenclatural errors (Siddall 1980), evidenced by a long list of synonymies (http://www.marine species.org/aphia.php?p=taxdetails&id=140483), complicating and obfuscating the geographical distribution of the species. The availability of molecular tools, beginning with DNA-based genetic analyses, enabled research into the geographic origins of species (Darling et al. 2017). Perna perna, originally described from the Straits of Magellan, South America (as Mya perna Linnaeus, 1758: 671), was long assumed to be indigenous to the Atlantic coast of South America, notwithstanding its near cosmopolitan distribution and association with harbors. Recently, a re-appraisal of zooarcheological data suggested it is a human-mediated introduction, possibly dating back to the 15th century African slave-trade shipping (de Souza et al. 2003; Silva et al. 2018), a premise corroborated by studies of haplotype diversity of the Brazilian and African populations (de Oliveira et al. 2017). Molecular tools argue that P. perna (erroneously described as P. indica) was introduced to southern India from Oman more than 100 years ago via maritime trade (Gardner et al. 2016). Perna perna was identified in fouling biota of a semi-submersible drilling rig, previously stationed in South Africa, and cleansed in-water in Tasman Bay, New Zealand. Specimens were found to be viable 2 months later when the defouled material was dredged up (Hopkins and Forrest 2010; Hopkins et al. 2011). It was likely introduced to the Corpus Christi Bay area, Texas, U.S.A., with vessels of a Venezuelan oil refinery (Hicks and Tunnell 1993). The live P. perna specimens of the earlier ephemeral introduction in Israel were collected on the outskirts of Tel Aviv, near the anchorage for fuel tankers that supplied the heavy fuel oil for the operation of the Reading power station. At the time, Israel imported the oil from Venezuela, South America (https://fs. knesset.gov.il/5/Committees/5_ptv_420046.pdf, viewed 14 October, 2020). The present extensive P. perna beds in Haifa Bay, home to Haifa port, fit the pattern of a vessel-transported introduction. However, the 10 specimens from Israel revealed a high haplotype diversity and 9 COI haplotypes (two are private). The non-private seven haplotypes belong to clades 1 and 2 (Cunha et al. 2014) of P. perna (97–100% identity), and all showed 94% similarity to P. perna, currently considered as the fourth clade, originating from the Oman region (Cunha et al. 2014). This clade has been introduced to southern India (Gardner et al. 2016), and was mistakenly described as P. indica (Thankakkon and Edward 2013). The high haplotype diversity of the samples collected off the Israeli coast (9 haplotypes in 10 samples), and their similarity to 7 different P. perna

Douek et al. (2021), BioInvasions Records 10(1): 136–148, https://doi.org/10.3391/bir.2021.10.1.15 143 Outbreak of Perna perna off Israel

haplotypes collected from widely spread locations (Table S1), suggest multiple propagules (from one or more sources) and precludes identification of the origin of this introduction. Mussels commonly serve as ecosystem engineers in intertidal habitats, supporting diverse communities by increasing habitat complexity and providing food and shelter (Arribas et al. 2014). An invading mussel may impact at least some features of the community structure. In Uruguay, where P. perna invaded beds of the smaller native rodriguezii (d’Orbigny, 1842), it forms a significant part of the biomass and outnumbered B. rodriguezii at the lower intertidal (Carranza and Borthagaray 2009). In southern Brazil, the native (Stramonita spp.) prey on P. perna, and it is probably a significant food resource for crabs and fishes (Fofonoff et al. 2018). Levantine beds of intertidal mytilids underwent a rapid shift in the 1990s (Galil 2007), when the invasive Erythraean (native to the Red Sea) (P. Fischer, 1870) displaced the previously abundant native mytilid minimus (Poli, 1795). Owing to its larger size compared with the native M. minimus (max. 12 mm shell length) and B. pharaonis (max. 40 mm shell length), P. perna, if established, may cause changes to the ecology of the Levantine mussel beds. Indeed, change is already apparent: the dense beds of P. perna off the Israeli coast enhanced the population of the local (Linnaeus, 1767) (pers. obs.) (Figure 5). Contemporaneous documentation of the invasion of P. perna into the Gulf of Mexico attest to its spread with the prevailing southeasterly currents from Port Aransas, Texas, to Veracruz, Mexico, a distance of 1300 km, within four years (Hicks and Tunnell 1995). As the prevailing inner shelf and wave-induced longshore currents along the Levant coast run northwards, it would be of interest to learn whether the mussel spreads further in the Levant. The mussel attaches to intertidal rocks as well as man-made marine and coastal infrastructure (e.g. marine outfalls, seawalls, jetties, and navigation buoys) (Rajagopal et al. 2003), and offshore infrastructure (e.g. offshore terminals, oil and gas platforms) (Silveira et al. 2007; Sheehy and Vik 2010), incurring heavy maintenance costs (Hicks and Tunnell 1995). The recently approved framework for the management and planning of the Israeli maritime area (Policy Paper for Israel’s Maritime Space/Mediterranean Sea 2018) outlines plans for massively expanded littoral, shelf and off-shore maritime infrastructure (from pipelines and desalination plants to large-scale platforms for facilities and artificial islands) and coastal defence (e.g. detached breakwaters, groins), conducive to the establishment of non-indigenous (mostly) fouling species, such as P. perna (Firth et al. 2016). Recently amended regulations decree that all molluscs (excepting ) in the waters under Israeli national jurisdiction are “Protected Nature Values”, including invasive non-indigenous species (https://www.gov.il/he/departments/ news/protected_species_list, viewed 20 December 2020). As the tally of marine

Douek et al. (2021), BioInvasions Records 10(1): 136–148, https://doi.org/10.3391/bir.2021.10.1.15 144 Outbreak of Perna perna off Israel

Figure 5. Stramonita haemastoma inhabiting the subtidal Perna perna bed off Havazelet HaSharon, Israel. Photo: K. Gayer.

non-indigenous species recorded off the Mediterranean coast of Israel is upward of 450, inclusive of 150 species of molluscs (Galil et al. 2020), the current policy and governance heedlessly enhance the proliferation of non- indigenous species. The national Mediterranean monitoring program, carried out since 1978, and overseen since 2019 by an inter-ministerial board comprising representatives from government ministries, the Israel Nature and Parks Authority, the Water Authority, and environmental organizations (https:// www.gov.il/en/Departments/Guides/marine_environment_monitoring? chapterIndex=2), has yet to focus efforts on monitoring for newly introduced species, abundance and spatial distribution of established non-indigenous species, and their impacts on nature and nature contributions to people.

Douek et al. (2021), BioInvasions Records 10(1): 136–148, https://doi.org/10.3391/bir.2021.10.1.15 145 Outbreak of Perna perna off Israel

Acknowledgements

This study is part of the National Israeli Marine Barcoding project (BIM) at IOLR and was supported by the Ministry of Energy project no. 218-17-009 and by the Israeli National Monitoring program in the Mediterranean Sea. We thank Henk Mienis for his preliminary identification and advice, Prof. Patrick Schembri for information on the species presence in Malta, and Eran Greenberg for collecting and delivering the Neurim samples. BSG is deeply grateful to the library of the American Museum of Natural History, New York, for the helpfulness of its staff and access to its rich resources.

References

Alves FA, Beasley CR, Hoeh WR, da Rocha RM, Simone LR, Tagliaro C (2012) Detection of mitochondrial DNA heteroplasmy suggests a doubly uniparental inheritance pattern in the mussel Mytella charruana. Revista Brasileira de Biociências 10(2): 176–185 Arribas LP, Donnarumma L, Palomo MG, Scrosati RA (2014) Intertidal mussels as ecosystem engineers: their associated invertebrate biodiversity under contrasting wave exposures. Marine Biodiversity 44: 203–211, https://doi.org/10.1007/s12526-014-0201-z Barash A, Danin, Z (1992) Fauna Palaestina: Mollusca I. Annotated list of Mediterranean molluscs of Israel and Sinai. The Israel Academy of Sciences and Humanities, Jerusalem, 372 pp Berry PF, Schleyer MH (1983) The brown mussel Perna perna on the Natal Coast, South Africa: utilization of available food and energy budget. Marine Ecology Progress Series 13: 201–210, https://doi.org/10.3354/meps013201 Blair D, Waycott M, Byrne L, Dunshea G, Smith-Keune C, Neil KM (2006) Molecular Discrimination of Perna (Mollusca: Bivalvia) species using the Polymerase Chain Reaction and species-specific mitochondrial primers. Marine Biotechnology 8: 380–385, https://doi.org/10.1007/s10126-005-6121-y Buccheri G, Palisano G (1976) Nouvi dati sulla distribuzione geografica di Perna (Perna) picta (Born, 1780) e considerazioni sistematiche sulla specie. Conchiglie 12: 143–156 Cachia C, Mifsud C, Sammut PM (2004) The marine Mollusca of the Maltese Islands. Part four. The classes: Caudofoveata, Solenogastres, Bivalvia, Scaphopoda & Cephalopoda. Backhuys, Leiden, The Netherlands, 270 pp Carranza A, Borthagaray AI (2009) The brown mussel Perna perna in the native mussel beds of Cerro Verde (Uruguay). Marine Biodiversity Records 2: E76, https://doi.org/10.1017/ S1755267209000608 Cole VJ, McQuaid CD (2011) Broad-scale spatial factors outweigh the influence of habitat structure on the fauna associated with a bioengineer. Marine Ecology Progress Series 442: 101–109, https://doi.org/10.3354/meps09403 Colgan DJ, McLauchlan A, Wilson GDF, Livingston SP, Edgecombe GD, Macaranas J, Cassis G, Gray MR (1998) Histone H3 and U2 snRNA DNA sequences and arthropod molecular evolution. Australian Journal of Zoology 46: 419–437, https://doi.org/10.1071/ZO98048 Cunha RL, Nicastro KR, Costa J, McQuaid CD, Serrão EA, Zardi GI (2014) Wider sampling reveals a non-sister relationship for geographically contiguous lineages of a marine mussel. Ecology and Evolution 4: 2070–2081, https://doi.org/10.1002/ece3.1033 Darling JA, Galil BS, Carvalho GR, Rius M, Viard F, Piraino S (2017) Recommendations for developing and applying genetic tools to assess and manage biological invasions in marine ecosystems. Marine Policy 85: 54–64, https://doi.org/10.1016/j.marpol.2017.08.014 De Min R, Vio E (1998) Molluschi esotici nell’alto Adriatico. Annales 13: 43–54 de Oliveira MJS, Beasley CR, Barros NGV, do Socorro Marques-Silva N, de Simone LRL, Lima ES, Tagliaro CH (2017) Two African origins of naturalized brown mussel (Perna perna) in Brazil: past and present bioinvasions. Hydrobiologia 794: 59–72, https://doi.org/ 10.1007/s10750-016-3082-2 de Souza RCCL, da Costa Fernandes F, da Silva EP (2003) A study on the occurrence of the brown mussel Perna perna on the sambaquis of the Brazilian coast. Revista do Museu de Arqueologia e Etnologia 13: 3–24, https://doi.org/10.11606/issn.2448-1750.revmae.2003.109462 Douek J, Barki Y, Gateño D, Rinkevich B (2002) Possible cryptic speciation within the sea anemone Actinia equina complex detected by AFLP markers. Zoological Journal of the Linnean Society 136: 315–320, https://doi.org/10.1046/j.1096-3642.2002.00034.x Firth LB, Knights AM, Bridger D, Evans AJ, Mieszkowska N, Moore PJ, O’Connor NE, Sheehan EV, Thompson RC, Hawkins SJ (2016) Ocean sprawl: challenges and opportunities for biodiversity management in a changing world. In: Hughes RN, Hughes DJ, Smith IP, Dale AC (eds), Oceanography and Marine Biology: an annual review. Taylor & Francis, pp 193–269, https://doi.org/10.1201/9781315368597-5 Fofonoff PW, Ruiz GM, Steves B, Simkanin C, Carlton JT (2018) National Exotic Marine and Estuarine Species Information System, http://invasions.si.edu/nemesis/ (accessed 9 October 2020)

Douek et al. (2021), BioInvasions Records 10(1): 136–148, https://doi.org/10.3391/bir.2021.10.1.15 146 Outbreak of Perna perna off Israel

Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994) DNA Primers for amplification of mitochondrial Cytochrome c Oxidase Subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology 3: 294–299, https://doi.org/10.1071/ZO9660275 Galil BS (2007) Loss or gain? Invasive aliens and biodiversity in the Mediterranean Sea. Marine Pollution Bulletin 55: 314–22, https://doi.org/10.1016/j.marpolbul.2006.11.008 Galil BS, Mienis HK, Hoffman R, Goren M (2020) Non-native species along the Israeli Mediterranean coast - tally, policy, outlook. Hydrobiologia, https://doi.org/10.1007/s10750- 020-04420-w García-Souto D, Sumner-Hempel A, Fervenza S, Pérez-García C, Torreiro A, González- Romero R, Eirín-López JM, Morán P, Pasantes JJ (2017) Detection of invasive and cryptic species in marine mussels (Bivalvia, Mytilidae): A chromosomal perspective. Journal for Nature Conservation 39: 58–67, https://doi.org/10.1016/j.jnc.2017.07.005 Gardner JP, Patterson J, George S, Edward JP (2016) Combined evidence indicates that Perna indica Kuriakose and Nair 1976 is Perna perna (Linnaeus, 1758) from the Oman region introduced into southern India more than 100 years ago. Biological Invasions 18: 1375– 1390, https://doi.org/10.1007/s10530-016-1074-9 Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41: 95–98 Hicks DW, Tunnell JW (1993) Invasion of the south Texas coast by the edible brown mussel Perna perna (Linnaeus, 1758). 36: 92–94 Hicks DW, Tunnell JW (1995) Ecological notes and patterns of dispersal in the recently introduced mussel, Perna perna, in the Gulf of Mexico. American Malacological Bulletin 11: 203–206 Hopkins GA, Forrest BM (2010) Challenges associated with pre-border management of biofouling on oil rigs. Marine Pollution Bulletin 60: 1924–1929, https://doi.org/10.1016/ j.marpolbul.2010.07.015 Hopkins GA, Forrest BM, Jiang W, Gardner JP (2011) Successful eradication of a non‐indigenous marine bivalve from a subtidal soft‐sediment environment. Journal of Applied Ecology 48: 424–431, https://doi.org/10.1111/j.1365-2664.2010.01941.x Jacobi CM (1987) The invertebrate Fauna associated with intertidal beds of the brown mussel Perna perita (L.) from Santos, Brazil. Studies on Neotropical Fauna and Environment 22: 57–72, https://doi.org/10.1080/01650528709360720 Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution 35: 1547– 1549, https://doi.org/10.1093/molbev/msy096 Lambert G, Steinke TD (1986) Effects of destroying juxtaposed mussel-dominated and coralline algal communities at Umdoni Park, Natal coast, South Africa. South African Journal of Marine Science 4: 203–217, https://doi.org/10.2989/025776186784461855 Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD (2007) Clustal W and Clustal X version 2.0. Bioinformatics. 23: 2947–2948, https://doi.org/10.1093/bioinformatics/btm404 Lê HL, Lecointre G, Perasso R (1993) A 28S rRNA-based phylogeny of the gnathostomes: first steps in the analysis of conflict and congruence with morphologically based cladograms. Molecular Phylogenetics and Evolution 2: 31–51, https://doi.org/10.1006/mpev.1993.1005 Linnaeus C (1758) Systema Naturae per regna tria naturae, secundum classes, ordines, genera, species, cum characteribus, differentiis, synonymis, locis. Editio decima, reformata [10th revised edition], Laurentius Salvius: Holmiae. vol. 1., 824 pp, https://doi.org/10.5962/bhl.title.542 Lourenço CR, Nicastro KR, McQuaid CD, Chefaoui RM, Assis J, Taleb MZ, Zardi GI (2017) Evidence for range wide panmixia despite multiple barriers to dispersal in a marine mussel. Scientific Reports 7: 1–16, https://doi.org/10.1038/s41598-017-10753-9 Mars P (1965) Faune marine des Pyrénées-Orientales: Mollusques aplacophores, polyplacophores, scaphopodes et bivalves. Masson et Cie, Paris, France, 156 pp Medlin L, Elwood HJ, Stickel S, Sogin ML (1988) The characterization of enzymatically amplified eukaryotic 16S-like rRNA-coding regions. Gene 71: 491–499, https://doi.org/10. 1016/0378-1119(88)90066-2 Micklem JM, Griffiths CL, Ntuli N, Mwale M (2016) The invasive Asian green mussel in South Africa: all that is green is not viridis. African Journal of Marine Science 38: 207–215, https://doi.org/10.2989/1814232X.2016.1180323 Mienis HK (2019) On the sudden appearance and disappearance of Perna perna on the Mediterranean coast of Israel (Mollusca, Bivalvia, Mytilidae). Triton 38: 1–3 Monterosato T (1878) Enumerazione e sinonimia delle conchiglie mediterranee. Giornale di Scienze Naturali ed Economiche 13: 61–115 Pérès JM, Picard J (1964) Nouveau manuel de bionomie benthique. Recueil des Travaux de la Station marine d’Endoume 31(47): 5–137 Pochon X, Bott NJ, Smith KF, Wood SA (2013) Evaluating detection limits of next-generation sequencing for the surveillance and monitoring of international marine pests. PLoS ONE 8: e73935, https://doi.org/10.1371/journal.pone.0073935

Douek et al. (2021), BioInvasions Records 10(1): 136–148, https://doi.org/10.3391/bir.2021.10.1.15 147 Outbreak of Perna perna off Israel

Policy Paper for Israel’s Maritime Space/Mediterranean Sea (2018) 2nd stage report - Planning for maritime space. Planning Administration, Israel Ministry of finance. 155 pp [in Hebrew] Rajagopal S, Venugopalan VP, Van der Velde G, Jenner HA (2003) Response of fouling brown mussel, Perna perna (L.), to chlorine. Archives of Environmental Contamination and Toxicology 44: 369–376, https://doi.org/10.1007/s00244-002-2098-y Rozas J, Ferrer-Mata A, Sánchez-DelBarrio JC, Guirao-Rico S, Librado P, Ramos-Onsins SE, Sánchez-Gracia A (2017) DnaSP 6: DNA Sequence Polymorphism Analysis of Large Data Sets. Molecular Biology and Evolution 34: 3299–3302, https://doi.org/10.1093/molbev/msx248 Sheehy DJ, Vik SF (2010) The role of constructed reefs in non-indigenous species introductions and range expansions. Ecological Engineering 36: 1–11, https://doi.org/10.1016/j.ecoleng.2009. 09.012 Siddall SE (1980) A clarification of the Perna (Mytilidae). Bulletin of Marine Science 30(4): 858–870 Silva EP, Souza RC, Lima TA, Fernandes FC, Macario KD, Netto BM, Alves EQ, Carvalho C, Aguilera O, Duarte MR (2018) Zooarchaeological evidence that the brown mussel (Perna perna) is a bioinvader of coastal Brazil. The Holocene 28: 1771–1780, https://doi.org/10. 1177/0959683618788670 Silveira NG, de Souza RCCL, da Costa Fernandes F, Silva EP (2007) Occurrence of Perna perna, Modiolus carvalhoi (Mollusca, Bivalvia, Mytilidae) and Megabalanus coccopoma (Crustacea, Cirripedia) off Areia Branca, Rio Grande do Norte state, Brazil. Biociências 14(1): 89–90 Teske PR, Papadopoulos I, Barker NP, McQuaid CD (2012) Mitochondrial DNA paradox: sex- specific genetic structure in a marine mussel-despite maternal inheritance and passive dispersal. BMC Genetics 13: 1–6, https://doi.org/10.1186/1471-2156-13-45 Thankakkon S, Edward P (2013) Molecular phylogenetic analysis of two closely related marine Indian mussels of genus Perna (Philipsson, 1788) based on mitochondrial (COI) and nuclear (ITS) genes. Journal of Aquatic Biology & Fisheries 1: 123–139 Wood AR, Apte S, MacAvoy ES, Gardner JP (2007) A molecular phylogeny of the marine mussel genus Perna (Bivalvia: Mytilidae) based on nuclear (ITS1&2) and mitochondrial (COI) DNA sequences. Molecular Phylogenetics and Evolution 44: 685–698, https://doi.org/ 10.1016/j.ympev.2006.12.019 Zaouali J (1973) Note sur la presence de Perna perna L.(= Mytilus africanus Chemnitz) dans la region de Bizerte (Tunisie). Bulletin de l’Institut national scientifique et technique d’océanographie et de pêche 2: 637–642 Zardi GI, McQuaid CD, Teske PR, Barker NP (2007) Unexpected genetic structure of mussel populations in South Africa: indigenous Perna perna and invasive Mytilus galloprovincialis. Marine Ecology Progress Series 337: 135–144, https://doi.org/10.3354/meps337135

Supplementary material The following supplementary material is available for this article: Table S1. Samples of Perna perna collected in Israel. Table S2. Sample IDs of Perna perna sequences from Israel. This material is available as part of online article from: http://www.reabic.net/journals/bir/2021/Supplements/BIR_2021_Douek_etal_Table_S1.pdf http://www.reabic.net/journals/bir/2021/Supplements/BIR_2021_Douek_etal_Table_S2.pdf

Douek et al. (2021), BioInvasions Records 10(1): 136–148, https://doi.org/10.3391/bir.2021.10.1.15 148