Mediterranean non indigenous species at the start of the 2020s: recent changes

The current amendments to the Mediterranean marine Non-Indigenous Species (NIS) inventory for the period 2017-2019 are the result of a continuous literature search and update of the Hellenic Centre for Marine Research (HCMR) offline database. They take into account recent findings, previously missed records, back-dated records based on the re-examination of existing material or phylogenetic studies and changes in nomenclature. During the period 2017-2019, 70 new species were added to the inventory of established species, 25 that had escaped our attention in the past and 23 newly introduced, which have already established self-sustaining populations. Meanwhile, 22 species previously known only with casual records have established viable populations and a total of 36 species have expanded their distribution into new Marine Strategy Framework Directive regions, primarily the Central Mediterranean and the Adriatic Sea. Intensified research efforts, prompted by the reporting obligations created by recent legislation, complemented by ever expanding networks and initiatives involving citizen scientists have certainly contributed to higher rates of discovery of alien species presences. However, the expansion of tropical and sub-tropical species into the cooler waters of the Aegean, the Adriatic and the western Mediterranean indicates that the warming of Mediterranean waters due to climate change is also facilitating the geographic expansion of NIS in the region. The rate of new introductions in this 3-year period is 8 species per year for the whole Mediterranean, without taking into account casual records or species with reporting lags. Only 4 species per year enter through the Suez Canal, while a considerable number of species are introduced through shipping vectors and the aquarium trade. Acknowledging the dynamic nature of invasions and the uncertainty inherent in compiling check lists, we consider the present work as the most accurate and up-to-date NIS list to inform policy, management and decision-making.


Introduction
Alien species inventories constitute a fundamental first step and a very important tool for the implementation of relevant policies and the delineation of management approaches (Vanderhoeven et al. 2017). They inform prioritization processes, e.g. Horizon Scanning (Roy et al. 2015;Tsiamis et al. 2020), the assessment of trends in introduction pathways (Korpinen et al. 2019) and the evaluation of existing management practices through the calculation of suitable indicators (Armon and Zenetos 2015;Rabitsch et al. 2016).
As management efforts/policy initiatives to tackle alien and invasive species are intensified at the global and regional level (CBD (Convention on Biological Diversity) 2011; IMAP 2017), it is imperative that such inventories are regularly updated and remain current, especially since the introduction rate of new alien species globally does not appear to level off (Seebens et al. 2017 but see Zenetos 2019 for the Mediterranean), while their spread into new areas is facilitated by increasing volumes of trade and global transport networks (Latombe et al. 2017;Seebens et al. 2015).
The inclusion of alien species descriptors and indicators in policy instruments has created reporting obligations at the national level (e.g. see Tsiamis et al. 2019) and triggered a surge of scientific activity dedicated to their detection, quantification, the exploration and mitigation of their impacts. Indicatively, the financial investments for alien species research and management have increased significantly in Europe since the early 1990s (Scalera 2010) and national monitoring schemes that cover NIS are in place in many Member States (ICES 2019). Additionally, Citizen Science (i.e. the involvement of the public in the production of scientific data - McKinley et al. 2017) has emerged as a powerful contributor to the early detection of new alien species as well as the surveillance of established invaders (Groom et al. 2019;Giovos et al. 2019) and novel genetic methods are more routinely employed, helping to clarify uncertainties with respect to species' identities and geographic origins (Bayha et al. 2017;Simon et al. 2019;Viard et al. 2019). As a result of this, the time lag between the first detection of a new alien species and the publication of the corresponding record has decreased in recent years, largely aided by the willingness of scientific journals to publish such biodiversity observations . The development of open data infrastructures, such as the European Alien Species Information Network (EASIN) (https://easin.jrc.ec.europa.eu/easin), that assure quality control of alien species information (Tsiamis et al. 2016) further enhances accessibility and analysis of the available information. The above notwithstanding, there are still information gaps and validation problems with alien species lists, that can only be overcome with the collaboration of local and taxonomic experts and the continuous update of regional/national databases. One such effort for Mediterranean marine alien species resulted in the critical revision and massive update of the alien species inventory for the Mediterranean Sea ). In the current review, we build on that study and report recent changes in the marine Mediterranean xenodiversity since 2016, aiming to present a comprehensive and up-to-date NIS checklist at the closing of the previous decade.

Methodology
The work of Zenetos et al. (2017), including the published species list as well as the underlying database (HCMR offline database), forms the baseline for the current work. Updates are based on an extensive literature search, complemented by personal communications with local experts where needed, to verify alien status and establishment success.
Updates presented herein include: i Each species presented in the results section was assigned its most likely primary pathway of introduction or secondary spread, as appropriate. Pathway descriptions follow the Convention on Biological Diversity (CBD) definitions (CBD (Convention on Biological Diversity) 2014) and the relevant abbreviations are as follows: CON = TRANSPORT-CONTAMINANT, including Contaminant on animals (except parasites, species transported by host/vector) and Parasites on animals (including species transported by host and vector), COR = CORRIDOR: Interconnected waterways/basins/seas (in the current context this refers to the Suez Canal), ESC = ESCAPE FROM CONFINEMENT, referring to the accidental escape or intentional release of aquarium species (see IUCN (International Union for Conservation of Nature) 2017), T-S: TRANSPORT-STOWAWAY, including Ship/boat ballast water, Ship/boat hull fouling and Other means of transport, UNK = Unknown.
Mediterranean subregions are defined as per the MSFD delineation (Jensen et al. 2017), namely (a) the Western Mediterranean Sea (WMED); (b) the Ionian Sea and the Central Mediterranean Sea (CMED); (c) the Adriatic Sea (ADRIA); and (d) the Eastern Mediterranean Sea (EMED), encompassing the Aegean and Levantine basins. Country names are abbreviated according to the ISO 3166-1 alpha-2 code system into a two-letter acronym.

Results-discussion
Excluding Foraminifera, a total of 666 established marine alien species are reported in the Mediterranean by December 2019 (Additional file 1). The continuous literature search and update of the HCMR offline database since December 2016 resulted in a total of 70 additions to the inventory of established marine alien species for the Mediterranean Sea (Additional file 1) and 112 new species in the Mediterranean NIS if casual records are considered. At the same time 16 species were deleted from the list (Additional file 2).

Species to be removed from the established NIS inventory
The presence of the bivalve mollusk Pseudochama corbierei (Jonas, 1846) in Greece was disputed by Crocetta et al. (2017). Its record in Israel is based on one shell from the beach (Barash and Danin 1972), while in Cyprus the record is not substantiated by a picture (Demetropoulos 1971). Therefore, the species is removed altogether from the list of Mediterranean NIS.
A new scyphozomedusa, sampled as bycatch in the Adriatic, was initially named by Piraino et al. (2014) as a new species, Pelagia benovici, and later placed in a new genus Mawia by Avian et al. (2016). Subsequently, Mawia was observed and sampled in several locations of the northern Adriatic and Ionian Sea (Chartosia et al. 2018). Bayha et al. (2017), in a comprehensive phylogenetic analysis of Pelagiidae, included two unidentified specimens collected from the west coast of Africa (Senegal), which genetic alanysis conclusively grouped together with M. benovici samples from the Adriatic. While this provides a potential source region for the species' introduction into the Mediterranean, the same study raises systematic questions about the phylogeny of the various species in the family and the erection of the new genus Mawia. Based on the discovery of paraphyly in the genus Chrysaora, Bayha et al. (2017) postulate that, in order to accept the validity of Mawia, as well as previously established Pelagia and Sanderia, additional genera would have to be erected within Pelagiidae in order to maintain monophyly of these generic groupings. Therefore, the species is assigned the Data Defficient status.
According to Faulwetter et al. (2017), the increasing discovery of cryptic species through molecular methods is creating uncertainty as to the diversity and distribution of polychaete species worldwide. Several of the of non-native polychaete species recorded in the Mediterranean were proven to be a complex of cryptic and pseudo-cryptic species. In these cases, it is likely that the name refers to a complex of cryptic species and Mediterranean material could belong to an overlooked native species, e.g Eurythoe complanata (Pallas, 1766), Lumbrineris perkinsi Carrera-Parra, 2001 that are now assigned to the cryptogenic category (Langeneck et al. 2020),  described from the Italian coast Streblosoma pseudocomatus which corresponds to Streblosoma comatus (Grube, 1859). Until there are further studies S. comatus is moved to the cryptogenic status.
As an example, Simon et al. (2019) based on phylogenetic analysis of DNA concluded that S. kraussii is a warm temperate/subtropical intertidal species restricted to South African coasts. It belongs to a globally distributed complex including some tropical fouling and invasive species. Yet, it is believed that the records of S. kraussii from the Levantine might actually correspond to other species/strains of unknown identity (Elena K. Kupriyanova, pers. commun) but surely an alien for the Mediterranean.

Missing records
Of these, 25 refer to species for which records pre-2017 had escaped our attention (missing records in Table 1). All these species are considered established but 9 of them are only established locally, i.e. they have documented populations in one location. The discovery of many of the missing records is owed to Servello et al. (2019), who confirmed the establishment of already recorded species in Italian waters, as well as to Abd El-Rahman (2005) who studied the zooplankton of the Mediterranean coast of Egypt and confirmed the establishment of alien copepods. Other notable mentions include Paracartia grani Sars G.O., 1904, a species that was previously assigned cryptogenic status but has now been reinstated as alien (see Tsiamis et al. 2019) and the parasite Livoneca redmanii Leach, 1818, which was found parasitizing Argyrosomus regius in Egyptian waters (Rania and Rehab 2015).
Cassiopea andromeda (Forskål, 1775), a long-established species in the Mediterranean (e.g. see Maggio et al. 2019), is included here to rectify an omission in the Zenetos et al. (2017) inventory. The same applies for the copepod Arietellus pavoninus Sars G.O., 1905, already established in the eastern Mediterranean, which was included in Zenetos et al. (2010) but was missed in Zenetos et al. (2017). If records of foraminifera were included in this list, the number would be much higher, however we opted to exclude them from the current work as the particular taxonomic group is due for a revision of its native/ alien species in a comprehensive article (Langer, in preparation).    (Krupnik et al. 2018). The same study reported U. chaugulii for the first time from the Mediterranean in 2015-2016, although the authors point out that, due to the high phenotypic plasticity of Ulva species, earlier records of U. linza Linnaeus (see Einav and Israel 2008) may represent one or both of these species (Krupnik et al. 2018). The establishment success of both U. tepida and U. chaugulii is currently unknown. Concerning B. diegensis, Viard et al. (2019), based on genetic studies and a re-examination of GenBank sequences from various Mediterranean locations, propose that the presence of this species may have been strongly underestimated in the Mediterranean region, as it is commonly confused for Botrylloides leachii (Savigny, 1816), such that its year and location of first introduction cannot be easily ascertained. Cotylorhiza erythraea on the other hand, has been observed off the Israeli coast since 2003 but its identity was only recently confirmed with the help of molecular analyses (Galil et al. 2017a). Not surprisingly, the countries with the highest number of newly recorded and established NIS were Israel (17 species in total / 6 established), Turkey (10/3) and Egypt (7/5). Nevertheless, the Suez Canal (i.e. the corridor pathway) is not the only important pathway of introduction for new species (n=19+7), where 19 is the number of species whose introduction is attributed exclusively to the corridor pathway and 7 are the species which could have entered also via alternative pathways. The transport-stowaway (T-S) pathway, representing ships ballast, hull fouling and other vectors, contributed almost equally to new species introductions reported in the 2017-2019 period (n=13+6), where 13 is the number of species whose introduction is attributed exclusively to the T-S pathway and 6 are the species which could have entered also via alternative pathways, while escape from confinement of pet species from aquaria (ESC) and transport-contaminant (CON), both as parasites and as hitchhikers on aquaculture transports, are considered responsible for the introduction of 8 and 7 new species respectively. Finally, the primary pathway of introduction remains undetermined for 12 of the 65 new species.

Species that have changed their establishment success
Twenty two species previously considered as casual records have established viable populations ( Table 3). The time spans between consecutive records for the species of this category vary considerably but are as long as 50-90 years in some cases. The amphipod Bemlos leptocheirus, recorded for the first time near the entrance of the Suez Canal almost a century ago (Schellenberg 1928), reappeared in the Mediterranean in 2015, recorded by Ulman et al. (2017) in two Greek marinas, and is now considered established. The bryozoan Hippopodina sp has been sporadically reported from the Eastern Mediterranean Sea as H. feegensis (Busk, 1884) since 1966 (Powell 1969). The species was recently re-described by Tilbrook (1999), leading Ulman et al. (2017) to conclude that the specimens found in Turkish and Greek marinas belong to the yet undescribed Hippopodina sp. collected by Tilbrook (2006) (Cervera et al. 2004), has now been identified by molecular analysis and reported from three different locations in southern Turkey (Yokeş et al. 2018). Genetic analyses were also instrumental in verifying the presence of the pomacentrid Abudefduf vaigiensis (Quoy & Gaimard, 1825) in the Mediterranean Sea, which had been excluded from the list of established species in Zenetos et al. (2017), after it was concluded by Tsadok et al. (2015) that the population in Israel actually belonged to A. saxatilis. Since then, established populations of A. vaigiensis have been reported from Malta (Vella et al. 2016) and Libya (Osca et al. 2020), in both cases supported by DNA sequencing.
A worrying development is the widespread establishment of Oithona davisae Ferrari and Orsi 1984, as already anticipated by Karachle et al. (2017). This cyclopoid copepod, previously only reported from Spain within the Mediterranean (Saiz et al. 2003), has now been recorded with considerable populations from the Aegean Sea ; Terbıyık Kurt and Beşiktepe 2019), the port of Venice (Zagami et al. 2018) and southern Italy (Vidjak et al. 2019), presumably carried with ballast waters and aquaculture transports. It displays an invasive character in San Francisco Bay (Ferrari and Orsi 1984) and the Black Sea (Seregin and Popova 2019) by competitive exclusion of native copepod species, while it is suspected that it can affect pelagic food webs through increased consumption of flagellates and interference with larval fish feeding (Karachle et al. 2017). The Indo-West Pacific rock shrimp Sicyonia lancifer (Olivier, 1811) is now confirmed as established in Israel, with multiple specimens having been collected by trawl fishermen in 2019 (Galil & Lubinevsky in Stern et al. 2019). The species was already present in a number of locations in Turkey and one in Israel by 2016 (Gönülal et al. 2016; Patania and Mutlu 2016) but due to the sporadic specimens and novelty of the invasion, it was not considered established in the Zenetos et al. (2017) update.
For the nudibranch Plocamopherus tilesii Bergh, 1877, local establishment in Turkish waters was confirmed by    (Bloch, 1787), which has spread along the Levantine coast from Israel as far as southern Turkey (Gürlek et al. 2019). While it was initially hypothesized that the species' introduction may have also resulted from an aquarium escape (Golani et al. 2010), recent genetic evidence indicates introduction through the Suez Canal as the most plausible pathway (Stern et al. 2019).
As much as the spread pattern of P. imperator is a typical example of natural dispersal, the case of Watersipora arcuata Banta, 1969 is exactly the opposite. A relatively recent introduction, this fouling bryozoan was only known from one location in the Ligurian Sea, Italy (Ferrario et al. 2015) up to the compilation by Zenetos et al. (2017). In a matter of 2-3 years the species was discovered in Spain, Malta, Sicily, Turkey (Ulman et al. 2017) and the Tyrrhenian Sea , always in marinas for recreational craft. Another species most likely spreading via hull fouling is the colonial scleractinian coral Oulastrea crispata (Lamarck, 1816). The species, which was only recorded once in Corsica in 2012 (Hoeksema and Ocaña 2014), has now been found in two additional locations in Spain (Mariani et al. 2018) and is considered established in the western Mediterranean.
The parasite Neoallolepidapedon hawaiiense Yamaguti, 1965, which was thus far recorded only once in Tunisia in 2007 from Fistularia commersonii Rüppell, 1838 (Merella et al. 2007), has also changed its establishment success in the Mediterranean. The species has now been also reported from Libya, in the same host, by Salem (2017), and is considered established in the central Mediterranean. Regarding the establishment status of parasites with complex life cycles, one could argue that establishment success can only be affirmed if the intermediate hosts have been found carrying the parasites as well; otherwise a plausible hypothesis would be that the findings belong to residual infections that took place before the definitive host's migration (Merella et al. 2010). Besides the fact that this is an overly strict criterion, given the scarcity of distributional information on many of these parasites, the alternative assumption would be that each alien parasite record represents a new introduction event, which does not seem plausible either. Thus, while high uncertainty in their establishment success is acknowledged, alien parasites follow the same criteria for establishment outlined in the methods section and in Zenetos et al. (2017) and Zenetos et al. (2008).
Lastly for this group, a small number of species which were considered established until Zenetos et al. (2017) have now been re-classified as casual, due to the absence of any further records.
These are: Papilloculiceps longiceps (Cuvier, 1829), an Indian ocean fish species reported only from Israel (Golani and Ben 1990); Parvocalanus latus Andronov, 1972, a species native in the Indian Ocean reported only from S. Turkey (Uysal et al. 2002); Terapon theraps Cuvier, 1829, an Indo-Pacific species with only two records of single specimens in Slovenia (Lipej et al. 2008) and Greece (Minos et al. 2012) and whose presence cannot be attributed to any pathway of introduction.

Species that have expanded their distribution into new MSFD regions
A total of 36 species have expanded their distribution into new MSFD regions, of which 21 are already established in the new locations (Table 4).
The main pathways implicated in their spread are natural dispersal from neighbouring areas (unaided pathway) and hull fouling (n=13), while ship-mediated pathways as a whole are considered responsible for the spreading of 24 species. The number of hull-fouling species records has undoubtedly been augmented since Zenetos et al. (2017) by the extensive study of Ulman et al. (2017), who sampled a large number of recreational marinas in the Mediterranean, targeting specifically alien species. It is also worth noting that the majority of large, conspicuous species, i.e. fishes and molluscs that disperse naturally (n=11), were recorded by citizen scientists and communicated to the authors through increasingly expanding initiatives and networks with national experts (e.g. expansion records of Pterois miles (Bennett, 1828 and , Etrumeus golanii in Table 4, but also Pomacanthus imperator and Charybdis feriata (Linnaeus, 1758) in Table 3).
The Central Mediterranean received the highest number of expanding species (n=19), which is not surprising, given its central position in the region, which is critical both for natural dispersal but also for its busy shipping routes and transshipment hubs Deidun et al. 2018). The Western Mediterranean was the recipient of only 7 expanding species, primarily through shipping vectors, with the exception of two Lessepsian fishes and one fish parasite. Etrumeus golanii, which has already attained commercial significance in the Eastern Mediterranean (DiBattista et al. 2012), has recently spread through the central basin all the way to the Alboran Sea (Tamsouri et al. 2019). Pteragogus trispilus, another Lessepsian immigrant, appears to be pushing the lower limits of its thermal envelope both in the Aegean (Yapici et al. 2015) and along the African coast, having been observed in the Gulf of Tunis in 2016 (Hamed et al. 2018).
Ten species expanded their Mediterranean distribution into the Adriatic Sea, most of them already widespread in two or three MSFD sectors of the basin. Of  The pathway column refers to the most likely pathway of secondary spread. Taxonomic group and Pathway abbreviations as in Table 2 the Biodiversity Strategy (EC (European Commission)  , have resulted in a large demand in relevant data production and reporting and are the driving force behind dedicated research projects and national monitoring schemes for NIS. Furthermore, the increased appreciation of stakeholder involvement  and the burst in citizen science initiatives and networks (e.g. Zenetos et al. 2013;Crocetta et al. 2017;Giovos et al. 2019) has undoubtedly improved the spatial scale and temporal resolution of alien species detection, monitoring and surveillance. The above notwithstanding, climate change is also expected to have played a role in this accelerated rate of alien species expansion. Warming of the Mediterranean Sea surface waters between  has been estimated at 0.04°C/year, leading to an overall SST increase of approximately 1°C for the eastern basin, with the Aegean Sea and the Adriatic among the hotspots of this warming trend (Nykjaer 2009). For marine species, geographic ranges conform closely to their thermal limits (Sunday et al. 2012;Payne et al. 2016), which in turn show higher plasticity at their lower end (Stuart-Smith et al. 2017). Expansion records of Indo-Pacific species discussed in previous sections indicate that a number of species are likely already favoured by increased temperatures, exhibiting their northernmost records in the Mediterranean (e.g. Sepioteuthis lessoniana Férussac [in Lesson], 1831, Biuve fulvipunctata (Baba, 1938), Haminoea cyanomarginata Heller & Thompson, 1983) or pushing their way into cooler western Mediterranean waters (e.g. Etrumeus golanii).
Attention needs to be paid to the fact that the observed rate of new introductions does not appear to be related to the enlargement of the Suez Canal, verifying the findings of Zenetos (2017Zenetos ( , 2019. When all potential pathways are considered for the new species introduced between 2017-2019, it is only 4 species per year that seem to enter the Mediterranean through the Suez Canal, a number much lower than the 7 species per year, reported by Zenetos et al. (2017) for the period 2015-2017. An appreciable number of alien species were introduced to Mediterranean waters via shipping pathways (7 in total), predominantly zoobenthic and planktonic species, as well as benthic plants. Aquaria releases have also contributed 7 species in total to the Mediterranean xenodiversity, all but one demersal fish with, as of yet, only casual records since 2017. A characteristic example is the high-valued aquarium species Abudefduf sexfasciatus (Lacepède, 1801), native to the Red Sea, which was however observed for the first time in Greek waters, too far away from the Suez Canal (Giovos et al. 2018). Chrysiptera hemicyanea (Weber, 1913) on the other hand, a typical coral-reef dweller, is not found in the Red Sea and is almost certainly released from an aquarium into Maltese waters . It remains to be seen whether some of these species will be confirmed as aquarium releases or will be proven Lessepsian migrants by additional records and/or genetic evidence, as was the case for Acanthurus sohal (Forsskål, 1775) (Bariche et al. 2019) and Pomacanthus imperator (Bloch, 1787) (Stern et al. 2019).
In agreement with Zenetos et al. (2017), we consider this a "provisional" list, presenting the changes in Mediterranean marine alien species since the end of 2016. We acknowledge that even the current update may contain errors, as species invasions are dynamic phenomena, where new information continually comes to light, whether from new observations or from re-examination of older material, changes in nomenclature and phylogenetic studies. Finally, we re-affirm the critical role of international collaboration and consultation with local and taxonomic experts in keeping these important policy tools as accurate and up-to-date as possible in order to provide well-informed management and policy advice.