Risk Assessment for the lionfish Pterois miles (Bennett, 1828)

Prepared in the framework of the EU LIFE project “Preventing a LIONfish invasion in the MEDiterranean through early response and targeted REmoval (RELIONMED-LIFE)” - LIFE16 NAT/CY/000832 EU NON-NATIVE ORGANISM RISK ASSESSMENT SCHEME

Name of organism: Pterois miles (Bennett, 1828)

Lead authors: Periklis Kleitou1, Ioannis Savva2, Demetris Kletou2

Contributing authors: Charalampos Antoniou2, Niki Chartosia3, Yiannis Christodoulides4,5, Maria Christou2, Louis Hadjioannou4, Margarita Hadjistylli5, Jason M Hall-Spencer1, Carlos Jimenez4, Sian Rees1

1 School of Biological and Marine Sciences, University of Plymouth, Plymouth, UK 2 Marine and Environmental Research (MER) Lab., Limassol, 4533, Cyprus 3 Department of Biological Sciences University of Cyprus P.O. Box 20537, 1678 Nicosia, Cyprus. 4 Enalia Physis Environmental Research Centre, Acropoleos 2, Aglantzia 2101, Nicosia, Cyprus 5 Department of Environment, Ministry of Agriculture, Rural Development and Environment, Nicosia, Cyprus

Risk Assessment Area: The geographical coverage of the risk assessment is the territory of the European Union (excluding outermost regions) and the United Kingdom

Peer review 1: Stelios Katsanevakis, Dr, Department of Marine Sciences, University of the Aegean, Mytilene, Lesvos Island, Greece Peer review 2: Ernesto Azzurro, Dr, Institute for Environmental Protection and Research (ISPRA) Livorno, Italy

Positive opinion by the Scientific Forum: 17/11/2020

1 | P a g e EU CHAPEAU

QUESTION RESPONSE COMMENT

Ch1. In which EU biogeographic region(s) Biogeographic region The lionfish (Pterois miles) is native to the Indo-Pacific but in or marine subregion(s) has the species been  Mediterranean Sea the past five years it has spread rapidly throughout the eastern recorded and where is it established? basin of the Mediterranean Sea (Bariche et al., 2013; Crocetta Subregion et al., 2015; Oray et al., 2015; Turan & Öztürk, 2015; Dailianis  Aegean-Levantine Sea (recorded et al., 2016; Kletou et al., 2016; Jimenez et al., 2016; Ali et al., and established) 2016; Azzurro & Bariche, 2017; Özbek et al., 2017). It is now  Ionian Sea and the Central established in the Aegean-Levantine Sea Marine Strategy Mediterranean Sea (recorded and Framework Directive (MFSD) subregion (Kletou et al., 2016; established) Jimenez et al., 2016; Azzurro & Bariche, 2017; Giovos et al.,  Western Mediterranean Sea 2018). It has been recorded in the Ionian Sea, the Central (recorded) Mediterranean (Azzurro et al., 2017) and the Western Mediterranean MSFD subregions (Yokeş et al., 2018). All lionfish records from the literature and the EU-LIFE RELIONMED project until July 2018 were compiled to produce the density map that is shown in Figure 1. It has continued to spread in 2019, with lionfish pairs recently sighted in the northern Ionian Sea (Cephalonia and Corfu islands, Greece) at the boundaries with the Adriatic Sea (iSea’s citizen-science database “Is it Alien to you…. Share it!!!”) (see Figure 2) .

2 | P a g e 3 | P a g e Figure 1. Non-native lionfish sightings density map based on 668 lionfish records published in scientific literature (Bariche et al., 2013; Crocetta et al., 2013; Turan et al., 2014; Oray et al., 2015; Turan and Öztürk, 2015; Dailianis et al., 2016; Kletou et al., 2016; Ali et al., 2017; Azzuro and Bariche, 2017; Azzuro et al., 2017; Özbek et al., 2017; Stern et al., 2018) and RELIONMED project from October 2012 to July 2018. The KERNEL density estimation algorithm based on the quartic kernel function was used with a search radius distance from the point at 5 km. Most RELIONMED data were acquired from the eastern part of Cyprus; thus the low abundance of lionfish illustrated in other areas might be due to the lack of adequate data rather than the limited presence of lionfish. No data are available on the abundance and spread of the pelagic juvenile stages of these fish. Source: Georgiou et al., in RELIONMED project (2018).

Figure 2. Lionfish sightings in Greece (until 14/07/2019) based on the iSea’s citizen-science database “Is it Alien to you…. Share it!!!” (https://isea.com.gr/activities/programs/alien-species/is-it-alien-to-you-share-it/?lang=en).

4 | P a g e Ch2. In which EU biogeographic region(s) Worst case scenario using the 15 oC In the Western Atlantic Ocean, non-native lionfish (Pterois miles or marine subregion(s) could the species criterion and Pterois volitans) have spread up to Long Island but seem not establish in the future under current climate Current climate to be able to reproduce in the cold winter waters at this latitude. and under foreseeable climate change? Biogeographic region: In north Carolina the lionfish has established where the winter  Mediterranean Sea mean seawater temperature is above 15 oC (NCCOS project). Subregion: Although the lethal temperature for lionfish is 10.5-11 oC  Western Mediterranean Sea (Kimball et al., 2004; Barker et al., 2018) they stop feeding  Southern Iberian coast below 16 oC (Kimball et al., 2004). Based on the observation of Atlantic lionfish invasion, we Foreseeable climate change assume that in the Mediterranean Sea, lionfish (Pterois miles and Biogeographic regions: Pterois volitans) can establish in areas where minimum water  Mediterranean Sea temperature is above 15°C (Figure 4).  Atlantic o Subregions: If we apply a mean winter temperature threshold of 15 C then  Western Mediterranean Sea all of the southern Mediterranean regions offer potential habitat for lionfish under the current climate, and are highly likely to  Adriatic Sea spread there through larval dispersal and adult movement.  Bay of Biscay and Iberian coast

 Macaronesia The probability of lionfish entering the Black Sea is thought to

be low due to the relatively high salinity and low temperature Best case scenario using the Species conditions encountered in this enclosed Sea. The lionfish could Distribution Models (SDMs) however cross the Gibraltar Strait and establish in the eastern Current climate Atlantic Ocean initially off Portugal and Morocco and then No other areas apart from those stated in moving north and south. Ch1. Remote sensing and dispersion modelling could also be used to Foreseeable climate change project the future lionfish niche in the Mediterranean under Southern Adriatic Sea current and foreseeable climate change, and scientists are working on this (e.g. Turan, 2019). Unpublished evidence from species distribution modelling (SDM) based on habitat suitability shows that the entire eastern Mediterranean is currently subject to one of the most rapid invasions ever recorded (Poursanidis et al., unpublished data; Figure 3). SDMs projections show that lionfish current habitat suitability is clearly limited to the Central and Eastern Mediterranean in areas

5 | P a g e where lionfish have already been recorded (Poursanidis, 2015; D’Amen & Azzurro, 2019; Poursanidis et al., unpublished). However, previous research has shown that tropical invaders may spread far beyond their native niches and that SDMs may underestimate the potential spread of invasive species (Parravicini et al., 2015). Indeed, Poursanidis (2015) SDM failed to predict lionfish invasion in current lionfish hotspot areas such as Cyprus. Climatic niche expansion (i.e. the environmental shift of species beyond their climatic limits in their native ranges) and niche unfilling (i.e. the presence of favorable climate in the invaded domain not yet occupied by the species) should be taken into consideration (Parravicini et al., 2015; Poursanidis et al., unpublished).

Further investigations such as ecophysiological models (e.g. Jørgensen et al., 2012; Marras, et al., 2015) and other SDMs (e.g. Bernal et al., 2015) could help to better assess which Mediterranean areas are suitable to the lionfish invasion under current conditions and CC scenarios.

For the purposes of this risk assessment, we use the SDMs projections as the best case scenario (i.e. lionfish will not expand to the western Mediterranean), and the 15oC criterion as the worst case scenario. However, P. miles distribution might end up being worst than our worst-scenario due to niche unfilling or expansion, and our distribution assessments are based on a low confidence. For instance, P. miles modelled niche unfilling (without climate change scenarios) using mean annual temperature and salinity (Poursanidis et al., unpublished) encompasses all the south Mediterranean but also colder Mediterranean sectors (i.e. the North Aegean Sea, the North Adriatic Sea and the Alboran Sea); corresponding to native P. miles distribution on the South African coast.

6 | P a g e

The Mediterranean Sea is warming faster than the global ocean (Burrows et al., 2011), especially the eastern basin (Marbà et al., 2015), helping the successful invasion and the establishment of many thermophilic non-indigenous marine species (Bianchi, 2007; Pancucci-Papadopoulou et al., 2011; Zenetos et al., 2012). Foreseeable sea surface temperature change based on Intergovernmental Panel on Climate Change (IPCC) Representative Concentration Pathway (RCP) scenario varies considerably between the emission scenarios ranging from about 1.2 °C ± 0.6 °C (RCP2.6) to 3.1°C ± 0.6 °C (RCP8.5) increase by the end of the century (Collins et al., 2013). Depending on the emission scenario, global ocean warming between 0.5°C (RCP2.6) and 1.5°C (RCP8.5) will reach a depth of about 1 km by the end of the century (Collins et al., 2013). Near-term sea surface temperature projection according to all scenarios is displayed in Figure 4. For the purposes of this Risk Assessment, we used RCP 6.0 which uses a high greenhouse gas emission rate and is a stabilization scenario where total radiative forcing is stabilized after 2100 by employment of a range of technologies and strategies for reducing greenhouse gas emissions. RCP 6.0 projects a global sea surface temperature increase of 1.9 ± 0.4 by end of this century. Different areas of the Mediterranean basin will respond differently to climate change and the projected changes based on RCP 6.0 are illustrated in Figure 5. Under these conditions and using the 15 oC scenario, the lionfish will expand its range in the northern Mediterranean (i.e. northern Aegean, Adriatic, Ligurian and northern Balearic Seas), hence almost the entire Mediterranean Sea, will become suitable for its establishment (Figure 4). On the other hand, according to D’Amen & Azzurro (2019) SDM, under 2050 scenarios P. miles is predicted to find suitable habitats not further west than the southern Adriatic Sea and Italian Ionian sea; although the species could eventually expand further in case of a niche

7 | P a g e expansion as seen in other tropical species (sensu Parravicini et al., 2015).

8 | P a g e

Figure 3. Distribution map of lionfish (probability of occurrence) using MaxEnt and 254 records published in the literature until 2017 (Turan et al., 2015; Mytilineou et al., 2016; Azzurro et al., 2017) (Poursanidis et al., unpublished).

9 | P a g e

Figure 4. Minimum temperature of the Mediterranean and adjacent seas, at present, near-term projection (2050) and long-term projection (2100). Data sets have been obtained from Bio-ORACLE v2.0 (Assis et al., 2017).

10 | P a g e

Figure 5. Near term global sea surface temperature change based on Representative Concentration Pathway (RCP) scenarios (Kirtman et al., 2013).

Ch3. In which EU member states has the Cyprus, Greece, Italy for sure, and possibly It has been recorded in Cyprus (2012), Greece (2015; Rhodes), species been recorded? List them with an Malta and Portugal. Italy (2016; Sicily) (Crocetta et al., 2015; Kletou et al., 2016; indication of the timeline of observations. Jimenez et al., 2016; Azzurro et al., 2017) and there is also an anecdotal report from Malta (2016) (https://www.um.edu.mt/newspoint/news/features/2016/07/ongoi ngresearchfindsnewalienspeciesinmaltesewaters.) P. Carmo (pers. comm., 2020) reported sightings of Pterois sp. since 2018 in Portugal, suspected release from aquaria.

11 | P a g e

Figure 6. Cumulative occurrences of Pterois miles in the Mediterranean Sea from July 1991 to October 2016. Data consisted of 230 georeferenced observations pooled by bibliographic sources. Source: Azzurro et al., (2017)

Ch4. In which EU member states has this Cyprus and Greece We consider species as established in an area when they are species established populations? List them reported more than twice, spread over time and place, even with an indication of the timeline of though there may be no evidence of reproduction. establishment and spread. 12 | P a g e The species has established in Cyprus in 2014-15 (Kletou et al., 2016; Jimenez et al., 2016) and Greece in 2016-2017 in Crete and Dodecanese islands, and later in 2018 in southern Peloponnese and 2019 in Ionian Sea (Giovos et al., 2018; iSea’s citizen-science database “Is it Alien to you…. Share it until 14/07/2019).

Ch5. In which EU member states could the Worst case scenario (using the 15 oC The areas where this species could establish in the species establish in the future under current criterion) Mediterranean Sea under current climate and foreseeable climate climate and under foreseeable climate Current climate change are described in Ch2. change? Cyprus, Greece, Italy, Malta, Spain, and Portugal. Warming of the Mediterranean Sea has already assisted the spread of many IndoPacific non-native species, including Foreseeable climate change lionfish. Foreseeable climate change will assist the spread of All the above plus Croatia, Slovenia and this species further unless its population can be controlled. France Ongoing seawater warming) in the Mediterranean will provide a wider area that is suitable for the metabolism and growth of lionfish (Kimball et al., 2004; Barker et al., 2018). The Best case scenario (using the SDMs) Mediterranean Sea is warming in deep (Bethoux & Gentili, Current climate 1996) as well as surface waters (Metaxas et al.,1991; Marba and Cyprus, Greece, Italy, Malta Duarte 1997; IPCC 2007; Lejeusne et al.,2010) throughout the region (Metaxas et al., 1991; Ulbrich et al., 2006; Diffenbaugh Foreseeable climate change et al., 2007; Marbà et al., 2015) including its western (Vargas- All the above Yáñez et al.,2008; Vargas-Yáñez et al.,2010a; Vargas-Yáñez et al.,2010b) and eastern limits (Samuel-Rhoads et al., 2010, 2013; Skliris et al., 2012; Sisma-Ventura et al., 2014). Given the fact that lionfish have a wide thermal tolerance and can survive temperatures as low as 10.5-11 °C (Kimball et al., 2004; Dabruzzi et al., 2017; Barker et al., 2018) and based on current climate change, the lionfish could easily spread and become established throughout EU member states in south and central latitudes of the Mediterranean Sea (i.e. Cyprus, Malta, southern Greece, southern Italy, southern Portugal and southern Spain) where winter sea surface water temperatures are >15 oC. Under 13 | P a g e foreseeable climate change and the criterion of 15°C (worst case scenario – see Ch2), lionfish are projected to spread and become established in to more northern parts of Italy, Greece, Portugal and Spain as well as into Croatia, Slovenia and France. The coastal waters of these northern Mediterranean EU member states currently have winter mean temperatures below 15 oC but under foreseeable warming scenarios their surface water temperature will increase above this value and will fall within the temperature niche of lionfish.

It should be highlighted that despite having a low chronic thermal minimum at 10.7 °C (Kimball et al., 2004), the Pterois volitans/miles complex has been detected up to New York’s Long Island (Betancur-R. et al., 2011), where temperature gets as low as 4 °C in winter. However, the juvenile fish that make it to Long Island do not seem able to cope with the cold water and so fail to grow or reproduce. Under current climate conditions lionfish apparently cannot become established at winter temperatures north of North Carolina (mean < 15 oC). It remains to be seen whether the lionfish will inhabit Mediterranean waters that have temperatures below this threshold.

On the other hand, using the best case scenario (SDM projections) (see Ch2), D’Amen & Azzurro (2019) SDM predicted that lionfish will be restricted to the central-eastern Mediterranean and until 2050, it will be able to find suitable habitats no further west than the southern Adriatic Sea and Italian Ionian sea. These predictions are made with high degree of uncertainty (for more information see Ch2).

Ch6. In which EU member states has this Cyprus, Greece During 2016-2019 lionfish populations have started to dominate species shown signs of invasiveness? rocky reefs and are now amongst the most abundant fish at 2-30 m depth in some areas off Cyprus. They have also invaded deep- water fragile habitats (Jimenez et al., 2019), including 14 | P a g e Dendrophyllia ramea coral communities at 130-150 m depth (Orejas et al.,2019). The RELIONMED LIFE+ project has documented a high abundance of lionfish, ranging from juveniles to large adults, at many locations around the island.

In a RELIONMED removal expedition in a Marine Protected Area (MPA) on November, 2017 about 75 lionfish were removed in a single day from an area less than 1 hectare. In 2019, RELIONMED scientists observed more than 50 lionfish in a 400 m2 rocky reef. In May 2019, RELIONMED organised a lionfish derby at Capo Greco and about 125 lionfish were removed by 27 divers each carrying out a single dive. Most of these invasive fish were removed from an area of about 200 m2. Another tournament organised in Famagusta Bay with 48 participants (39 free divers and 9 scuba divers) removed 315 lionfish in about two hours (Jimenez et al., 2018). In July 2019, RELIONMED organised a removal event with approximately 25 divers, and after two dives at the popular Zenobia wreck (178 m length), around 120 lionfish were removed.

In Greece, the lionfish recently started to expand in numbers (summer 2019) in areas off Crete and the southern Aegean such as Rhodes, with divers sending photographs to the citizen- science project (“Is it Alien to you…. Share it!!!”) with up to 7 lionfish aggregated together on just one rocky structure. The high densities of lionfish in some areas of Greece is evidence that the species has become invasive and substantially impacts the local biota.

Ch7. In which EU member states could this Worst case scenario (using the 15 oC The species has the potential to become invasive in all areas species become invasive in the future under criterion) where it becomes established - see Ch5 for more information. current climate and under foreseeable Current climate climate change? Cyprus, Greece, Italy, Malta, Spain, and 15 | P a g e Portugal.

Foreseeable climate change All the above plus Croatia, Slovenia and France

Best case scenario (using the SDMs) Current climate Cyprus, Greece, Italy, Malta

Foreseeable climate change All the above

Distribution Summary:

EU Member States and the United Kingdom

Recorded Invasive / Invasive/Established Invasive/Established Comments Established (future) – Worst (future) – Best case (currently) case scenario (15 oC scenario (SDMs) criterion) Belgium - - - Future scenarios are Bulgaria - - - based on a low Croatia - - Yes confidence due to Cyprus Yes Yes Yes Yes niche unfilling and Denmark - - - expansion of Estonia - - - lionfish in the Finland - - - Mediterranean (see France - - Yes Ch2 for more Germany - - - information) Greece Yes Yes Yes Yes

16 | P a g e Ireland - - - Italy Yes - Yes Yes Latvia - - - Lithuania - - - Malta Probably - Yes Yes Netherlands - - - Poland - - - Portugal Probably - Yes Romania - - - Slovenia - - Yes Spain - - Yes Sweden - - - United - - - Kingdom

EU biogeographic regions

Recorded Established Established Comments (currently) (future) Alpine - - - Future scenarios are Atlantic Probably – based on a low only in the confidence due to niche - - worst-case unfilling and expansion scenario of lionfish in the Black Sea - - - Mediterranean (see Ch2 Boreal - - - for more information) Continental - - Mediterranean Yes Yes Yes Pannonian - - - Steppic - - -

17 | P a g e EU marine regions and subregions

Recorded Established Established Established Comments (currently) (future) (future) Worst case Best case scenario (15 scenario oC criterion) (SDMs) Baltic Sea - - - Future scenarios Black Sea - - - are based on a low North-east Atlantic Ocean - - - confidence due to Bay of Biscay and the Iberian Coast - - Yes niche unfilling and Celtic Sea - - - expansion of Greater North Sea - - - lionfish in the Mediterranean Sea Yes - - Mediterranean (see Adriatic Sea Yes (only Ch2 for more - - Yes southern) information) Aegean-Levantine Sea Yes Yes Yes Yes Ionian Sea and the Central Mediterranean Yes Yes Yes Yes Sea Western Mediterranean Sea Yes - Yes

18 | P a g e

SECTION A – Organism Information and Screening

Organism Information RESPONSE COMMENT

A1. Identify the organism. Is it clearly a The lionfish P. miles is a clear single Scientific name: Pterois miles (Bennett, 1828) single taxonomic entity and can it be taxonomic entity. Synonyms: Scorpaena miles Bennett, 1828; Pterois muricata Cuvier, adequately distinguished from other 1829. entities of the same rank? Hybridization with other Pterois spp. Subfamily: Pteroinae is possible. Family: Scorpaenidae Order: Scorpaeniformes Class:Actinopterygii Common names: Common lionfish (GB), Devil firefish (GB), Rotfeuerfisch (DE), Pez león soldado (ES), Pez fuego diablo (ES), Pez escorpión (ES), Poisson lion (FR), Pesce diavolo di fuoco (IT), Λεοντόψαρο (CY), Λιονταρόψαρο (GR).

The lionfish Pterois miles has a fusiform shape, moderately compressed, with no interrupted lateral line. Its colouration ranges from alternating reddish to tanned and grey or white narrow stripes, with frequent dark bands across its head and main body. It has 13 dorsal spines, 9-11 dorsal soft rays, 3 anal spines, 6-7 anal soft rays. The tentacle above the eye can be faintly banded. The adults also bear tiny spines across the cheek and dark or white spots on the median fins. The average gape size of adults (specimens >15 cm total length) from Mediterranean specimens is ~11 cm2. Its otoliths are as small (~ 0.5 cm in length) and located within the auditory capsule of its cranium. Unless startled, Pterois miles is a slow moving fish commonly found within reef communities (Schultz 1986; Whitfield et al., 2007; Biggs & Olden, 2011). It usually resides within holes or under crevices and ledges in an up-side down position during the day and becomes active at dusk and remains so through the night until dawn.

19 | P a g e

Pterois miles can be easily distinguished from sister species based on the morphology. An exception is the congeneric P. volitans, which looks very similar and is distinguished primarily through small variations in meristic counts. For instance, P. miles has one lower count of dorsal and anal fin rays than P. volitans (Morris et al., 2008). The taxonomic distinction of P. miles and P. volitans is supported by reciprocal monophyly of mtDNA sequences divergent between 4% and 11% depending on the marker (Freshwater et al., 2009). However, the ambiguous (or “mixed”) meristic characters of the two species in their invaded Atlantic range is evidence of interbreeding in their invaded range (Freshwater et al. 2009).

Recently, Wilcox et al. (2018) analyzed mtDNA COI and 2 nuclear introns (S7 RP1 and Gpd2) from 229 lionfish (44 P. miles, 91 P. volitans, 31 Pterois lunulata, and 63 Pterois russelii) from 10 locations in their native range and found out that all P. volitans specimens in the western Atlantic were hybrids between the Indian Ocean P. miles and a Pacific lineage encompassing P. lunulata/russelii, a conclusion supported by both genetics and morphology.

A2. Provide information on the existence Pterois volitans looks very similar to, Scientific name: Pterois volitans (Linnaeus, 1758) of other species that look very similar and known to hybridise with, P. miles Synonyms: Gasterosteus volitans Linnaeus, 1758 in the invasive range in the Western Family: Scorpaenidae Atlantic. There is also a hybrid Subfamily: Pteroinae between P. miles and P. russelii. Order: Scorpaeniformes Class: Actinopterygii The only morphological difference of Common English names: Red lionfish (GB), Perutýn ohnivý (CZ), P. volitans and P. miles is that the Pazifische Rotfeuerfisch (DE), Pez león Colorado (ES), Rascasse latter has one lower count of dorsal volante (FR), Peixe-leão-vermelho (PT), Κόκκινο λεοντόψαρο (CY), and anal fin ray. Κόκκινο λιονταρόψαρο (GR).

Lionfishes have spectacular fins and are striped/banded making them 20 | P a g e easily distinguishable. Misidentifying with another local or alien species is highly unlikely.

The lionfishes (subfamily Pteroinae) currently comprise 27 recognized species in 5 genera. There are 11 species described in the Pterois genus. In alphabetical sequence, P. andover, P. antennata, P. brevipectoralis, P. lunulata, P. miles, P. mombasae, P. paucispinula, P. radiata, P. russelii, P. sphex and P. volitans (FishBase accessed August 2018).

Four Pterois spp. that look very similar are the P. volitans, P. miles and the two closely related congeners P. lunulata and P. russelii. Phylogeographic analyses revealed only two major lineages within these species. An Indian Ocean lineage consisting of P. miles and a Pacific Ocean lineage consisting largely of P. lunulata and P. russelii (Wilcox, 2014). Genetic evidence suggests that the latter two are the same species and the classification of P. lunulata into a different species is dubious and that P. volitans has emerged from the hybridisation of P. miles and P. russelii (Wilcox et al., 2018).

Genetic studies of the Mediterranean lionfish specimens (Bariche et al., 2017; Stern et al. 2019; RELIONMED project data – Dimitriou et al., 2019) have found only P. miles in the Mediterranean Sea.

Gurlek et al., (2016), Gökoğlu et al., (2017), and Ayas et al., (2018) reported that P. volitans is also present in the eastern Mediterranean Sea. However, the studies that recorded P. volitans were only based on morphological characteristics. A genetic re-evaluation should be conducted to validate these findings. A3. Does a relevant earlier risk Yes No earlier risk assessment has been carried out pursuant to Article assessment exist? (give details of any 5(1) of the EU Regulation 1143/2014 on invasive alien species. previous risk assessment and its validity in relation to the EU) Assessments of lionfish invasion in the Western Atlantic have been carried out widely to assess risks associated with the invasion 21 | P a g e (USFWS, 2014), ecological, socioeconomic and management implications (e.g. Hare & Whitfield, 2003; Morris & Whitfield, 2009) as well as additional risks, e.g. lionfish presence in the aquarium trade (Lyons, 2018; Lyons et al., 2019). Other assessments have been carried out in EU and neighbouring countries that aimed to assess the potential invasiveness and impacts of lionfish in the Mediterranean. In 2015 and prior lionfish establishment, Roy et al., (2015) conducted a horizon scanning of 95 species (both terrestrial and marine) that represented high risk of arrival, establishment, spread and threat to biodiversity and associated ecosystem services across the EU within the next ten years, and lionfish was ranked as the second most important species for consideration. Filiz et al., (2017) assessed the invasiveness risk of Pterois miles in the eastern Mediterranean using the recently- developed Aquatic Species Invasiveness Screening Kit (AS-ISK) and found a high risk of invasiveness due to factors such as high climate match, tolerance of a wide range of environmental conditions, flexibility in utilising food resources, high fecundity, small size at maturity, high reproductive effort and high invasiveness potential elsewhere. Galanidi et al., (2018) used the Environmental and Socio- Economic Impact Classification of Alien Species (EICAT and SEICAT) and assessed the potential impacts of lionfish together with other six invasive marine fish species that are considered of “high risk” for the Mediterranean region. By systematically reviewing the published literature and scoring the demonstrated impacts, Galanidi et al., (2018) showed that P. miles had the highest environmental impacts among the species examined.

A4. Where is the organism native? Indian Ocean Pterois miles is of Indo-Pacific origin but restricted to the Indian Ocean (Kulbicki et al., 2012), specifically from the Red Sea all the way down to the eastern South Africa, in Arabian Sea, Persian Gulf, Gulf of Oman, Laccadive Sea, Bay of Bengal, Andaman Sea and Indonesian region. Around Indonesia P. miles, P. volitans and P. russelii populations overlap. Pterois miles is mostly found in the depth 22 | P a g e range of 0 to 50 m (Schultz 1986), but lionfish individuals were also found at 20 to 80 m depth on a mid and outer-continental shelf off the east coast of United States (Whitefield et al., 2002) and up to more than 300 m depth (Gress et al., 2017; Andradi-Brown, 2019). They prefer rocky reefs with crevices or underwater caves to hide during the day. They are also attracted to underwear artificial structures such as wrecks. The average surface seawater temperature (SST) (2002-2012) in its native environment ranges from 20 to >30 °C (Locarnini et al., 2013), where maximum sea surface temperature (SST) is observed during the summer season towards the equator. The minimum SSTs are observed in the subtropics in winter in the northern Red Sea and the northern Persian Gulf (Locarnini et al., 2013). Although lionfish can live in estuaries in the open sea the salinity in its native environment varies ranges between 34-43 PSU (Han, 2001; Zweng et al., 2013). The eastern Indian Ocean has the lowest salinity levels, whereas the Red Sea has the highest (Zweng et al., 2013). The optimum conditions for development and reproduction across its native environment are as yet unknown (Morris et al., 2008) and most of today´s knowledge on its biology and ecology comes from studies in its invasive range in the western Atlantic Ocean. In the 1960s however, prolonged cold weather reduced winter shallow reef temperatures in Wakayama Prefecture, Japan to 10.1 °C for three weeks, and resulted in mortalities to the native there congeneric P. volitans population (Araga & Tanase, 1968).

A5. What is the global non-native Non-EU Mediterranean countries: Pterois miles has recently invaded several countries close to those of distribution of the organism (excluding Turkey, Lebanon, Syria, Israel, Libya, the EU, namely Turkey (Turan et al., 2014; Turan & Öztürk, 2015), the Union, but including neighbouring Tunisia Lebanon (Bariche et al., 2013), Syria (Ali et al., 2016), Tunisia European (non-Union) countries)? (Dailianis et al., 2016; Azzurro et al., 2017), Israel (Golani & Sonin, North America countries: 1992; Stern et al., 2018) and Libya (Al Mabruk & Rizgalla, 2019). Bermuda, Mexico, USA Lionfish complex (Pterois miles/volitans) was introduced in the Central America and Caribbean: North-western Atlantic in late 1980s and expanded throughout the Anguilla, Bahamas, Barbados, Belize, region, northwards along the east coast of the USA reaching as far as 23 | P a g e British Virgin Islands, Costa Rica, Rhode Island, eastwards to Bermuda, and southwards throughout the Cuba, Dominican Republic, Gulf of Mexico, Central America, South America, Caribbean and Guadeloupe, Haiti, Honduras, Brazil (Morris et al., 2009; Morris, 2012; (Goodbody-Gringley et al., Jamaica, Netherlands Antilles, 2019). It is with high uncertainty to know where each species of the Nicaragua, Panama, Puerto Rico, complex is present due to their similar appearance and the need for Saint Kitts and Nevis, Turks and genetic analyses to distinguish them. Caicos islands, United States Virgin Islands During the last years, genetic analyses suggested that although both Pterois volitans and P. miles were introduced in the NW Atlantic, P. South America: volitans is the most ubiquitous species (Hamner et al., 2007; Betancur Colombia, Venezuela et al., 2011; Freshwater et al., 2009). Until recently, it was believed that Pterois miles dispersal was limited to the US coasts and that their population didn’t cross across the Florida Strait (Freshwater et al., 2009). However, more recently, it has been shown using genetic barcoding that the species has now extended its distribution into the Caribbean basin and Mexico; albeit in low numbers (Guzmán-Méndez et al., 2017). Morphological characters, life cycle, habits, and dispersal potential of this species are very similar to those of Pterois volitans, and this is the reason that many studies treated both species as a single one (i.e. Pterois complex) and it is highly uncertain in what countries only Pterois volitans might exist. For this reason, we hereby list the Western Atlantic countries that Pterois complex was established (after Invasive Species Compendium – CABI Pterois volitans list https://www.cabi.org/isc/datasheet/109158#1A100CB0- DC2B-4DDA-8E47-167764A2B1E2).

A6. Is the organism known to be invasive Yes. It is considered one of the most The lionfish is a generalist mesopredator feeding mainly on a wide (i.e. to threaten organisms, habitats or damaging invasive species in the variety of herbivorous fish in its non-native range of the western ecosystems) anywhere in the world? World since these fish colonised the Atlantic. It destabilizes coastal marine communities through a series western Atlantic Ocean and now is of cascading effects (Albins & Hixon, 2008; Morris & Akins, 2009; proliferating in many countries of the Albins & Hixon, 2011). Mediterranean. It is considered invasive in areas where it is The introduction of the red lionfish P. volitans (Linnaeus, 1758) and established (see Q.A5) the devil firefish P. miles (Bennett, 1828) in the western Atlantic is 24 | P a g e one of the fastest and most ecologically harmful marine fish introductions to date (Albins & Hixon, 2013). Lionfish have several traits that enable rapid spread and invasion across large expanses of coastal ecosystems (Morris & Whitfield, 2009). These include: long pelagic larval phase, quick sexual maturity, continuous reproduction and high fecundity, high survival of eggs and larvae, a wide ecological niche, they are generalist predators and able to cross environmental barriers (e.g. thermal, salinity and deep-water barriers), and are highly competitive with native fish for space and resources (Côté et al., 2013a).

Lionfish are considered invasive in the entire tropical western Atlantic, Caribbean Sea and Gulf of Mexico (Schofield, 2010). All the identified impacts in the literature on its invasive range are attributed to both P. miles and P. volitans (described as the P. volitans/miles complex) and not P. miles alone. The impacts of the lionfish complex to date are mostly evident in the Bahamas (Albins & Hixon, 2008; Lesser & Slattery, 2011; Green et al., 2012; Benkwitt, 2015; Raymond et al., 2015) and south-eastern United States (Ballew et al., 2016). The impacts of the lionfish complex to this region are associated to habitat modification (Morris & Whitfield, 2009; Lesser & Slattery, 2011) and impacts on the native fauna communities. The predation rate of the lionfish complex reduces the abundance of native fish fauna (Albins & Hixon, 2008; Green et al., 2012; Albins, 2013; Benkwitt, 2015; Rocha et al., 2015; Ballew et al., 2016; Kindinger & Albins, 2017; Tuttle, 2017) and outcompetes the native predators (Albins, 2013; Raymond et al., 2015).

Empirical evidence shows that lionfish are already proliferating in many countries of the Mediterranean Sea (i.e. Cyprus, Greece, Israel, Lebanon and Turkey) (Azzurro et al., 2017; Giovos et al., 2018) (see Ch6). Although ecological studies that prove its impacts in the Mediterranean have not yet been carried out, it is inevitable that lionfish high densities in these areas pose a threat to local biota and

25 | P a g e disturb the ecosystem food chain; possible to cause impacts to other predators due to competition. MPAs of the Mediterranean have been found to host higher biomass of non-indigenous species (including lionfish) compared to adjacent unprotected areas (Giakoumi et al., 2019a) and presence of non-indigenous species can diminish potential beneficial effects of fisheries reduction in those MPAs (Corrales et al., 2018). Lionfish has been found to invade and become more prevalent in areas where prey fish density and biomass is more available (Goodbody- Gringley et al., 2019), posing an additional invasion threat to high- biodiversity areas that can be used as MPAs for increasing resilience against climate change and human stressors.

While socio-economic impacts have yet to be fully evaluated, lionfish can reduce the abundance of small native fish by up to 95 % (Côté et al., 2013a) and further lower fisheries yields of economically important fish either through direct predation on important commercial species or through over-consumption of native small- sized fish and competition with native predators for food and territory (Johnston et al., 2017).

A7. Describe any known socio-economic Potential uses: There is little known about the benefits of lionfish in the risk benefits of the organism in the risk Seafood, assessment area. A survey in Cyprus revealed that few stakeholders assessment area. Jewellery, experienced positive effects from the lionfish invasion; namely Aquarium trade, increase in diving tourism and food source (Kleitou et al., 2019). Therapeutic and medicine properties There is much to be learned from the western Atlantic where lionfish have densities 5-15 times greater than its native range; this is Socio-economic benefits: attributed to several reasons such as naïve prey, lack of predators, low Raise revenue in local communities, levels of parasitism and low levels of disease (Green & Côté, 2009; Healthy nutrition, Morris & Whitfield, 2009; Darling et al., 2011; Kulbicki et al., 2012). Market diversification with jewellery, There, divers and snorkellers demonstrate mixed preferences and Physical and intellectual interaction heterogeneous attitude against lionfish presence. For instance, Alemu et al. (2019) survey on reefs in Tobago showed that snorkelers are intrigued by the beautiful appearance of lionfish and favour some 26 | P a g e lionfish on reefs relative to none while recreational divers perceived all lionfish levels as negatives and were willing to pay more than snorkelers for high quality reef attributes. Similar results were found by Malpiza-Cruz et al. (2017) who surveyed visitors in the Mexican Caribbean and found that casual divers and snorkelers preferred reefs with lionfish and accepted their impacts on the reefs, while in contrary, committed divers disliked lionfish and associated impacts and would elect to dive elsewhere if such impacts were high.

In the Western Atlantic, local authorities established management strategies to counteract the threat and to create localised benefits linked to control mechanisms. Exploring and initiating commercial market niches is a current management strategy among the seafood industry, distributors, chefs, researchers, fishers and conservationists in the Atlantic invasive range. Considering its high nutritional profile and low ciguatoxin content (the leading cause of non-bacterial seafood poisoning associated with fish consumption), lionfish consumption is widely promoted in the western Atlantic (Morris & Breen, 2011; Chapman et al., 2016; Hardison et al., 2018) so that cost-effective targeted removals remain feasible and endure over time, but also to establish a positive impact on the socio-economic sector. Removed individuals, especially small-sized, which are considered of low economic value in fisheries sector, are also being utilised for jewellery (Ali, 2017). Specifically, artists take advantage of the unique; ornate beautifully patterned spines, rays and tails of lionfish to make and/or sell an assortment of jewellery from them (Ali, 2017).

After socioeconomic surveys in the first year (year 2018) of RELIONMED project implementation in Cyprus, 30 fishers active in lionfish-hotspot areas mentioned that they never sold a fish but used them for personal consumption (54%), discarded them (42%) or gave them to friends (4%). The major reasons for discarding lionfish were that it was dangerous (15%), the fisherman did not eat lionfish (8%) or because the lionfish caught were juveniles (8%).

27 | P a g e From surveys with 10 popular fish restaurant owners of the area, it was revealed that only half of them knew how to prepare a lionfish, while only one restaurant bought a lionfish for the price of about €6 per kg. Lessons learnt from the western Atlantic invasion need to be used to increase lionfish value and motivate people in removing lionfish from the ecosystems.

The therapeutic and medicinal properties of the lionfish venom are being explored. The venom’s complex includes a diversity of chemical compounds, which makes it complicated, expensive and arduous towards pharmaceutical companies when it comes to patent processing, testing, and getting a drug to the market. A single study on mice however, provided evidence that its venom can potentially be used in cancer treatment (Sri Balasubashini et al., 2006). More specifically, the study showed that lionfish venom peptides reduced tumour burden and ameliorated oxidative stress in Ehrlich’s ascites carcinoma xenografted mice, which provides preliminary evidence that its venom may be utilised in further cancer research and potentially in cancer therapy. In addition to the study on mice, there have been tests on rabbits and frogs, both of which showed neurotransmission and cardiovascular effects to all (Bellis et al., 2012). A more recent study has shown potential of phospholipase A2 from fresh venomous parts of lionfish (Pterois volitans) to become anti-HIV substances (Sommeng et al, 2019). Overall, research on fish venom is slow and still in the early stages, but it is a field that is actively being explored and is sparking the interest of current and rising scientists.

There is no evidence that the implementation of control mechanisms for lionfish has stimulated a market or consumer demand. However, creating market niches is believed to be key to ensure the sustainability of the removal activity. Commercializing lionfish products can increase awareness and financially sustain fishermen or lionfish cullers. Direct beneficiaries include lionfish cullers, fish

28 | P a g e mongers/markets, seafood restaurants, jewel crafters, souvenir shops and of course those buying the product.

Given the high reliance of the Mediterranean economy on fisheries, tourism and recreation, coupled with the known ecological impact of lionfish in the western Atlantic, the potential risk (to maritime economies) of an uncontrolled lionfish invasion is high. Any social or economic benefits from lionfish in the Mediterranean should arise from the creation of market niches for removal of lionfish without encouraging a sustainable supply. Education and awareness about the negative impacts of lionfish and importance of tackling its invasion should be prioritized.

SECTION B – Detailed assessment Important instructions:  In the case of lack of information the assessors are requested to use a standardized answer: “No information has been found.”  For detailed explanations of the CBD pathway classification scheme consult the IUCN/CEH guidance document.  With regard to the scoring of the likelihood of events or the magnitude of impacts see Annex.  With regard to the confidence levels, see Annex.

PROBABILITY OF INTRODUCTION and ENTRY

Important instructions:  Introduction is the movement of the species into the EU.  Entry is the release/escape/arrival in the environment, i.e. occurrence in the wild. Not to be confused with spread, the movement of an organism within Europe.  For organisms which are already present in Europe, only complete this section for current active or if relevant potential future pathways. This section need not be completed for organisms which have entered in the past and have no current pathway of introduction and entry.

29 | P a g e QUESTION RESPONSE CONFIDENCE COMMENT 1.1. How many active pathways are relevant to the few high The possible active pathways are primarily the Suez potential entry of this organism? Canal with an additional vector represented by aquarium release. Transport by ballast waters is unlikely but cannot be completely ruled out.

1.2. List relevant pathways through which the organism - Corridor The main pathway of introduction is the movement of could enter. Where possible give detail about the specific (Interconnected P. miles through the Suez Canal and into the Eastern origins and end points of the pathways as well as a waterways/basi Mediterranean Sea. However, according to Bariche et description of the associated commodities. ns/seas - Suez al., (2017), other invasion pathways should not be Canal) excluded including: - Release in  Fertilised eggs/larval transport via ballast nature (other water through the Suez Canal. intentional  Release in nature (other intentional release – release – aquarium hobbyist) aquarium hobbyist) RELIONMED project results indicate that the - Transport - pathways stowaway (ballast) and escape (aquarium), Stowaway intentional aquarium releases should not be excluded (Ship/boat (Dimitriou et al., 2019). ballast water contaminated The Suez Canal pathway is considered to be the most with P. miles important as it is a major artificial seaway that larvae) connects the Red Sea to the Mediterranean with no biosecurity measures currently in place. There has been a major immigration of non-indigenous Indo- Pacific species through the Canal into the 30 | P a g e Mediterranean Sea and it serves as a continuous pathway for introduction.

P. miles are native to the Red Sea where they are common (Schultz 1986). Molecular evidence from fish collected in the Mediterranean indicates that the primary invasion pathway is the Suez Canal, whereby the lionfish followed a common invasion pattern, typical to Lessepsian immigration in the Eastern Mediterranean Sea, the stepping stones pattern (Bariche et al., 2017).

In the North-West Atlantic lionfish escaped from aquaria and displayed one of the worst fish invasions reported so far (Schofield 2009).

Molecular analyses carried out in RELIONMED project from 56 lionfish individuals has found the same haplotypes with the neighbouring Red Sea and Gulf of Aqaba, confirming the findings of the previews studies (Bariche et al., 2017; Stern et al., 2018) that support the arrival of the species through the Suez Canal. Phylogenetic analyses and haplotype networks derived from RELIONMED indicate that a few individuals captured off Cyprus may stem from populations in the Indian Ocean and South Africa (Dimitriou et al., 2019).

Lionfish are a popular aquarium fish as they are highly durable and attractive with rich and vivid colours. A recent survey of nine aquaria in Turkey revealed that the aquarium trade in lionfish is common and that most companies imported their specimens from Indonesia and Kenya while more

31 | P a g e recently, some aquaria started to exhibit lionfish caught in the southeast part of Turkey (Gülenç, 2019). RELIONMED project surveys of six pet shops across Cyprus showed that five of them were selling lionfish prior the 2015-19 lionfish invasion of local waters with prices ranging from €35 to €65 per individual. At the time of the survey (2018), pet shops were importing lionfish from Singapore, Indonesia and the Gulf of Eilat off Israel.

Therefore, as well as the main invasion pathway through the Suez canal, Mediterranean lionfish invasion via ship ballast waters or from release of aquarium fish originating from the Red Sea, Indonesia and South Africa cannot be excluded.

Pathway name: Corridor: Interconnected waterways / basins/ seas (Suez Canal)

1.3a. Is entry along this pathway intentional (e.g. the Unintentional very high The introduction of P. miles in the Mediterranean Sea organism is imported for trade) or accidental (the is considered by most to be unaided and organism is a contaminant of imported goods)? unintentional. It is well documented that the Suez Canal has been a pathway for a large number of organisms in the Mediterranean (Galil et al., 2014) and molecular work has shown high levels of gene flow between the populations of the Red Sea and the Mediterranean (Hassan et al., 2003; Bariche & Bernardi, 2009). As the Suez Canal was expanded, it provides the opportunity for an increase in species migration from the Red Sea to the Mediterranean (Galil et al., 2014).

Driven by the general surface circulation at the eastern Mediterranean (Gerin et al., 2009), the lionfish sightings indicate that the species followed a 32 | P a g e similar pattern of invasion as observed in other Lessepsian immigrations; initially following an anticlockwise direction, i.e. along the coastline of southern Turkey, towards the islands of the SE Aegean Sea, and then northwards in the Aegean and westwards towards the Ionian Sea, further westwards along the Italian coastlines and western Mediterranean and also southwards to the North African countries (Hassan et al., 2003; Galil, 2008; Katsanevakis et al., 2013) (Figure 7).

Figure 4. General spread pattern of Lessepsian species in the Mediterranean – the example of Fistularia commersonii. Source: Katsanevakis et al., 2013.

1.4a. How likely is it that large numbers of the organism likely low A review of genetics flux from Red Sea to will travel along this pathway from the point(s) of origin Mediterranean failed to reconstruct a uniform pattern over the course of one year? for Lessepsian invaders since their genetic variability could span between opposites, from the absence of genetic loss to severe bottleneck (Bernardi et al., 2010). Nevertheless, even extreme bottlenecks (such as the case of F. commersonii) did not preclude population growth and rapid geographical expansion (Golani et al., 2007). The overview concluded that

33 | P a g e there is enough information to support that the passage used by larvae and/or adults to enter the Mediterranean (the Suez Canal) had the potential to sustain great numbers (Bernardi et al., 2010). Above all, the warming trend of the Mediterranean is providing more suitable ecological conditions for Lessepsian immigrants which are typically thermophilic, with tropical or subtropical origin (CIESM, 2008). Coupled with the recent widening and deepening of the canal seems to have caused the crossing of a tipping point for invasion of lionfish individuals.

A recent study on the lionfish invasion pathways suggested that the increasing number of individuals in the Mediterranean Sea is because of successful reproduction of a few individuals that crossed the Suez Canal (Bariche et al., 2017). The haplotype frequency found by Bariche et al. (2017) (2 out of 14 samples) is similar to a genetic study conducted in the native range of P. miles, in which 13 haplotypes were found among 88 specimens from the native region (Northern Red Sea and the Gulf of Aqaba; Kochzius & Blohm, 2005). The authors argue that the normal expectation that an invasive species should experience a reduction in genetic diversity has rarely been the case with Lessepsian immigrations due to multiple entries of organisms via the Suez Canal, which acts as a permanent open gateway.

The results of this study were further strengthened by Stern et al. (2018) who analysed a larger sample size (n = 86). The authors revealed the presence of six haplotypes, for which only four were shared among 34 | P a g e the invaded studied regions (Turkey, Cyprus and Israel). On this basis, the authors concluded that lionfish invasion in the Eastern Mediterranean is a product of multiple invasion in the form of a continuous invasion process or single multi-haplotype founding event. The same conclusions were derived from the RELIONMED project, which analysed 56 lionfish individuals and identified six different haplotypes for COI and three for CR genes. Furthermore, shared haplotypes were identified between Cypriot lionfish and from Lebanon, Greece and Italy, which is further evidence that the population in the Mediterranean shares common ancestors and is the result of the founder effect. RELIONMED genetic study (Dimitriou et al., 2019) suggest that Mediterranean Sea lionfish genetic divergence is more likely to be the result of multiple introductions than of random genetic drift after a unique introduction event.

It is expected that lionfish adult individuals and/or egg/larvae will continue to enter the Mediterranean Sea via the Suez Canal unless biosecurity measures such as a saline lock are put in place, although the rate of entry is largely unknown. The numbers of the organism moving from the Suez Canal to the Mediterranean Sea may further increase as warming of the Sea is expected to accelerate.

. 1.5a. How likely is the organism to survive during passage likely medium Since the opening of the Suez Canal in 1869 lionfish along the pathway (excluding management practices that have possibly managed to cross this corridor multiple would kill the organism)? times. They were first recorded in 1991 based on a single specimen which was collected by trawling at 35 | P a g e 35 m depth, north of Tel Aviv, Israel, likely after an introduction through the Suez Canal (Golani & Sonin, 1992). Following this record, no other specimens were reported until 2012, when lionfish was sighted again off Lebanon (Bariche et al., 2013).

The second invasion event has been confirmed using molecular tools to be likely the result of Lessepsian immigration through the Suez Canal. Molecular analyses carried out in RELIONMED (Bariche et al., 2017; Stern et al., 2018) support that the Suez Canal corridor is the main pathway of the species in the Mediterranean.

The high salinity levels in the Great Bitter Lake in the Suez Canal, probably acted as a natural barrier against the migration of Lessepsian species in the Mediterranean Sea, but the salinity barrier is reduced as the canal was enlarged (Katsanevakis et al., 2013; El-Serehy et al., 2018). Specifically, the surface salinity decreased from 50–52‰ to 43–44‰ while the bottom salinity decreased from 68–80‰ to 45– 46‰ (El-Serehy et al., 2018).

It is hard to assess whether the recently enlarged canal and accelerated warming are increasing the rate of lionfish entry through the corridor. Lionfish however has all the probability to survive along its passage along the Suez Canal. 1.6a. How likely is the organism to survive existing very likely very high There is currently no existing management to control management practices during passage along the pathway? P. miles in its passage along the Suez Canal. On the contrary widening and deepening of the Canal provides greater opportunity for the migration of species as this has removed very high salinity areas 36 | P a g e along the canal which used to be called the ‘bitter lakes’ and acted as a barrier to species intolerant of hypersaline waters (Galil & Zenetos 2002; Galil et al., 2017).

1.7a. How likely is the organism to enter Europe very unlikely high Empirical evidence shows that lionfish are already undetected? proliferating throughout the Mediterranean Sea (i.e. Israel, Turkey, Lebanon which are close to the Suez Canal as well as European Countries Cyprus and Greece). Its introduction into the Mediterranean Sea and its presence in the Eastern Mediterranean countries was noticed at its very early stages. This is largely due to the fact that the lionfish is a spectacular fish with unique fins and colour patterns that is easily spotted and draws attention. It is also caught by fishermen using traditional fishing methods (Kletou et al., 2016). Its appearance in other EU member states would be quickly and readily detected. The spread of lionfish in the eastern Mediterranean Sea has increased vigilance from both recreational divers and fishers as well as researchers across the Mediterranean basin. Most are familiar with its morphology and are able to report it in local/global media platforms/fora.

RELIONMED project socioeconomic surveys in Cyprus have found that almost 100% of the stakeholders (including relevant sea users such as fishers and divers) have heard about lionfish and they would recognize it if they see it in a picture, live or on television (Kleitou et al., 2019). Lionfish have become a common species in the eastern Mediterranean so it is expected that the vast majority of sea users of neighbouring countries will be able to 37 | P a g e recognize them.

Detection in some European waters may be delayed depending on monitoring efforts, the popularity of recreational diving or specific fisheries restrictions. Lionfish are known to live up to 300 m deep where they are unlikely to be detected easily but experience gained in the RELIONMED project has shown that they are concentrated in shallow waters where productivity is higher especially during the warm season, so they are readily detected.

1.8a. How likely is the organism to arrive during the likely high As the Suez Canal is permanently open, P. miles can months of the year most appropriate for establishment? arrive at any time. Since it is a thermophilic tropical fish it is probably limited by winter seawater temperatures in the coolest parts of the Mediterranean Sea. Temperature controls many metabolic processes, including reproduction (Brown et al., 2004). With the current and ongoing seawater warming occurring in the Mediterranean Sea, high water temperatures will favour P. miles establishment and increase its reproduction rates.

The mechanisms of lionfish reproduction enhance the chances of egg/larvae masses drifting from the Suez Canal to the Mediterranean, particularly during the warmer periods. Pterois miles individuals reach sexual maturity within the 1st year of age (Gardner et al., 2015). Drifting of lionfish egg/larvae is the major way of introduction through the Suez Canal. Both males and females are capable of spawning year- round (Gardner et al., 2015) According to information derived from the Atlantic invasive range, females are able to spawn 1800–41945 eggs per spawning event 38 | P a g e (the larger the female the more eggs are released) at a rate of 2-3 days (Gardner et al., 2015) or over 2 million of eggs per female per year (Morris, 2009). The eggs and/or embryos are encased and protected in a hollow gelatinous matrix, and later on, the embryos become free floating (Morris et al., 2008, 2011; Gardner et al., 2015) with their planktonic larval stage in the Bahamian Archipelago lasts on an average 26.2 days (Ahrenholz & Morris, 2010); thus enhancing their ability of drifting, introduced and spread.

RELIONMED studies have shown lionfish reproduces year-round off Cyprus but their average gonadosomatic index is significantly higher during warm months which can arguably indicate that reproduction is more frequent and intense during summer-autumn period. Combined with its rapid maturity and growth, the mass production of eggs per female per spawning event (Gardner et al., 2015), and the anticipated shortened larval stage (Côté & Green, 2012), the lionfish individuals that cross the Suez Canal in the spring-autumn period, when the sea is warmer and there is plenty of prey, are expected to have increased likelihood of surviving, establishing and spreading to EU member states.

1.9a. How likely is the organism to be able to transfer very likely very high The movement of individuals to the Mediterranean from the pathway to a suitable habitat or host? Sea could be largely aided by larval dispersal through ocean/sea currents over great distances (Morris et al., 2008; Côté et al., 2013a) and thus, P. miles can be transported from the pathway to suitable habitats. The survivor juveniles will settle down on benthos and usually occupy a small area. Lionfish is a generalist species, able to thrive in various habitats 39 | P a g e and a large range of depth. Once adults, rocky or hard substrate surfaces (Biggs & Olden, 2011) are their more preferred habitat. In the Mediterranean, lionfish are mostly detected on hard substrata (including artificial reefs) and over seagrass (Posidonia oceanica) habitats with less abundances (RELIONMED data). These preferred lionfish habitats are representative of the majority of the Mediterranean coastlines.

1.10a. Estimate the overall likelihood of entry into Europe very likely very high This pathway has been documented to be the pathway based on this pathway? of a large number of species that have entered the eastern Mediterranean. P. miles follows the pattern exhibited by other Lessepsian immigrants strongly suggesting that this is the pathway used (Galil et al., 2015). Genetic analysis shows haplotypes found in Red sea populations and suggest a number of invasion events (Bariche et al., 2017; Stern et al., 2018; Dimitriou et al., 2019). While the organism has already entered in the Mediterranean Sea and is proliferating, the likelihood of continuous entry through the Suez Canal is considerably high, unless a more effective management measure is put in place. Lionfish will mainly continue to spread in the EU waters through secondary dispersal from the entry point (see Q2.2a).

Pathway name: Release in nature (other intentional release – aquarium hobbyist)

1.3b. Is entry along this pathway intentional (e.g. the intentional high The entry can occur by intentional releases into the organism is imported for trade) or accidental (the sea by hobbyists, fishermen, pet owners etc. organism is a contaminant of imported goods)? 1.4b. How likely is it that large numbers of the organism likely very high Lionfish are imported into many Mediterranean 40 | P a g e will travel along this pathway from the point(s) of origin countries by the aquarium trade from the Indo-Pacific. over the course of one year? RELIONMED surveys indicate that the majority of pet-shops in Cyprus are selling lionfish and the situation is similar in other Mediterranean countries such as Turkey (Gülenç, 2019), Lebanon (Bariche, pers. communication), and Italy (Tiralongo, pers. communication). Other aquarium species have been intentionally released in Cyprus when their size was too large for the tank (Kousteni et al., 2019). Until recent times, aquarium release of this species was not so likely (since the fish was observed only once before becoming a Lessepsian invader), but this pathway has increased much in importance and should be seriously monitored. A potential release could enhance substantially the genetic structure and variability of lionfish established in the basin; thus enhancing the invasion.

If P. miles is listed as an IAS of Union Concern (Regulation 1143/2014), then its trade into and within EU will be banned. Management measures and/or awareness among citizens of the non-EU countries should be also promoted to deactivate this potential pathway for the lionfish. Countries such as Turkey are already using lionfish captured in their coasts to sell them in the aquarium/petshop trade (Gülenç, 2019). In Cyprus, lionfish captured alive by free divers was sold in numerous occasions to pet shops (Jimenez unpublished data). Trade of the lionfish from the invasive Mediterranean range to countries where lionfish has not yet established can potentially lead to accidental spread of the lionfish in the unoccupied region.

41 | P a g e 1.9b. How likely is the organism to be able to transfer likely high According to recent SDMs, current suitable habitats from the pathway to a suitable habitat or host? are limited to the central and eastern Mediterranean (Figure 3) (D’Amen & Azzurro, 2019, Poursanidis et al., unpublished). Based on the 15 °C scenario a larger Mediterranean area is available for its establishment (see Ch2). If intentionally released, P. miles will likely be able to successfully be transferred to a suitable habitat (e.g. See Q1.9a), and if it reproduces with other escaped larva or adults, then it can spread far and wide.

1.10b. Estimate the overall likelihood of entry into Europe likely medium The pet trade is a pathway responsible for at least 18 based on this pathway? (from approximately 100) fish species introductions in the Mediterranean up to date (Giovos et al.,2018). RELIONMED genetic studies have shown that its entry through the aquarium trade is a possible scenario for the lionfish invasion in the Mediterranean (Dimitriou et al., 2019).

Introduction (i.e. movement of the species into the EU) through this pathway is very likely but entry (release in the environment) is not as likely, especially given the high awareness outreach of the RELIONMED project about the invasive character and potential impacts of lionfish. Even if lionfish adults are introduced through this pathway, there is a possibility that they will be released in unsuitable habitats for its establishment (see Q1.9b). Nevertheless, special caution is needed to avoid intentional releases.

In areas where lionfish is abundant, it is possible that citizen residents might believe that release of lionfish will not cause any effects; thus likelihood of release 42 | P a g e increases. New introductions can increase genetic pool and consequently invasive success of lionfish. It is important that biosecurity and precautionary measures are implemented and for people to be aware and environmentally conscious enough to avoid intentional release of individuals to the marine ecosystem. Pathway name: Transport – stowaway (Ship / boat ballast water) 1.3c. Is entry along this pathway intentional (e.g. the Unintentional very high Transportation of lionfish larva or eggs via ballast organism is imported for trade) or accidental (the water from shipping will be unintentional. organism is a contaminant of imported goods)?

(If intentional, only answer questions 1.4, 1.9, 1.10, 1.11) 1.4c. How likely is it that large numbers of the organism unlikely low It is well documented that ballast waters can transfer will travel along this pathway from the point(s) of origin organisms from one area to another when loading and over the course of one year? unloading cargo or in rough seas (Nunes et al., 2014).

It is estimated that approximately 1 species every 25 days is introduced in European Seas through shipping-mediated pathways (Katsanevakis et al., 2013) and this number is expected to increase as a result of our increasing reliance on maritime trade (Mannino et al., 2017). The number of fish species recorded worldwide from ballast waters as overviewed by Wonham et al. (2000) is very low, ca. 50 species, and mostly of other families, especially gobiidae and blenniidae. Filtered water and conditions in ballast waters are not favourable for fish species, thus we consider the chances of lionfish travelling through this pathway low, especially over the course of a year. However, high uncertainty needs to be acknowledged; since fishes might have been under sampled in ballast-water studies.

43 | P a g e

Indeed, most ballast water sampling studies revealed organisms of small body sizes, predominantly up to a few millimeters (Gollasch et al., 2002). However, larger organisms were also found. During one comprehensive German shipping study (Gollasch & David, 2019 and references therein), fishes up to 15- cm body length and larger juvenile decapods with a carapace width of >2 cm were found at the bottom of ballast water tanks. As most ballast water systems of ships have strainers and coarse grid filters with opening sizes of 5 mm to protect the ballast water pump from larger debri in the water, findings of larger organisms in ballast tanks do therefore indicate that species were either pumped on board as eggs or younger larvae and were growing inside a tank, or that reproduction has possibly even occured inside a ballast tank. In fact, Gollasch et al. (2000) found by daily samplings of the same ballast tank on an approximately 3-week voyage an increasing number of copepods in a tank and they concluded that reproduction occurred inside the tank during this voyage. In another ballast water sampling study where tank covers, the so-called manholes were opened for sampling, large fish of about 10-cm body length freely swimming inside the tank were observed. These were not observed in samples taken from the ballast water pipework of this vessel during ballast water uptake nor during discharge (Gollasch & David, 2019 and references therein). Therefore, there are evidences that macroorganism as well, are found in ballast tanks, but they are largely unknown and neglected aspect (Gollasch & David, 2019 and references therein).

44 | P a g e

Other fish species such Tridentiger trigonocephalus (Goren et al., 2009), Omobranchus punctatus (Golani, 2004) and Bregmaceros atlanticus (Goren & Galil, 2008), Leiognathus berbis (Alshawy et al., 2016) Chlorurus rhakoura (Insacco & Zava, 2017) have been suspected to be introduced in the Mediterranean via this pathway.

After Maclsaac et al., (2016): “Routine pore size of screens on the sea chests (that prevent large individuals and debris from entering ballast tanks) have holes 15–25mm diameter or slots 20–35mm width (Coutts et al., 2003) and can equal or exceed the size of many larval fishes, including lionfish whose reported larval stages range from 1.5 to 11mm (Mito, 1956; Imamura & Yabe, 1996; Vásquez-Yeomans et al., 2011). Cross-sectional area of these fishes is the more relevant metric and would be even smaller. Owing to their small size, it is anticipated that larval lionfish could be drawn into sea chests without injury when taken up prior to settlement, which occurs between 20 and 35 days after hatching [Ahrenholz & Morris, 2010]. These individuals could easily fit through sea chests and their associated coarse-sized grates. For example, a 15–16 day old larval individual is ~ 8mm standard length (Vásquez- Yeomans et al., 2011), and at settlement is between 10 and 12mm (Fishelson, 1975)., Given that lionfish can reproduce every 2–4 days and produce up to 25,000 eggs per reproductive bout or approximately two million eggs per year (Gardner et al., 2015), it seems likely that larval lionfish could be taken up through

45 | P a g e sea chests at some frequency”

Lionfish larva or eggs could be transferred from the Red Sea or even further to the Mediterranean. Since the Suez Canal is for shipping then it is possible that a number vessels may have brought ballast water with lionfish larvae or eggs, thus providing large numbers of individuals over the course of one year (Galil et al., 2015). A large number of vessels are moving directly from the Suez Canal or eastern Mediterranean to western Mediterranean countries thus a possible transportation directly to the central/western Mediterranean could substantially increase the spread pattern of the lionfish in the basin.

There are no records or information on successful introduction of scorpaenids via ballast-water transport but two individual scorpaenids were recorded in ballast waters in the literature (Wonham et al., 2000). Additionally, lionfish are reported from several harbour areas (Schultz, 1986) and coupled with the high tolerance of lionfish in various conditions; introduction or spread via ballast water cannot be excluded.

Although transfer via ballast waters could be possible for some propagules, it is unlikely that large numbers can be transferred in just one year. Specific further research would be needed to assess the risk of lionfish transportation in ballast water.

1.5c. How likely is the organism to survive during passage unlikely low As exhibited in other species it is likely that larvae along the pathway (excluding management practices that and eggs survive in ballast waters and can be

46 | P a g e would kill the organism)? transferred to different areas (Nunes et al., 2014). The majority of organisms taken-up in ballast water expire at an exponential rate during the first 3–5 days in a ballast tank due to a wide range of conditions that occur within them. Ballast tanks are, for most organisms, unfavourable habitats, there is an absence of light and there can be limited resources such as oxygen, food, lack of shelter and the varying temperatures that may take place during a voyage that may considerably differ from the ballast water uptake area. . 1.6c. How likely is the organism to survive existing unlikely high According to management practices during passage along the pathway? http://www.emsa.europa.eu/implementation- tasks/environment/ballast-water.html “At present there is no direct EU Law on Ballast Water, however Regulation (EU) No 1143/2014 on the prevention and management of the introduction and spread of invasive alien species recognises the BWM Convention as one of the possible management measures for invasive species of concern. The level of Invasive Alien Species and their environmental impact is also one of the many descriptors for assessing Good Environmental Status under the Marine Strategy Framework Directive”.

The BWM convention that came into force in 2017 and requires ships to manage their ballast water to remove, render harmless, or avoid the uptake or discharge of aquatic organisms and pathogens within ballast water and sediments, based on a ship-specific ballast water management plan. All ships must carry a ballast water record book and an International Ballast Water Management Certificate. 47 | P a g e

The BWM convention enforces ships to comply with two different standards, corresponding to:

D-1 standard: requires ships to exchange their ballast water in open seas, away from coastal waters. Ideally, this means at least 200 nautical miles from land and in water at least 200 metres deep. When this is not possible, the BWE shall be conducted at least 50 nm from the nearest land and in waters at least 200 metres in depth or in a designated ballast water exchange area. By doing this, fewer organisms will survive and so ships will be less likely to introduce potentially harmful species when they release the ballast water.

D-2: a performance standard which specifies the maximum amount of viable organisms allowed to be discharged, including specified indicator microbes harmful to human health.

New ships must meet the D-2 standard from today while existing ships must initially meet the D-1 standard. An implementation timetable for the D-2 standard has been agreed, based on the date of the ship's International Oil Pollution Prevention Certificate (IOPPC) renewal survey, which must be undertaken at least every five years. Existing ships, who are subject to the phased implementation schedule, have potentially (depending on the renewal of their ship certificates) until the 8th September 2024, by which time all ships will comply with the D2 standard. For most ships, this involves installing

48 | P a g e special equipment.

Ships conducting ballast water management under the strict D2 standard, must discharge fewer than 10 viable organisms per cubic metre that are greater than or equal to 50 micrometers in minimum dimension and fewer than 10 viable organisms per millilitre that are less than 50 micrometers in minimum dimension and greater than or equal to 10 micrometers in minimum dimension; and discharge of the indicator microbes must not exceed the specified concentrations.

Lionfish eggs are planktonic and can ride the ocean currents and cover large distances for about a month before they settle (Ahrenholz & Morris, 2010). If eggs are discharged from ballast waters despite management measures, they have high chances to successfully survive even if they are released far from the shore; however, the chances of surviving the management practices along this pathway are low

1.7c. How likely is the organism to enter Europe very unlikely high See Q1.7a undetected?

1.8c. How likely is the organism to arrive during the likely high There is no available literature about the spawning months of the year most appropriate for establishment? frequency of P. miles in its native environment. Considering however, the findings from the Atlantic invasive range, which is characterised by similar temperature regimes (e.g. Bermuda), the lionfish there

49 | P a g e appear to have an abbreviated reproductive season but batch fecundity estimates are similar to other areas of the Atlantic invasion (Eddy et al., 2019). Lionfish invasion in the Mediterranean might share similar characteristics, and RELIONMED project data has shown that lionfish reproduces year-round but there is higher reproduction during the warm summer-autumn period.

Taking this into account the entry of fertilised eggs/larvae into ballast waters, their transportation to the Mediterranean Sea is likely to occur at any time of the year but individuals arriving at the months when seawater temperature is higher (summer, early autumn), may have higher survivability and more success to establishment.

The score (‘likely’) assumes that the lionfish manages to enter via ballast waters in the Mediterranean which is an unlikely scenario (see Questions above).

1.9c. How likely is the organism to be able to transfer likely medium See Q1.9a from the pathway to a suitable habitat or host? Suitable habitats only exist in the warmer Mediterranean where the temperature is not too cold for the lionfish (worst-case scenario) or just in south central and eastern Mediterranean (best-case scenario).

The score (‘likely’) assumes that the lionfish manages to enter via ballast waters in the Mediterranean which is an unlikely scenario (see Questions above).

1.10c. Estimate the overall likelihood of entry into Europe possible medium There are a number of studies showing entry of 50 | P a g e based on this pathway? species through ballast waters (Nunes et al., 2014) and even a model showing how lionfish can survive in ballast waters and establish in a new area (Maclsaac et al., 2016).

Evidence from the early stages of the second invasion event exclude ballast waters as an active pathway since lionfish were not present at major port locations (Galil et al., 2015; Bariche et al., 2017), however, possibility for future introductions cannot be completely excluded.

End of pathway assessment, repeat as necessary.

1.11. Estimate the overall likelihood of entry into Europe very likely medium P. miles is already proliferating through natural based on all pathways in relevant biogeographical regions dispersal within the Mediterranean Sea (currently in current conditions (comment on the key issues that lead moving westwards), and therefore in the marine to this conclusion). ecoregions of the EU. The organism already established populations in neighbouring countries of the Suez Canal (Israel and Lebanon), with Cyprus being the first EU member state that has been infested and displaying the largest concentrations and lionfish hotspot aggregations among EU countries. An intentional release from aquarium trade or transfer of fertilized eggs/larvae via ballast waters can be also potential pathways but less possible Such pathways should be also seriously considered and strictly controlled within EU, as new genetic input from other areas, may further increase its genetic diversity and structure, and hence its viability as a species and invasion success.

To conclude, the major pathway that should be given attention and priority is the Suez Canal. The aquarium 51 | P a g e trade should also be controlled and measures for education/awareness can be promoted. Transport by ballast waters is unlikely, especially with the expected BWM measures which can be considered adequate for prevention.

1.12. Estimate the overall likelihood of entry into Europe very likely medium P. miles is a species of Indo-Pacific origin and was based on all pathways in relevant biogeographical regions sighted for the first time in the Mediterranean Sea in in foreseeable climate change conditions? 1991 in Israel. Since then, the species had not been detected until 2012, when it made its re-appearance off Lebanon. The evidence so far, point out the Suez Canal to be the primary introduction pathway. The flow of individuals in the Mediterranean Sea might be continuous, but the rate of entry is yet to be uncovered. Although there is no empirical evidence, the re-appearance of P. miles in 2012 can be linked to more suitable conditions (e.g. climate change, enlargement and widening operations of the Suez Canal, etc.).

The current and projected seawater warming is likely to benefit its establishment in the Mediterranean Sea, allowing it to expand westwards and northwards mainly through natural larval dispersal from Mediterranean established populations and not through the pathways described. Even if current habitat suitability (based on SDMs) seems to be limited to the EMED, the species could expand its ecological niche further and colonize novel areas and climates.

52 | P a g e

PROBABILITY OF ESTABLISHMENT

Important instructions:  For organisms which are already established in parts of the Union, answer the questions with regard to those areas, where the species is not yet established. If the species is established in all Member States, continue with Question 1.16.

QUESTION RESPONSE CONFIDENCE COMMENT

1.13. How likely is it that the organism will be able to very likely very high Although the Mediterranean Sea is cooler than the establish in the EU based on the similarity between Red Sea, particularly in winter, P. miles has a climatic conditions in Europe and the organism’s current broad thermal tolerance spectrum, which allows it distribution? to survive down to 10.7 °C (Kimball et al., 2004; Dabruzzi et al., 2017; Barker et al., 2018). Even if current modelling evidenced suitable areas only in the Eastern Mediterranean, possible niche expansion could allow the species to spread in other European countries. This hypothesis is also reinforced by the spread dynamics of the complex P. volitans/ miles in the western Atlantic where it reached up to North Carolina at Cape Hatteras (Barker et al., 2018), where winter temperatures fall to an average of 15 °C (Sea temperature, 2018). This relatively cool water tolerance is also evident by its current establishment in the entire Levantine basin and the progressive expansion westwards (Azzurro et al., 2017).

Given that the lionfish is already established in EU, the likelihood of establishment is scored very high with very high confidence. The EU marine subregions where lionfish are currently established are Cyprus and Greece (the southern Aegean 53 | P a g e islands, Cyclades and the Ionian Sea).

It is uncertain where the lionfish will expand and if it manages to establish in the western Mediterranean and expand to northern regions under current or foreseeable climate change (for more information see Ch2). 1.14. How likely is it that the organism will be able to Western Low Little is known about the abiotic conditions of establish in the EU based on the similarity between other Mediterranean Sea Pterois miles in the Mediterranean Sea. Modelling abiotic conditions in Europe and the organism’s current unlikely based on the work prior the invasion indicated that distribution? SDMs Mediterranean habitats such as in Cyprus were very likely based on the unsuitable for lionfish expansion (Poursanides, 15 oC 2015) Unfortunately these simulations were wrong and lionfish have since become established in Adriatic Sea medium great densities at the eastern Mediterranean. unlikely (except the very southern Adriatic Studies from the Western Atlantic Ocean Sea where is likely) demonstrated that lionfish is a habitat generalist with great biological and ecological versatility, Ionian Sea and the high inhabiting coral reefs, seagrass beds, mangroves, Central Mediterranean human-created habitats, deep slopes and the ocean Sea floor at depths as great as 300 m (Barbour et al., very likely 2010; Biggs & Olden, 2011; Lee et al., 2011; Claydon et al., 2012; Côté et al., 2013a; Gress et Aegean-Levantine Sea very high al., 2017). In addition to these, Pterois spp. and very likely specifically P. volitans can withstand a wide range of salinities, from as low as 7 ‰ (Jud et al., 2015) Overall very high to as high as in its native range (43 ‰). This has very likely also been confirmed by Jud et al., (2011), who found P. volitans/miles complex in an estuarine system in Florida, at salinity levels between 5.8 to 38.6‰.

Considering the above information and the 54 | P a g e availability of similar habitats in the rest of the Mediterranean Sea where lionfish has not yet recorded, P. miles will likely spread across the Mediterranean basin but the areas prone to invasion cannot be estimated with certainty due to niche unfilling/expansion (see Ch2)

1.15. How likely is it that the organism will become very likely high Lionfish are commonly kept in private aquaria established in protected conditions (in which the through pet trade. Many exotic fish species environment is artificially maintained, such as wildlife distributors promote P. miles for trade with guides parks, glasshouses, aquaculture facilities, terraria, for special care (e.g. zoological gardens) in Europe? https://www.liveaquaria.com/product/3339/miles- lionfish?pcatid=3339&c=15+36+3339). In fact, the oldest known lionfish was 30-33 years old and was kept in captivity (Potts et al., 2010). Evidence of successful reproduction within aquaria are limited. 1.16. How widespread are habitats or species necessary widespread very high Habitats necessary for the survival, development for the survival, development and multiplication of the and multiplication of P. miles are widespread in organism in Europe? Europe and its current invasive range in the Mediterranean Sea. It lives and reproduces in a variety of coastal habitats within a wide range of salinities and temperatures, including estuaries, muddy and rocky bottoms on shallow and deep areas (down to more than 300 m). 1.17. If the organism requires another species for critical NA P. miles does not require another species to stages in its life cycle then how likely is the organism to complete its life cycle. become associated with such species in Europe?

1.18. How likely is it that establishment will occur despite very likely very high The species competes strongly and successfully competition from existing species in Europe? with existing species in Europe. P. miles may even be outcompeting juvenile groupers, particularly Epinephelus marginatus which use the same habitat and food resources. In its Atlantic invasive 55 | P a g e range, the lionfish complex outcompetes Caribbean spiny lobsters and local groupers (Curtis-Quick et al., 2013; Raymond et al., 2015). The fact that lionfish share significantly better traits compared to other mesopredators (e.g. matures in 1 year compared to 4-7 years for a grouper) gives it an advantage for its expansion. Lionfish interactions with native predators did not appear to influence their colonization or establishment in the Caribbean since they are able to avoid trophic competition by adopting a unique predatory tactic and by changing behaviour when competitors are present (Hackerott et al., 2013; Raymond et al., 2015)

1.19. How likely is it that establishment will occur despite very likely very high P. miles predators are generally scarce thanks to its predators, parasites or pathogens already present in antipredator venomous spine defences, although Europe? several fish have been documented to prey on lionfish. Large groupers may serve as a biocontrol agent against the lionfish, as they prey on small- sized lionfish (Bernadsky and Goulet, 1991; Mumby et al., 2011). Predation from the bluespotted cornetfish Fistularia commersonii and moray eels is known in its native range (Bernadsky and Goulet, 1991; Bos et al., 2017). In the Mediterranean Sea, aside from one questionable observation, where a dusky grouper (Epinephelus marginatus) was reported to swallow a lionfish (Turan et al., 2017), there is evidence in Cyprus of groupers preying on lionfish in depths between 40 and 70m (Jimenez et al., 2018). Natural predation of lionfish may be more widespread than suspected. Even if so, these predators are overfished thus they are unlikely to pose any 56 | P a g e serious pressure to lionfish expansion unless efforts are made to rebuild their populations and adult sizes, for example through well managed MPAs. There remains hope that in well managed marine protected areas, healthy populations of natural predators may control lionfish populations in EU Countries. Presence of natural predators has the potential to control distribution and abundances of introduced species and damp their potential induced trophic cascade (DeRivera et al., 2005; Carlsson et al., 2011; Papacostas et al., 2019). (Carlsson et al. 2011; DeRivera et al. 2005; Papacostas and Freestone 2019)

Pterois volitans/miles complex are generally resistant to pathogens. In fact, the lionfish hosts their own unique external microbiota communities, which can provide resistance against pathogens something that has being observed from specimens collected in invasive (Atlantic, Caribbean) and native range (Stevens et al., 2016).

As for parasites, the lionfish is a host of several ecto-parasites observed from native and invasive range both in the Atlantic and Mediterranean (in Morris et al., 2008; in Ramos-Ascherl et al., 2015; Antoniou et al., 2019) as well as endo-parasites (Diamant et al., 2004: RELIONMED data). Pterois volitans/miles complex have lower parasite prevalence than the native reef species of North Carolina and Bahamas (Morris et al., 2008). In the Mediterranean, there are no studies investigating the frequency of lionfish and parasites associations. Lionfish culled as part of the

57 | P a g e RELIONMED project often have Cymothoidae isopod parasites (Nerocila bivittata). Antoniou et al., (2019) document these native individuals as reproductive adults and juveniles on the skin and inner branchial cavity of lionfish hosts off South Cyprus. More recently, copepods of the order Siphonostomatoida were found in the mouth of lionfish specimens. A lack of abundant cleaner fish in the Mediterranean may allow adapted ectoparasites such as copepods and isopods to harm invasive lionfish but they will very unlikely prevent its establishment. Further research is clearly needed to determine the sources and effects of lionfish parasites.

1.20. How likely is the organism to establish despite very likely very high Existing management practises currently running existing management practices in Europe? within EU did not prevent lionfish establishment. The species is already established in Mediterranean and further unaided expansion is expected despite any current or future management measures. Management actions can only control its populations but not eradicate it or prevent its further expansion in European waters.

In its Atlantic invasive range one of the most common management practices are to remove lionfish (Barbour et al., 2011; Harms-Tuohy et al., 2018). The frequency of lionfish removals (either in the form of coordinated diving removals or fishing pressure) may in fact allow for population control to as minimum as possible and mitigate lionfish impacts in priority areas, but it is not considered an ultimate tool for preventing its establishment (Barbour et al., 2011; Côté & Smith, 58 | P a g e 2018). For instance, multiple removals off Little Cayman Island at irregular intervals over 7 month period, restricted the size frequency distribution towards smaller individuals, which allowed decreased predation on ecologically and economically important fish (Frazer et al., 2012). Furthermore, a study on Bonaire and Curaçao, in southern Caribbean, revealed significant reduction in both lionfish densities and biomass compared to sites that were not targeted for culling (León et al., 2013). Similar results were observed in Puerto Rico. The removals decreased the lionfish densities and re-colonization to the targeted area at the initial densities was gradual and took about 9 months (Harms et al., 2018). Use of trophic dynamic model of West Florida and Gulf of Mexico has shown that small increases in lionfish harves can reduce peak biomass by up to 25% (Chagaris et al. 2017). Even though culling efforts have proven to be an effective eradication practice at local scales and showed to reverse the declines of reef fish (Green et al., 2014), it has its limitations. In fact, intense culling practices could ultimately alter the lionfish’s behaviour by turning them more vigilant and as a result to increase the removal effort per se (Côté et al., 2014a). On the other hand, partial culling does not remove all individuals within a local area but it reduces the time and effort by 30% and is still as effective as a complete local eradication (Côté et al., 2014a; Côté et al., 2014b). According to Barbour et al., (2011) and (Morris et al., 2011), if 15- 65% per year or 25% per month, respectively of adult population is eliminated, then

59 | P a g e it would be enough to drive population declines.

Many of the controlling practices in the western Atlantic Ocean were carried out through various removal techniques and tools. The most effective low cost removal practices include spearfishing (polespear or speargun), vinyl/mesh hand-netting and Hawaian slings for large individuals (Akins 2012). Many of these are restricted to divers, while other removal techniques that can be accessed by fishers include traps and hook and line. The former showed to catch lionfish as by-catch in lobster and fish traps (Frazer et al., 2012), whereas the latter is effective at deeper waters (91-183 m depth) with the inclusion of squid as cut-bait (Akins 2012). Other fishing techniques such as trawling and seining are deemed to be ineffective and plausibly to negatively impact the populations of other susceptible species (Côté et al., 2013a).

Undoubtedly monitoring requires continuous efforts, raising awareness, decent organization, citizen participation and stakeholder engagement (Akins 2012; Scyphers et al., 2015). To achieve monitoring and many of the controlling practices, most of the removals in the Atlantic Ocean were in the form of organized events such as tournaments, daily derbies and monthly contests, which in some cases allowed the removal of 1,408-2,000 lionfish in Bahamas and Mexico within a single day (Akins 2012). The contestants were composed by recreational spearfishers previously trained on how to collect/handle the lionfish underwater and would compete against other teams to earn a prize.

60 | P a g e The development of several targeted lionfish fisheries, was another idea established in Florida Keys and were composed by divers to keep lionfish populations down either in marine protected areas or other selected sites of interest (Akins 2012).

Given the fact that many lionfish were harvested and removed out of the Atlantic waters, none of the individuals has gone to waste. Instead, they were utilized by science – mainly fo morphometric measurements and diet studies – to further understand its biology and ecology, as well as being promoted to the market for consumption or for jewel-crafting. Unlike Lagocephalus sceleratus, the toxins of the lionfish are located in the spines. When the spines are removed, the rest of the body is edible and often yields a 30% fillet (out of the total biomass) with great palatability, mild flavour and rich in saturated and omega-3 fatty acids (Morris and Breen, 2011). Many of these controlling actions are critical steps and chapters in a successful management plan to ameliorate the invasion of lionfish, conserve the biodiversity and habitats of the Mediterranean Sea as well as to boost the local economy.

Due to its sentinel location near the Suez Canal, Cyprus is the first EU-country to directly be affected by the Lessepsian immigrations through the Suez Canal. Its strategic position should be used for early warning / response, monitoring the impacts of invasive species at an early stage of their introduction, and understanding the cost-

61 | P a g e effectiveness of potential management measures. The efficiency of removals are currently being studied as part of the RELIONMED project in the Mediterranean and some useful conclusions have been extracted. Following some experiments which are undergoing, preliminary results of the RELIONMED project indicate that manual removals of lionfish by divers can be effective in controlling their population in priority areas. Experiments were conducted during summer periods and have shown that lionfish recolonize fast depending on the habitat complexity and the area of removal. It has been shown that extensive homogeneous areas require higher commitment (i.e. removals by many people and/or very frequent removals, e.g. weekly) for any management to be successful. On the other side, removals in hard substrate habitats with prominent erosional features (crevices, depressions, dents, ledges) have been found to be more effective and feasible with small groups of divers (e.g. 2-3 divers). Habitat complexity has been found to drive aggregating behaviour in the invasive Caribbean lionfish (Hunt et al., 2019) and should definitely be taken into account when considering lionfish management actions.

Cost-effectiveness of manually removing lionfish from selected locations can only be assured if divers (citizens) are allowed to remove lionfish under special permits. Targeted removals during peaks of lionfish numbers and reproduction season (i.e. summer period) can cause more impact to their population.

62 | P a g e

1.21. How likely are existing management practices in possible high Current existing managements in EU, particularly Europe to facilitate establishment? artificial reefs and Marine Protected Areas (MPAs) may in fact facilitate the establishment of P. miles. Artificial reefs are being deployed widely in the EU waters as a way to improve species recruitment and recover the ecosystems, while at the same time promote alternative sustainable sea uses (e.g. dive ecotourism) (Jensen et al., 2012). Recent research has shown that artificial reefs have the potential to facilitate tropical species at range edges and assist species range expansion (Paxton et al., 2019). In Cyprus, artificial reefs have been found to be some of the most favourite habitats of lionfish where it is found in great densities.

MPAs have been proposed as the solution to increase complexity of ecosystems, improve ecosystem resilience and fight against the increasing global pressures (Sala & Giakoumi, 2017). However, MPAs of the Mediterranean have been found to host higher biomass of non- indigenous species compared to adjacent unprotected areas (Giakoumi et al., 2019a). Beneficial effects of fisheries reduction in MPAs may be dampened by the impact of climate change and alien species when acting together (Corrales et al., 2018). For these reasons, encouragement for targeted removal and commercial and/or recreational utilization of non-indigenous species has been identified by scientists as the most applicable physical measure for invasive species in the Mediterranean (Giakoumi et al., 2019b).

63 | P a g e In the Caribbean Sea, the density and biomass of the invasive lionfishes (Pterois complex) have been found to be lower in MPAs than in unprotected areas, and this was attributed to the restoration of predators (Mumby et al., 2011), and targeted removal (by spearfishing) within MPAs (Hackerott et al., 2013). The fishing protection offered within MPAs in the area will benefit the growth of juvenile individuals into adult stages. On a local scale, targeted removals and long-term commitment to removals are effective at shifting direct impacts of invasive lionfish away from highly vulnerable species (Barbour et al., 2011; Côté et al., 2014). Thus manually removing lionfish from MPAs can be potentially used to protect MPAs role towards enhancing the resilience of the ecosystems and benefiting the wildlife.

1.22. How likely is it that biological properties of the very likely very high The biological properties of P. miles will certainly organism would allow it to survive eradication campaigns allow it to survive eradication campaigns. The in Europe? organism exhibits rapid growth rates, early sexual maturity, and a considerably high reproductive output (RELIONMED project data). Moreover, the species can occupy areas beyond the limits of SCUBA diving, up to more than 300 m depth (Gress et al., 2017; Andradi-Brown, 2019) where manual control is extremely challenging.

Eradication campaigns would allow for population control in some areas (i.e. control of lionfish below levels that can cause damage to the other biota). Lionfish eradication will not be possible now in the Mediterranean and larval export is expected to 64 | P a g e occur continuously, hence it is very unlikely that the lionfish will be eliminated. More information about the results of management activities for lionfish in the Mediterranean and abroad have been described in Q1.19 while biological characteristics are mentioned below (Q1.23).

1.23. How likely are the biological characteristics of the very likely very high For the reproductive characteristics and life organism to facilitate its establishment? history/dispersal capacity of the species see Q1.4.

One of the primary reasons of why P. miles is so successful in the Mediterranean Sea is because of its biological characteristics and the enemy release hypothesis; both have been discussed in the case of the Atlantic invasion (Côté et al., 2013a; Sandel et al., 2015). Furthermore, their morphological peculiarities together with their behaviour enhance their success as predators in two ways and might partially explain why lionfish exhibit higher consumption rates than similarly sized native predators occupying the same habitats (Albins & Hixon, 2013; Azzurro et al., 2014). First, their slow movements, cryptic coloration, and elongated fin rays give them the appearance of a tuft of seaweed, a crinoid, or a tube-worm, perhaps a case of masquerade mimicry as well as camouflage and second, while stalking prey, lionfish flare their large, fan-like pectoral fins and slowly herd small fish, which are typically cornered then rapidly consumed. Atlantic prey fishes have not encountered such a predator in their evolutionary history, and native prey seem to take no evasive action.

65 | P a g e Pterois miles exhibits rapid growth rates, early maturity, a considerably high reproductive capacity (Côté et al., 2013a; Johnson & Swenarton, 2016) and a great longevity, which is estimated as long as 30-33 years (Potts et al., 2010). In fact, the organism reaches sexual maturity within the 1st year of age (Gardner et al., 2015) with females releasing two buoyant egg masses which contain about 1800-41945 eggs per spawning event (Morris et al., 2008, 2011; Gardner et al., 2015). The spawning frequency occurs at a rate of 2-3 days year-round, with higher Gonadosomatic index (GSI) during periods of stable warm and cool temperatures (Gardner et al., 2015), and is less frequent during the cold months (Morris, 2009). It is estimated that the planktonic larval stage in the Bahamian Archipelago lasts on an average 26.2 days (Ahrenholz & Morris, 2010) and it is expected to shorten under a projected seawater warming scenario (Côté & Green, 2012).

Preliminary observations from the RELIONMED project suggest that Mediterranean populations may be characterised with similar biological characteristics, since specimens collected showed to grow fast and reach maturity at the first year of their life, spawn throughout the year, despite the prevalence of low temperatures during the winter. It should be noted that few individuals were spawning capable during the winter and most were in immature and early developing gonadal stages. Females were more reproductive during the summer period.

66 | P a g e Stomach content analyses of the RELIONMED project and other research activities in the Mediterranean (Zannaki et al., 2019) indicate that lionfish is mainly feeding on a wide prey variation in the Mediterranean Sea, ranging from mainly small native teleosts to different species of crustaceans (including crabs and shrimps. Species of the Pomacentridae family (e.g. Chromis chromis) appear as their favourite prey in Cyprus (RELIONMED data) and Greece (Giovos et al., 2019), a family of species that was found to reveal lack of risk perception and being naïve against lionfish in the Caribbean (Haines & Côté, 2019).

In the western Atlantic, P. miles is a generalist mesopredator and eventually becomes more piscivorous at larger sizes (Côté et al., 2013b; Dahl & Patterson, 2014; Peake et al., 2018). Its growth rate and consumption rate was found significantly higher than the native predators in the Caribbean, with individuals of an approximate weight of 340 g consuming ~13 g d-1 of prey (Côté & Maljković, 2010; in Côté et al., 2013). Its preference on different types of prey and the effective anti-predatory defences allows it to establish faster in in its new environment. In fact, the lack of predators in the Mediterranean Sea and the scarcity of pathogens and parasitism (Côté et al., 2013a; Côté & Smith, 2018), are believed to be another factor to its successful proliferation and establishment.

1.24. How likely is the capacity to spread of the organism very likely very high There is no published information about the to facilitate its establishment? migration of the lionfish adults in the 67 | P a g e Mediterranean but individuals usually stay at one location for many days before they move to another. However, the spread of lionfish is likely facilitated by the larval dispersal and not the adults’ movement. Johnston & Purkis (2014) model found that connectivity among potential lionfish habitats in the Mediterranean is low and oceanographic conditions unfavourable for wide dispersion of lionfish larvae. However, lionfish spread in the Mediterranean ended up being one of the fastest ever recorded (Poursanidis et al. unpublished) and it is inevitable that the characteristics and capacity of the species to spread facilitated its establishment in the basin. As described in previous sections (Q.1.4, Q1.8 & Q1.23) the organism exhibits a high reproductive output and spawning frequency. These combined with the long planktonic larval duration (i.e. on average around 26 days) and egg dispersal over large distances facilitate spread and establishment in new marine regions. Mediterranean lionfish dispersal has been very rapid, similar to what was observed in the lionfish invasion at the western Atlantic.

1.25. How likely is the adaptability of the organism to very likely very high Pterois miles is very tolerant species to a wide facilitate its establishment? range of salinities (Jud et al., 2015), and has a large thermo-tolerance spectrum (Dabruzzi et al., 2017; Barker et al., 2018), which may reflect its capability in acclimating fast in the habitats and the environment of the Mediterranean Sea. Present records from the Mediterranean Sea (Azzurro et al., 2017) strengthen this hypothesis, showing that P. miles effectively acclimates and gradually 68 | P a g e establishes in the Mediterranean Sea; despite SDM initial projections (e.g. Poursanidis, 2015). It is possible that the species expanded its ecological niche to pioneer new climates in the invaded areas, as observed with other Lessepsian immigrants (Parravicini et al., 2015); thus justifying lionfish high plasticity in the Mediterranean. Also the possibility of a rapid evolutionary change, as observed in other Lessepsian invaders (e.g. the bluespotted cornetfish Fistularia commersonii) cannot be excluded (Bernardi et al., 2016). Further genetic, physiological and SDM studies can help to better understand lionfish spread and adaptation in the basin.

1.26. How likely is it that the organism could establish very likely very high The standard expectation that a NIS should despite low genetic diversity in the founder population? experience a reduction in genetic diversity has rarely been the case with Lessepsian immigration, the cause could be the multiple entry possibility via the Suez Canal, which acts as a permanent open gateway (Bariche et al., 2017). Even a low genetic diversity is not a constraint for biological invasions of Lessepsian species (see e.g. Golani et al., 2007). Studying the very first individuals that were introduced in the Mediterranean, Bariche et al., (2017) found two haplotypes (out of 14 samples), a similar haplotypes to samples ratio as observed in the Red Sea (Kochzius & Blohm, 2005).

Stern et al., (2018) analysed a larger sampling size (n = 86) and found six haplotypes, for which only four were shared among the invaded studied 69 | P a g e regions (Turkey, Cyprus and Israel). On this basis, the authors concluded that lionfish invasion in the Eastern Mediterranean is a product of multiple invasion in the form of a continuous invasion process or single multi-haplotype founding event. The same conclusions were derived from the RELIONMED project which analysed 56 lionfish individuals and identified six different haplotypes for COI and three for CR genes. RELIONMED genetic results suggest multiple introductions and possible pathways of lionfish invasion in the Mediterranean.

The different haplotype number found between the former and the two latter studies, may suggest sampling bias in the first study or the inflow of new haplotypes through the Suez Canal over time. Despite this, low genetic diversity does not seem to be a constraint in P. miles establishment within the Mediterranean Sea as reflected in the early stages of its invasion as well as in the case of the Atlantic Ocean (Betancur-R. et al., 2011).

1.27. Based on the history of invasion by this organism very likely very high The fish is already established in the southeastern elsewhere in the world, how likely is it to establish in Europe and its range is expanding fast. Europe? (If possible, specify the instances in the comments box.) Taking into account the Atlantic invasion, which is characterised as the worst marine bioinvasion to date (Morris & Whitfield, 2009; Albins & Hixon, 2013; Côté et al., 2013a; Côté & Smith, 2018), Lionfish have several traits that enable rapid spread and invasion across large expanses of coastal ecosystems (Morris & Whitfield, 2009), including long pelagic larval phase, quick sexual 70 | P a g e maturity, continuous reproduction and high fecundity, high survival of eggs and larvae, a wide ecological niche, they are generalist predators and able to cross environmental barriers (e.g. thermal, salinity and deep-water barriers) (Côté et al., 2013a).

P. miles has the potential to establish in more parts of the Mediterranean the rest of the Mediterranean Sea, mainly explained by its biological characteristics, the venomous spines that protect it from predators and potentially the multiple introductions of individuals in the Mediterranean Sea that are discussed in the previous questions (i.e. 1.4, 1.23, 1.26). It is yet uncertain however, if in the future lionfish will further expand in the western Mediterranean Sea, northern Mediterranean areas (e.g. Adriatic Sea), Eastern Atlantic Ocean by crossing through the strait of Gibraltar, and the Black Sea. SDMs based on lionfish occurrences in other areas show that expansion to the abovementioned areas is unlikely; however niche expansion of lionfish could prove them wrong (for more information about SDMs and projections about lionfish establishment in the Mediterranean and EU countries see Ch2). The rate of lionfish dispersal in the Mediterranean leaves no doubt that it will likely establish in other EU countries soon (e.g. Italy and Malta) if habitats are suitable.

1.28. If the organism does not establish, then how likely is very likely very high Given the potential flow of individuals via the it that casual populations will continue to occur? Suez Canal and the offspring being produced from established populations in the Levant Sea, casual 71 | P a g e populations will be maintained. In some locations where sea temperature goes below 15 °C in the winter (e.g. Adriatic Sea), it is possible that lionfish will not be able to establish or be present there year-round (i.e. might move southern for the winter season or to reproduce). For example, off North Carolina lionfish across a depth-temperature gradient were only found at mesophotic coral ecosystem (MCE) depths in areas that maintained winter mean temperatures of ≥15.3 °C (Whitfield et al.,2014). Furthermore, in Bermuda, lionfish have been reported at higher densities on MCEs than shallow reefs, which may in part be driven by water temperatures since Bermudian shallow inshore marine habitats experience lower winter minimum temperatures than offshore habitats, with temperatures dropping as low as 14 °C (Coates et al., 2013; Andradi-Brown, 2019). Lionfish might be recorded but not establish in certain areas of the basin if their niche is not expanded (e.g. western and northern Mediterranean) (see Chapeau 2 for more information).

1.29. Estimate the overall likelihood of establishment in very likely very high Based on the invasion history in the Atlantic relevant biogeographical regions in current conditions Ocean and the current situation in the (mention any key issues in the comment box). Mediterranean Sea, where biotic and abiotic parameters do not seem to pose any constraint to its survival, its establishment in the risk assessment area is high but differs between regions of the Mediterranean; with the eastern basin being the most prone to the lionfish (see Ch2 for more information). Additionally, the current conditions are not conducive for the species to establish in the EU Atlantic biogeographical regions and perhaps 72 | P a g e neither in the Black Sea. Abiotic factors are still too restrictive.

1.30. Estimate the overall likelihood of establishment in very likely low Under foreseeable climate change (warming) and relevant biogeographical regions in foreseeable climate based on the lionfish traits and invasion change conditions characteristics described above (e.g. eco- physiological and biological traits as well as the enemy release hypothesis) (See also Q2 of the EU Chapeau, Q1.14, Q1.19 and Q1.23), P. miles might be further favoured by a projected climate change scenario of temperature increase and expand its range further north in the Mediterranean Sea, where present temperatures can reach below its feeding cessation and critical minimum temperature (Kimball et al., 2004). On the other hand, SDM studies have shown that western and northern Mediterranean regions might not be subject to lionfish establishment even in the case of foreseeable climate change (see Ch2 for more information). It is possible that lionfish will spread far beyond their native niches and that SDMs may underestimate its potential spread but future studies are needed to elucidate it.

73 | P a g e

PROBABILITY OF SPREAD

Important notes:  Spread is defined as the expansion of the geographical distribution of an alien species within the assessment area.  Repeated releases at separate locations do not represent spread and should be considered in the probability of introduction and entry section.

QUESTION RESPONSE CONFIDENCE COMMENT

2.1. How important is the expected spread of this major very high Pterois miles is a demersal fish, living most of its time organism in Europe by natural means? on hard substrata-surfaces. Its movement is limited, thus, adults moving over a large geographical area constitutes an unlikely scenario but cannot be excluded, especially given the fact that large individuals were found in Italy and Tunisia. Like many other marine fish species, Pterois miles is capable of dispersing over great distances as part of its reproductive strategy (Morris et al., 2008). The organism releases buoyant gelatinous eggs on the sea surface, through which are dispersed via surface currents (Betancur-R. et al., 2011). The pelagic larval stage have a ~26 day duration (Ahrenholz & Morris, 2010), after which, they settle down on the benthos. Surviving individuals will surpass the juvenile stage and become reproductive adults within one year of age. At this point, the individuals that were born in the Mediterranean are able to reproduce. Based on research in the Atlantic invasive range, it was estimated as frequent as 3-4 days, with two major spawning events occurring during warm and cool stable temperatures (Gardner et al., 2015). The spawning becomes less frequent in cold months (Morris, 2009), which might be the case in the

74 | P a g e Mediterranean Sea during the winter. In the Mediterranean case, individuals can also be spawning capable throughout the year, but only one major reproductive event has been observed during the summer, coinciding with peak of seawater temperature (RELIONMED data).

Pterois miles was capable of spreading from Lebanon (1st record in 2012 after the one observed in 1991) to Sicily, Italy in only four years. This leads to an approximate westwards progression expansion of -1 ~1062.82 km yr if stepping stone proliferation was achieved via the African’s Mediterranean coastline, or ~872.98 km yr-1 in case it bypassed the Gulf of Sirte and Gulf of Gabès or ~580.41 km yr-1 if proliferation was achieved via Lebanon/Israel to Cyprus, then to Crete and eventually to Sicily.

Due to the generalist niche of P. miles, the absence of temperature, salinity, parasites/pathogens and predator constraints (see Q1.19, Q1.23, and Q1.25), the organism’s spread by natural means across the Mediterranean is further enhanced and safeguarded, especially when available niche are still unfilled (Poursanidis et al., unpublished).

2.2. How important is the expected spread of this minor medium Purchasing specimens for private and public aquaria organism in Europe by human assistance? either from the RA area or from elsewhere, increases the risk of spread to other areas if individuals escape or are released from captivity. This will additionally facilitate new genetic input in the already invading population, especially when released individuals are native to Indo-Pacific region, other than the Red Sea.

75 | P a g e P. miles dispersion can be further enhanced via shipping ballast waters; although unlikely (see Q1.6c). An aquarium release could accelerate the invasion geographical dynamics if occurs in suitable areas that lionfish is not yet established.

The probability of fisheries discards contributing to human assistance spread is considered low in P. miles case. When fishers catch P. miles as bycatch, the fish has a high probability of dying after being entangled in the nets. Even if it survived the fishing, hauling, and disentangling process, discards are commonly thrown back to the same location to which they are caught or taken to land for personal consumption.

Since the species is already established in the basin, its expected spread is largely facilitated by unaided dispersal (secondary dispersal) and the impact of human-assistance but a (relatively unlikely) aquarium release/ballast water transfer is not of high importance in terms of facilitating its spread.

2.2a. List and describe relevant pathways of spread. Unaided (natural For unaided (natural dispersal) pathway see Q1.4 Where possible give detail about the specific origins and dispersal): rapid and end points of the pathways. massive Trade of living Pterois miles increases the risk of spread to other areas given that individuals are For each pathway answer questions 2.3 to 2.9 (copy and - Release in intentionally released from captivity. This scenario is paste additional rows at the end of this section as nature (other more related if individuals are caught from the wild in necessary). intentional release – the Mediterranean Sea and sold in private and public aquarium hobbyist): aquaria. It is estimated that aquaria trade is possible responsible for the movement of ~24 million Transport individuals of ~1500 fish species (Katsanevakis et al., Stowaway (Ship 2013) from their native region to elsewhere. /boat ballast water): Presently, aquarium trade is recognised as a 76 | P a g e not very likely but significant invasion pathway in marine systems also possible (Katsanevakis et al., 2013) with many examples of species being released in a new environment after confinement (Padilla & Williams, 2004; Semmens et al., 2004). Examples are the Pterois volitans/miles complex in the Atlantic Ocean (in Betancur-R. et al., 2011) and Caulerpa taxifolia in the Mediterranean Sea (Wiedenmann et al., 2001). The probability to which lionfish may establish a successful reproduction after their escape, depends on prevailing abiotic factors, the numbers of individuals released and the presence of both sexes.

Shipping is to date a recognized pathway and one of the most important marine invasions vectors in European seas, with at least 130 marine alien species being introduced either via hull fouling or ballast waters (Katsanevakis et al., 2013). Ballast waters seemed a less prominent pathway than hull fouling prior to 1950 in European seas, but this pattern has changed the following decades (Katsanevakis et al., 2013). Shipping ballasts are capable of transporting holoplanktonic or mesoplanktonic organisms, seeds or resting stages such as eggs or cysts (Katsanevakis et al., 2013) from the area of loading to an area where the cargo is unloaded.

There are no records or information on successful introduction of scorpaenids via ballast-water transport but two individual scorpaenids were recorded in ballast waters in the literature (Wonham et al., 2000). Additionally, lionfish are reported from several harbour areas (Schultz, 1986) and coupled with the high tolerance of lionfish in various conditions; a

77 | P a g e spread via ballast water cannot be excluded.

Pathway name: Unaided: Natural dispersal from established populations

2.3a. Is spread along this pathway intentional (e.g. the unintentional very high organism is released at distant localities) or unintentional (the organism is a contaminant of imported goods)? 2.4a. How likely is it that large numbers of the organism very likely very high See Q1.4 will spread along this pathway from the point(s) of origin Taking into account its continuous reproduction over the course of one year? throughout the year in the Mediterranean (RELIONMED data), high fecundity and reproductive strategy as observed in the Western Atlantic (Côté et al., 2013a), large numbers of P. miles are very likely to spread along continuous coastlines and likely across open continuous waters as far as 3000 km with depths of more than 7000 m (in Côté et al., 2013a). This is additionally supported by the spatio-temporal dynamics, which illustrate a progressive westward expansion of the species along the basin (see Azzurro et al., 2017; Figure 1) with isolated records reported as far as Italy and Tunisia.

2.5a. How likely is the organism to survive during passage very likely very high See Q1.5 along the pathway (excluding management practices that would kill the organism)?

2.6a. How likely is the organism to survive existing very likely very high Existing management practices are taking place in the management practices during spread? framework of the RELIONMED project but these are focused in small-priority areas aiming to demonstrate their feasibility. Management should be coordinated at the regional level to control lionfish populations and to help local communities to adapt. It seems that it is too late to attempt total eradication of the species in the Mediterranean. 78 | P a g e

2.7a. How likely is the organism to spread in Europe unlikely very high See Q1.7 undetected?

2.8a. How likely is the organism to be able to transfer to a very likely very high See Q1.9, Q1.14 and Q1.16 suitable habitat or host during spread?

2.9a. Estimate the overall likelihood of spread into or very likely very high The lionfish is already established in the south-eastern within the Union based on this pathway? Europe and already expanding westwards in the Mediterranean Sea, reaching the central basin. The RELIONMED project scientists project that it will not be long until it proliferates towards the western parts of the Mediterranean and becomes abundant throughout the basin, especially if a complete lack of biosecurity in the region continues.

End of pathway assessment, repeat as necessary.

Pathway name: Release in nature (other intentional release – aquarium hobbyist)

2.3b. Is spread along this pathway intentional (e.g. the intentional very high The spread (release in the environment) can occur by organism is released at distant localities) or unintentional intentional releases into the sea by hobbyists, (the organism is a contaminant of imported goods)? fishermen, pet owners etc.

2.4b. How likely is it that large numbers of the organism unlikely medium Pterois miles is a common aquarium species will spread along this pathway from the point(s) of origin worldwide and it is possible that specimens could be over the course of one year? collected from the wild (either from Mediterranean Sea or elsewhere) and sold to private and public aquaria such as already recorded from the southeast part of Turkey (Gülenç, 2019) and in Cyprus (Jimenez unpublished data).

Not many lionfish are required to achieve a major 79 | P a g e invasion event. Few individuals (including both sexes) can successfully establish viable populations, as has been observed in Florida, where the founding population consisted of possibly 3 females (in Whitfield et al., 2007). The spread is then succeeded via fertilised eggs/larval dispersal. For larval dispersal see Q1.4. Over the course of one year, it is quite unlikely that large numbers will cross through that pathway.

2.5b. How likely is the organism to survive during very likely high Pterois miles and generally Pterois spp. are very passage along the pathway (excluding management common ornamental fish in aquaria, which makes practices that would kill the organism)? them very adaptable in such environments. In fact, a considerable amount of online material provides guides on the proper maintenance for Pterois spp. in aquaria and artificial conditions (e.g. https://secure.liveaquaria.com/category/36/lionfish?c= 36&qv=1).

Survival will be high in intentional releases (e.g. to the shore by a hobbyist). If the fish is released in a non-marine environment or unsuitable temperature, then its chances of survival are very low, even if lionfish can survive in low-saline environments such as estuaries.

2.6b. How likely is the organism to survive existing likely high Management of lionfish in the aquarium-trade management practices during spread? industry exists only in the form of voluntary code of ethics, strategies and educational material against releasing live fish from aquariums (Penning et al., 2009). These measures cannot affect survival of lionfish during spread through this pathway.

For the moment, Florida is the only place to prohibit 80 | P a g e the importing of lionfish due to a number of measures proposed in 2014 by Florida Fish and Wildlife Conservation Commission (FWC) (Farquhar, 2017). FWC allows people to have lionfish in the aquariums but only if the specimens have originated from Florida waters. Adoption of such measures can eliminate aquarium-trade as a potential spread pathway of lionfish in the EU waters.

2.7b. How likely is the organism to spread in Europe very unlikely high See Q1.7; 2.7a. undetected? Trade information and imports of marine ornamental species from outside EU are recorded through the trans-European veterinary health agreement (Trade Control and Expert System TRACES) by the Common Veterinary Entry Document (CVED) and it’s not compulsory to list the species (Biondo, 2017). As no import declarations or licences are required within EU regarding the movement of goods, which increases the risk of P. miles being traded in EU aquarium/pet shops undetected. However, it is highly unlikely that the species will spread in the wild undetected.

2.8b. How likely is the organism to be able to transfer to a likely high The transfer of P. miles during the transport process suitable habitat or host during spread? to a suitable habitat is controversial, as it has to do with the future hosting aquaria (private or public) conditions.

Release from these in the natural environment is considered a secondary introduction event; the implications with finding suitable habitat are discussed in Q1.9, Q1.14 and Q1.16. 2.9b. Estimate the overall likelihood of spread into or possible high See Q1.4b to Q1.10b. 81 | P a g e within the Union based on this pathway?

End of pathway assessment, repeat as necessary.

Pathway name: Transport - Stowaway (Ship /boat ballast water) 2.3c. Is spread along this pathway intentional (e.g. the organism is released at distant localities) or unintentional unintentional very high (the organism is a contaminant of imported goods)? 2.4c. How likely is it that large numbers of the organism unlikely medium Hundreds of thousands of vessels weighing more than will spread along this pathway from the point(s) of origin 100 tonnes each, cross through the Mediterranean Sea over the course of one year? annually, with 2000 merchant ships operating continuously in the Mediterranean (Galil, 2006). The probability of translocating large numbers of P. miles fertilized eggs or larvae from one region of the Mediterranean to another per year, increases (Maclsaac et al., 2016), especially when taking into account its continuous reproduction, high fecundity (Côté et al., 2013a) and small larval size i.e. larval stages until settlement size ranges between 1 mm to 12 mm (Mito & Uchida, 1958; Ahrenholz & Morris, 2010; Vásquez-Yeomans et al., 2011), which is less than the routine pore size of cargo ships (i.e. holes 15–25 mm diameter or slots 20–35 mm width) (Coutts et al., 2003).

This becomes even more relevant when ballasting occurs in ports, where lionfish may be present within or in nearby areas. To date, P. miles has been sighted multiple times close to the major port of Limassol, Cyprus.

2.5c. How likely is the organism to survive during passage Unlikely medium Shipping ballast waters are proven to sustain species along the pathway (excluding management practices that survival during transportation. For instance Wonham would kill the organism)? et al., (2000) identified 32 fish species that were 82 | P a g e transported and introduced to a new location via ballast water, 24 of which established viable populations at the new site. While there is no literature to support this regarding P. miles in the Mediterranean Sea, Maclsaac et al., (2016) expressed that it’s not an impossible scenario.

Reproduction through this pathway is unlikely, as the potential individuals transported are still in larval stage.

2.6c. How likely is the organism to survive existing Likely high See Q1.6c management practices during spread?

2.7c. How likely is the organism to spread in Europe very likely high Also see Q1.7c and Q2.7b..It very likely to spread undetected? undetected during the transportation in ballast-waters but very unlikely to spread undetected in the wild. 2.8c. How likely is the organism to be able to transfer to a Possible very high The ballast waters may serve as a convenient habitat suitable habitat or host during spread? for P. miles at its very early life stages, when it’s still in planktonic phase and exhibits planktonic feeding. It is unclear however, if the same scenario accounts also for post settlement stage. Deballasting in another area and new environment is then considered a secondary introduction event and the implications with finding suitable habitat are discussed in Ch2, Q1.9, Q1.14 and Q1.16. 2.9c. Estimate the overall likelihood of spread into or Possible medium See Q1.4c to Q1.10c. within the Union based on this pathway?

End of pathway assessment.

2.10. Within Europe, how difficult would it be to contain very difficult high Containing or even reversing the lionfish invasion is the organism? an unlikely scenario. This especially accounts for species with high dispersal abilities (Carlton, 1996). 83 | P a g e However, controlling the lionfish populations in certain locations (e.g. priority sites such as Marine Protected Areas or artificial reefs) might be possible. Removals of lionfish in the project RELIONMED in Cyprus were promising in terms of reducing lionfish abundance as observed in the Western Atlantic (for comprehensive information about the management techniques that have been used in the Western Atlantic see Q1.20). To be effective in small targeted areas however, the consistency and frequency of removal efforts needs to be assured. Furthermore, management should be guided by tools that will enable to determine the cost effectiveness of any measures, and their feasibility in preventing lionfish impacts to the native communities. For the cost-effectiveness and sustainability of the removals to be assured, we advise that strict removal permits need to be given to dive centres.

Alternative removing gear/techniques needs to be developed/used for catching juvenile lionfish or lionfish from areas below depths of recreational diving. Many patents have been developed and currently being tested for these purposes (e.g. patent application US 20190021297A).

On another level, lionfish market could be promoted to increase its value as a product so that more fishers have incentives to target it using specific fishing gear and techniques. Valorisation techniques to enhance the properties and quality of lionfish could be further explored (e.g. Jiménez-Muñoz et al., 2019). Appropriate measures to ensure that lionfish do not spread further via the commercial pathway should be

84 | P a g e adopted; such as inspecting and not allowing movement of live specimens across areas.

2.11. Based on the answers to questions on the potential Worst case scenario medium See Q1, Q2 of the Chapeau (Ch1, Ch2). for establishment and spread in Europe, define the area (using the 15 oC endangered by the organism. criterion) Present conditions: Levantine Sea, southern Aegean Sea, Ionian Sea, southern Balearic Sea, Alboran Sea. Future conditions: Adriatic Sea, northern Aegean Sea, Ligurian Sea, northern Balearic Sea.

Best case scenario low (using the SDMs) Present conditions: Levantine Sea, southern Aegean Sea, Ionian Sea Future conditions: Southern Adriatic

2.12. What proportion (%) of the area/habitat suitable for Worst case medium Considering the present records so far in Cyprus, establishment (i.e. those parts of Europe were the species scenario (using the Greece, and Sicily (Italy) (Azzurro et al., 2017), only could establish), if any, has already been colonised by the 15 oC criterion) EU waters of the Western Mediterranean Sea have not organism? 60-80% been invaded yet.

Best case scenario 85 | P a g e (using the SDMs) 80-90% 2.13. What proportion (%) of the area/habitat suitable for 80-100 high Considering the rate of westwards spread mentioned establishment, if any, do you expect to have been invaded in Q2.1 as well as taking into account the additional by the organism five years from now (including any risk of spread through various pathways. the lionfish current presence)? may succeed spreading far and beyond and establishing in coastlines that have their minimum temperatures above 15oC (worst case scenario), such as Spain and Southern Italy and other locations of Greece within 5 years from now. Based on SDM scenario (best case) the southern Adriatic will be likely invaded and almost 100% of the suitable lionfish areas will be invaded. For conservative purposes, we estimate the proportion (%) of the area/habitat suitable for establishment to have been invaded by the organism five years from now to be within the range of 80-100 %.

2.14. What other timeframe (in years) would be 10 very high With the current rate of spread, it is estimated that appropriate to estimate any significant further spread of within 10 years from now, P. miles will strengthen its the organism in Europe? (Please comment on why this presence in the already invaded areas (i.e. Cyprus, and timeframe is chosen.) Greece) and establish population in almost all areas that offer suitable habitats under present conditions The Northern parts of the Mediterranean Sea (Adriatic, French coasts and Northern Aegean Sea) may not serve as suitable habitats by then, and neither other European northern seas (i.e. Black Sea, Bay of Biscay region, etc.).

2.15. In this timeframe what proportion (%) of the 70-80 medium In this timeframe, lionfish will likely establish in endangered area/habitat (including any currently occupied ~100% of all areas that are currently suitable and areas/habitats) is likely to have been invaded by this endangered by its invasion (i.e. <15 °C). The lionfish organism? will likely invade endangered areas that are not currently suitable for its reproduction under current 86 | P a g e climate conditions (e.g. Adriatic Sea, northern Aegean Sea) but not establish there.

2.16. Estimate the overall potential for spread in relevant very likely very high Based on the invasion history in the Atlantic Ocean biogeographical regions under current conditions for this and on the westwards spread rate (Q2.1) in the organism in Europe (using the comment box to indicate Mediterranean Sea, where biotic and abiotic any key issues). parameters do not seem to pose any obstacles to its survival, its spread westwards under current conditions is estimated to be rapid. The species will likely spread in most parts of the Mediterranean but it is unlikely to form established populations in climatically unsuitable areas under present conditions (see Q2.11). Lionfish spread and establishment partially depends on finding suitable habitats in the Mediterranean. We based our assessment on two scenarios that show mismatch – first that lionfish will follow the results of the SDMs without any niche expansion (best-case scenario), and second that lionfish will establish in all areas with minimum temperature more than 15 C° as it was found to control its spread in the western Atlantic (for more information see Ch2.). SDMs indicate that lionfish will not spread western than the central Mediterranean and northern than the southern Adriatic Sea even in the case of climate change, while the 15 C° indicate that lionfish will be able to spread to all the southern Mediterranean (including western region) under present conditions, and expand to its northern regions (e.g. Northern Aegean and Adriatic Sea) under climate change (see Ch2).

2.17. Estimate the overall potential for spread in relevant very likely very high It is foreseen that P. miles will continue to spread in biogeographical regions in foreseeable climate change climatically suitable areas (Aegean-Levantine Sea, conditions Ionian and the Central Mediterranean Sea) and maybe 87 | P a g e uncolonized areas of the Western Mediterranean (see two scenarios in Question above).. Under a projected climate change, the spread of P. miles might be further favoured in more areas (see Ch2; Q2.11; Q.2.16).), where lionfish will be able to form established populations.

88 | P a g e

MAGNITUDE OF IMPACT

Important instructions:  Questions 2.18-2.22 relate to environmental impact, 2.23-2.25 to impacts on ecosystem services, 2.26-2.30 to economic impact, 2.31-2.32 to social and human health impact, and 2.33-2.36 to other impacts. These impacts can be interlinked, for example a disease may cause impacts on biodiversity and/or ecosystem functioning that leads to impacts on ecosystem services and finally economic impacts. In such cases the assessor should try to note the different impacts where most appropriate, cross-referencing between questions when needed.  Each set of questions above starts with the impact elsewhere in the world, then considers impacts in Europe separating known impacts to date (i.e. past and current impacts) from potential future impacts (including foreseeable climate change).  Assessors are requested to use and cite original, primary references as far as possible.

QUESTION RESPONSE CONFIDENCE COMMENTS

Biodiversity and ecosystem impacts 2.18. How important is impact of the organism on Massive very high Lionfish invasion in the Atlantic Ocean, is considered biodiversity at all levels of organisation caused by the one of the worst marine bioinvasions at global scale. organism in its non-native range excluding the Union? The lionfish observed in invasive range reefs were found larger and more abundant than those observed in the native range (Darling et al., 2011). The invasion is characterised by an exponential growth, rapid spread and establishment across the coastline of Central and Northern America (Betancur-R. et al., 2011).

The large densities of P. volitans/miles complex in the western Atlantic Ocean exerted a great amplitude of damage on biodiversity at local scales in Caribbean region (Albins & Hixon, 2008; Lesser & Slattery, 2011; Green et al., 2012; Benkwitt, 2015; Raymond et al., 2015) and south-eastern United States (Ballew et al., 2016). P. volitans/miles complex was found responsible for local habitat modification (Morris & Whitfield, 2009; Lesser & Slattery, 2011), negatively affected

89 | P a g e native communities by reducing the abundance of native ichthyofauna (Albins & Hixon, 2008; Green et al., 2012; Albins, 2013; Benkwitt, 2015; Rocha et al., 2015; Ballew et al., 2016; Kindinger & Albins, 2017; Tuttle, 2017) and outcompeting the native predators either space-wise or prey-wise (Albins, 2013; Raymond et al., 2015).

2.19. How important is the impact of the organism on Aegean- low To date, the impacts of lionfish in the Mediterranean biodiversity at all levels of organisation (e.g. decline in Levantine Sea: region are anecdotal as research is still in progress. A native species, changes in native species communities, moderate to recent modelling study by Michailidis et al., (2019) hybridisation) currently in the different biogeographic major which used data from the period 2015-2017 did not regions or marine sub-regions where the species has show evidences of negative impacts by lionfish. established in Europe (include any past impact in your However, preliminary results from the RELIONMED response)? project, show that densities of lionfish over the years low increased drastically, which is an indicative sign of Ionian and potential foreseeable decrease in the native fauna and Central displacement of native predators (e.g. Epinephelus Mediterranean: marginatus). Despite this, without any further empirical minor evidence, the degree of impact in EU region cannot be low estimated with certainty. Understanding biodiversity Overall: impacts in the marine environment is usually very major complex, especially in environments such as the Mediterranean, which faces many pressures and includes many confounders in a potential analyses of the impacts. Impacts will take some time to be detected but given the dense lionfish population at some areas of the eastern Mediterranean (i.e. 30-50 lionfish at natural reefs of ~25 m2) impacts to the native communities are inevitable. Impacts are likely to be major in the south Aegean-Levantine Sea where lionfish are found in great densities but with low confidence, given that empirical data are yet unavailable. Impacts in the Ionian and the Central Mediterranean Sea are expected to be minor 90 | P a g e given that lionfish are yet found in very low densities.

See Q2.18. 2.20. How important is the impact of the organism on Western low Combining current knowledge from the western biodiversity at all levels of organisation likely to be in the Mediterranean Atlantic Ocean case and the recent invasion in the future in the different biogeographic regions or marine Sea (if Mediterranean Sea, P. miles is expected to have a major sub-regions where the species can establish in Europe? established) effect on coastal communities of most of the major Mediterranean in the near future, taking into account the considerably fast growth, early maturation, high Adriatic Sea (if low consumption rates and competition (Côté & Maljković, established) 2010; Curtis-Quick et al., 2013; in Côté et al., 2013; moderate Raymond et al., 2015; RELIONMED data, 2019). low Lionfish are expected to cause long-term irreversible Ionian Sea and ecosystem changes, which spread beyond local areas the Central but its impact might be different in each Mediterranean Mediterranean regions. Sea major Like reported in the Western Atlantic, an increase in lionfish abundance at certain locations will likely Aegean- coincide with a decline in the biomass of local fish Levantine Sea high species (Green et al., 2012; Côté et al., 2013) with the major impacts likely to be felt at a regional scale (Ballew et al., 2016). Biodiversity decrease will lead to decrease of population and ecosystem genetics. Overall: high major In terms of ecosystem changes, lionfish have been found able to drive an overall shift in invertebrate assemblage composition (Layman et al., 2014) and mesophotic habitats (e.g. shift to algal dominated habitats) (Lesser & Slattery 2011). It likely that the presence of lionfish will skew food webs towards a loss of higher trophic groups and a gain in lower order consumers as reported for other human disturbances (Byrnes et al., 2007) 91 | P a g e

With the expected spread throughout the Mediterranean Sea and the subsequent establishment of high dense populations, indigenous community structure may alter significantly, but not at the same way as in the Caribbean, where shifts in habitat forming species have been observed (i.e. from corals to macroalgae) (Lesser & Slattery, 2011).

P. miles consequences on ecosystem functioning may be reflected on alterations of trophic interactions (Arias- González et al., 2011). There is a general consensus where generalist invasive species replace specialists and subsequently leading to homogenization of the communities with potential changes on ecosystem functioning and productivity, as well as result in the deterioration of ecosystem goods and services (Clavel et al., 2011).. Competition for space between macroalage and other sessile organisms including hydroids and sponges as part of herbivory release due to P. miles should not be excluded, although the probability of such occurrence is low, considering the significant impacts of Siganus spp. on macrophytic communities (Vergés et al., 2014). 2.21. How important is decline in conservation value with moderate low The species is currently established in only few areas of regard to European and national nature conservation the risk assessment area (Cyprus, a few parts of Greece legislation caused by the organism currently in Europe? and recorded in southern islands of Italy), but no impacts based on empirical data are yet revealed. The increasing densities, however, observed over the years will soon be followed by potential decrease in the native fauna population and direct competition with native mesopredators both for resources and habitat. If this scenario is true, then predators which are protected by national or regional legislation will be affected, e.g. 92 | P a g e Epinephelus marginatus is included in the Annex III of Protocol SPA/BIO Barcelona convention and Annex III Bern Convention and is considered as Endangered according to the IUCN red list. lionfish will likely decline ; thus the conservation value set by European and national nature conservation legislations (e.g. nature reserves, MPAs, no take zones, etc.) will decline.

2.22. How important is decline in conservation value with major medium P. miles has already been observed in Natura 2000 sites regard to European and national nature conservation and Marine Protected Areas of Cyprus (Cape Greco and legislation caused by the organism likely to be in the Nisia) as well as in artificial reefs (e.g. Zenobia), which future in Europe? became part of EU’s Habitat Directive. The numbers of lionfish observed were considerably high. For instance, 74 specimens were caught on November 2017 within a single removal expedition in an MPA at Paralimni in one of RELIONMED removal expeditions. In 2018 a removal event produced a total catch of 315 lionfish (Jimenez et al.,2018). In 2019, about 120 lionfish were removed from a single removal event at the Zenobia wreck (see Q.6 of the EU Chapeau). The increasing abundance (if not kept low) poses a serious threat to these protected marine communities and habitats. Based on stomach content analyses of lionfish in the Mediterranean, demersal teleosts and crustaceans are the most directly affected taxa (RELIONMED data; Savva et al. under review; Zannaki et al., 2019) by lionfish. Decrease of their biomass is expected which will diminish the ecosystem status of Marine Protected Areas as well as further impacting the native competitive (meso)predators such as those belonging to of Serranidae and Sparidae family (see Q2.21 for impacts on species that are protected by conservation

93 | P a g e legislation).

Furthermore, increase of lionfish presence will likely diminish the goals of Action 16 of Target 5 of the EU 2020 Biodiversity Strategy and Aichi Target 9 of the Strategic Plan for Biodiversity 2011-2020 under the Convention of Biological Diversity (i.e. “priority invasive species controlled or eradicated by 2020”), as well as of the Descriptor 2 of the Marine Strategy Framework Directive (MSFD), i.e. “Non-indigenous species introduced by human activities are at levels that do not adversely alter the ecosystems.”

Ecosystem Services impacts 2.23 How important is the impact of the organism on massive very high Although the socioeconomic impacts of the lionfish provisioning, regulating, and cultural services in its non- invasion are not fully evaluated in the western Atlantic native range excluding the Union? Ocean, it is estimated that they are substantially high since they have the potential to cause deleterious impacts to the reef communities, diminishing local communities and degrading important habitats such as coral reefs (Albins & Hixon, 2013, see Q.A6 and Q2.20).According to the literature, P. volitans/miles complex may have a significant impact on fishery yields, hence affecting provisioning services for nutrition (Burke & Maidens, 2004; Morris & Whitfield, 2009); i.e. wild animals and their outputs (according to the Common International Classification for Ecosystem Services). With the significant impacts on biodiversity (both species and habitats see Q2.20) reported in the western Atlantic, other ecosystem services (ES) that are likely affected include provisioning ES related to materials such as genetic materials from all biota and animal based resources. Impacts caused by lionfish 94 | P a g e invasion likely affects indirectly the Regulation & Maintenance ES related to mediation of waste, toxics and other nuisances by filtration/sequestration/storage/accumulation, and impacts on maintaining nursery populations and habitats.

Lionfish invasion strongly affects cultural ES related to physical and intellectual interaction (both positively with lionfish as the ‘model’ species and negatively with impacts on other biota); affecting services such as experiential use in environmental settings, physical use in environmental settings, educational services since lionfish is a subject matter of education about invasive species both on location and via other media, Specifically, lionfish is very popular attraction for divers, especially for photographs due to its beautiful appearance. However, dense populations of lionfish are likely to affect the diving industry due to possible envenomation on recreational divers, especially in caves/wrecks, causing fear and reduction of diving destinations attractiveness (see also QA.7 for different attitudes of divers and snorkelers reported in the western Atlantic. In general, impacts on cultural services (e.g. physical and intellectual interactions) can be a likely consequence of socioeconomic impacts (e.g. revenue gain or lose) which can affect differently particular stakeholders such as small-scale fishers, recreational divers and MPAs. Positive impacts should be also evaluated if any.

Johnston et al., (2015) estimated that if populations of lionfish were left uncontrolled, total service losses to

95 | P a g e recruitment and biomass functions of 26.67 and 21.67 DSUYs (i.e. one DSUY equivalent to the entire quantity of services provided by one area unit of the damaged or replacement system for a given year) per km2 of a Bahamian reef were expected.

2.24. How important is the impact of the organism on minor Low No impact has been officially assessed yet, neither on provisioning, regulating, and cultural services currently in fishery yields or diving industry. Nevertheless, results the different biogeographic regions or marine sub-regions from questionnaire surveys both from Jimenez et al., where the species has established in Europe (include any (2017) and RELIONMED project (Kleitou et al.,2019) past impact in your response)? on the perception of recreational divers and spear- fishers regarding the presence of lionfish in Cyprus’ waters are controversial. A recent decrease in fishery yields is attributed to several factors according the fishermen interviewed in RELIONMED project, lionfish invasion could be one of them. Divers are worrying that access to some sites (e.g. caves and wrecks) is not safe due to the lionfish presence (RELIONMED project). Surveys with divers has shown that many divers like to see 1-2 lionfish (but not more) during their dives (Kleitou et al., in prep Impacts on services can be a consequence of socioeconomic impacts felt by each stakeholder group (see QA.7 for different attitudes observed in the western Atlantic between snorkelers and scuba divers). 2.25. How important is the impact of the organism on major Low Similar impacts as mentioned above for the western provisioning, regulating, and cultural services likely to be Atlantic (Q.23) are expected in the Mediterranean; in the different biogeographic regions or marine sub- especially on provisioning ES after impacts on fishery regions where the species can establish in Europe in the industry, as well as on cultural ES with impacts on the future? diving industry and fishery.

If P. miles manages to reduce the biomass of high economically commercial fish within the risk assessment area, fishery yields will decline further with 96 | P a g e severe implications on the socio-economic sector. Additional impacts of lionfish on fishery activities are related to its venomous spines, which require special handling and might slower fishery operations. According to FAO (2018) marine capture fisheries in the Mediterranean and Black Sea produce an estimated annual revenue of USD 2.8 billion and directly employ just under a quarter of a million people (248000) onboard fishing vessels. Revenue calculations, based on official data on value at first sale, represent only a small part of the total economic impact of fisheries, which is estimated to be at least 2.6 times larger (approximately USD 7.3 billion). Lionfish is likely to strongly affect those stocks and revenue, particularly related to reef species. On the other hand, specific fishery gear (e.g. fishery adapted pots) might be developed for catching lionfish which might end up being profitable for a species specific fishery.

Negative impacts on the ecosystem structure and integrity will decrease the value of regulatory services related to the maintenance of physical, chemical, and biological conditions (e.g. maintaining nursery populations and habitats) as well as cultural (e.g. recreational diving/fishing) services. The increased abundance may or may not enhance the diving tourism in the risk assessment area, depending if recreational divers are willing/attracted to dive in a fish homogenized and a lionfish infested area or consider it a safety hazard. As lionfish abundance increases there is a growing worry by divers, especially of wreck/cave divers. With over 27 million divers worldwide, and around a million annual certificates from PADI alone (PADI 2019), scuba diving is a mass leisure activity. No

97 | P a g e figures are available for the Mediterranean diving industry but it is inevitably a very popular activity. Beach tourism is a major economic driver in the Mediterranean, with an established value chain (e.g. hotels, banks, restaurants). The tourism industry may be affected as well if bathers are stung by lionfish and then bathers start to fear entering the water at lionfish infested sites. Lionfish have been increasingly detected in shallow waters (<2 m) and it is likely that an incident will occur in the future. Mediterranean tourism is usually defined as “3S tourism” – the three S’s standing for sea, sand, and sun. Impact on beach attractiveness can substantially influence touristic activity of an area.

Economic impacts 2.26. How great is the overall economic cost caused by major very high No official quantitative data relevant to economic losses the organism within its current area of distribution, are yet available from nations affected by the invasion including both costs of damage and the cost of current of P. volitans/miles complex in the western Atlantic management Ocean, apart from only one study in Jamaica (Binet & Smidt, 2015). The authors stated that the lionfish invasion degrades the value and services provided by coral ecosystems, leading to heavy economic losses. These ecosystem service were estimated at 60 million euros in the French West Indies, which are now directly impacted by the lionfish (Binet & Smidt, 2015). Despite the lack of additional studies, the scale of impact is thought to be substantially high. The main impacts are linked to high consumption rates and decrease of native high economically important species (up to 95% decrease of small native fish species was recorded in some invaded areas, Côté et al., 2013), the effort/time of fishermen to sort out the catch when lionfish are caught in the nets, and the loss of a fisher’s time and resources if envenomation occurs. Lionfish impacts in 98 | P a g e the Mediterranean have not yet assessed but RELIONMED, a four-year EU LIFE project which aims to tackle lionfish invasion in the Mediterranean, has a budget of 1,676,077.00 €.

2.27. How great is the economic cost of damage* of the minor low No economic cost impact has been assessed yet within organism currently in the Union (include any past costs in the EU. your response)?

*i.e. excluding costs of management 2.28. How great is the economic cost of damage* of the massive low This depends on the P. miles foraging behaviour and organism likely to be in the future in the Union? interaction with the ecosystem in the Mediterranean Sea. If similar patterns are observed as in the case of the *i.e. excluding costs of management western Atlantic Ocean, the economic costs linked to fishing, recreation and tourism could potentially be high given also the alarming rate of lionfish spread and establishment.

2.29. How great are the economic costs associated with major very high RELIONMED is an EU funded project to tackle the managing this organism currently in the Union (include lionfish invasion in the Mediterranean and total budget any past costs in your response)? equal to 1,676,077.00 € with at least 40% of it being devoted to develop a surveillance system for lionfish, identify best cost-effective management measures and develop guides and tools for managers.

2.30. How great are the economic costs associated with major medium Management costs might include national action plans, managing this organism likely to be in the future in the establishment of a surveillance system, awareness Union? campaigns, human adaptation techniques, citizen science programs, control/eradication actions as part of derbies and championships with prizes, promotions for lionfish consumption in restaurants and jewelcrafts purchases, so that a lionfish fishery is developed and sustained on the long-term, or even compensations to fishers for lionfish catch. 99 | P a g e RELIONMED, a four-year EU LIFE project which aims to tackle lionfish invasion in the Mediterranean, has a budget of 1,676,077.00 € but includes the development of surveillance system, management guides, dissemination, modelling, removals in priority areas and other. Adaptation measures and monitoring of lionfish can be conducted with cost-effective techniques such as those identified by the EU Interreg project MPA-Adapt including utilization of Local Ecological Knowledge and citizen science techniques (e.g. such as the protocols developed by MPA-Adapt to monitor abundance of target species in permanent transects or other citizen science schemes to monitor presence in certain locations such as EASIN and MedMIS platforms).

Control of lionfish in all the Mediterranean is not possible nor economic, but control of lionfish in certain priority areas can be achievable if removals are applied consistently (RELIONMED data). The effort required in each area depends on its characteristics but it is estimated that at least 5,000 € are needed to organise removal events each month to a specific small area (e.g. a popular wreck) for a year. However, if legal framework is reformed to allow more divers to cull lionfish, then eco-touristics schemes might be developed and removals can become sustainable with little economic burden for authorities. In national parks of the Carribean such as in Bonaire, there are well established marine conservation program the main body of which is run by the national park authority, and funds the non-resident visitors a dive fee of $45 per calendar year for scuba diving, and $25 for

100 | P a g e other water activities. Actions funded by this fee includes a lionfish hunting program, patrols to enforce fishing restrictions, and coral reef monitoring (Roberts et al. 2018).

Any legal reframes should be adopted after careful examination of all parameters and more enforcement should be secured to prevent potential illegal activities.

Social and human health impacts 2.31. How important is social, human health or other moderate high P. miles is a highly venomous scorpaenidae fish, impact (not directly included in any earlier categories) producing its venom via glands within long and slender caused by the organism for the Union and for third spines located at the dorsal (12 to 13), anal (3) and countries, if relevant (e.g. with similar eco-climatic pelvic (2) fins. The venom is delivered to the wound conditions). when the ray of the fin penetrates the skin of the victim. During this process, the epithelial sheath that covers the spine is pushed down, and venom is released from the glandular tissue upwards (Haddad et al., 2015). Scorpaenidae fish are still venomous up to 48 hours after death (Rensch & Murphy-Lavoi, 2018).

Symptoms from lionfish envenomation include excruciating local pain that increases over time throughout the affected limb, marked inflammation, which causes important erythema, edema and local heat and in some cases local cyanosis, paleness, vesicles and blisters are observed (Garyfallou & Madden, 1996; Vetrano et al., 2002; Schult et al., 2017). Rarely, the sting site presents skin necrosis. Additionally, it may usually cause nausea, vomiting, cold sweating, fever, dyspnea, convulsions, abdominal pain, syncope, cardiac effects and affect blood pressure (Saunder & Taylor, 1959; Garyfallou & Madden, 1996; Vetrano et al., 2002; Schult et al., 2017). There are no published 101 | P a g e reports of death, and the only severe case to our knowledge was a near-fatal incidence caused by P. volitans (Saunder & Taylor, 1959). The development of anaphylaxis and severe infections can also occur (Haddad et al., 2015).

The majority of the envenomation reports occur in aquaria during lionfish handling (Haddad et al., 2015), however it has been reported during recreational diving, spear-fishers, and fishermen during netting (RELIONMED interviewees with divers and fishermen; also in Haddad et al., 2015 and Diaz, 2015). No incidence has been reported yet by beachgoers in the Mediterranean invasive range, although it is possible.

There is no particular antidote, though the most common treatment is the immersion of the injured area in heated water to denature the venom (Diaz, 2015). At the emergency care, patients should receive tetanus prophylaxis as indicated and antibiotic prophylaxis for most wounds other than those considered minor. Antibiotic prophylaxis is not standardized although general guidelines recommend broad-spectrum antibiotic prophylaxis (Rensch & Murphy-Lavoi, 2018).

Based on the above information, P. miles affects the health and the safe access to the activities of identifiable groups. First-aid training of relevant stakeholder groups (e.g. life-guards) can be facilitated.

For social impacts see Q2.23 and Q2.24.

2.32. How important is social, human health or other moderate low Significant effects will be noticed in the sectors impact (not directly included in any earlier categories) mentioned above (Q2.31, i.e. fishery and tourism). With 102 | P a g e caused by the organism in the future for the Union. P. miles spreading and establishing all across the Mediterranean Sea in the near future, as well as the further increase of already established populations, the envenomation cases are expected to increase. For more social impacts see Q2.23 and Q2.24.

Other impacts 2.33. How important is the impact of the organism as minimal low Parasitic infestations (mostly ectoparasites) are common food, a host, a symbiont or a vector for other damaging in P. miles in both native and the invaded range organisms (e.g. diseases)? (Diamant et al., 2004; in Morris et al., 2008; in Ramos- Ascherl et al., 2015; Antoniou et al., 2019). However, no information is available about being a vector of other damaging diseases or parasites. The only parasitism association reported in the literature to date from the Mediterranean is by native ectoparasites Nerocila bivittata (Antoniou et al., 2019). Endoparasites have been also found in the stomach contents of lionfish examined as part of the RELIONMED project.

2.34. How important might other impacts not already minimal medium Most known lionfish impacts are summarised above. To covered by previous questions be resulting from our knowledge no other lionfish impacts exist. introduction of the organism? (specify in the comment box)

2.35. How important are the expected impacts of the major high The magnitude of expected impacts can be considerably organism despite any natural control by other organisms, high despite the possible existence of a few organisms such as predators, parasites or pathogens that may already that may adapt to feed on lionfish (see also Q1.19 for be present in Europe? more information on natural enemies of lionfish in the Mediterranean).

Nevertheless, evidence shows that some native predators are able to feed on and potentially control certain alien species without requiring “adaptive” time- lag periods (Giakoumi et al.,2019c). 103 | P a g e The lionfish possesses venomous spines that provide protection from predators. It is yet uncertain if natural control will take place in the Mediterranean Sea invasive range (see Q1.19). Groupers have been known to eat lionfish in the Caribbean and in Cyprus. However, groupers and other predators in the Mediterranean are overfished and the biotic resistance of the Mediterranean ecosystems against non- indigenous species is likely low (Kimbro et al.,2013). Strengthening top predators protection and conservation can be important, especially for species which can adapt and have a major role in controlling introduced NIS including lionfish. Examples are that of France, that prohibited the commercial, spearfishing, and recreational hook-and-line fishing of groupers (Epinephelus spp. and Mycteroperca rubra) until at least 2023, or of Israel, where elasmobranch’s commercial fishing is prohibited.

104 | P a g e

RISK SUMMARIES

RESPONSE CONFIDENCE COMMENT S1. Summarise Entry very likely very high Already entered the eastern Mediterranean Sea and exhibits a westwards expansion. S2. Summarise Establishment very likely very high Already established in countries such as Cyprus and Greek islands including Crete and islands of the Dodecanese. It will also likely establish in Italy and Malta. Its establishment in more countries depends on niche unfilling/expansion (see Ch2 for more information).

S3. Summarise Spread very rapidly very high Lionfish was able to spread very rapidly i.e. in 3-4 years after the first record in the Mediterranean Sea, it reached central basin (e.g. Tunisia, Sicily, and Greek Ionian islands) and expanding fast in the eastern region. It demonstrates one of the fastest invasive species spread recorded to date in the Mediterranean Sea.

S4. Summarise Impact major medium Significant ecological and socio-economic impacts observed in the western Atlantic invasive range, and similar pattern is expected in the Mediterranean Sea. Major impacts are expected for the biodiversity, massive for ecosystem services and economy, while moderate impacts are expected from lionfish as public health threat.

S5. Conclusion of the risk assessment high High There is high confidence that there is a high degree of risk (social, ecological and economic) associated with the future development of the lionfish invasion in the Mediterranean. Empirical evidences from the Western Atlantic show that lionfish has the potential to cause

105 | P a g e devastating impacts. Initial evidence from RELIONMED in the Mediterranean shows increased abundance over time and shows the potential to cause detrimental damage to local marine communities, including iconic endangered species and protected organisms and habitats in NATURA 2000 sites and MPAs. Socio-economic impacts are also predicted for coastal communities, especially for the vulnerable sector of small-scale fishery and tourism.

106 | P a g e

ADDITIONAL QUESTIONS - CLIMATE CHANGE 3.1. What aspects of climate change, if any, are most Seawater medium The NCCOS research project in North Carolina, started likely to affect the risk assessment for this organism? warming in 2004 and is still ongoing. It monitors lionfish at the northern point of establishment in the invasive range. Long-term observations indicate that there is a thermal threshold for invasive lionfish off North Carolina: Lionfish were present in locations where mean winter water temperature was above 15°C. This threshold can be used for predicting lionfish presence in the Mediterranean. Areas in the Mediterranean with mean winter temperatures below 15 °C include the Northern Aegean, Adriatic Sea, North Western Mediterranean, Iberian coast and Bay of Biscay. If they warm up by 2 oC they might offer suitable habitat for the establishment of lionfish throughout the whole Mediterranean. On the other hand, D’Amen & Azzurro (2019) SDM predicted that under 2050 climate change scenarios, lionfish will be restricted to the central-eastern Mediterranean. Future expansion depends on niche filling/unfilling (see Ch2) of lionfish and complementarty studies such as ecophysiological models, SDMs and genetics can help to better anticipate changes.

3.2. What is the likely timeframe for such changes? 80 years High For the purposes of this Risk Assessment, we used RCP 6.0 which projects a global sea surface temperature increase of 1.9 ± 0.4 by end of this century (Collins et al., 2013) .

3.3. What aspects of the risk assessment are most likely to Habitat low change as a result of climate change? suitability Proliferation 107 | P a g e Establishment Increased l impacts

ADDITIONAL QUESTIONS – RESEARCH 4.1. If there is any research that would significantly Reproductive high Much of this risk assessment is based on empirical strengthen confidence in the risk assessment, please traits. evidence derived from the western Atlantic Ocean summarise this here. Trophic invasive range, which is a considerably different marine ecology. system, and some preliminary research conducted as Niche. part of the EU-RELIONMED project. To strengthen the Interspecific confidence of the risk assessment related to the invasion interactions. in the Mediterranean Sea, additional studies are Genetic required, focusing on the impacts of lionfish on research. ecosystem services and goods, as well as on the socio- Modelling economic sector. approaches. More precisely, multi-dimensional studies should Socio- examine biology and ecology of lionfish such as economic dispersal capabilities and connectivity patterns, impacts. demography, interspecific interactions (i.e. predation, competition, parasitism), intraspecific interactions (i.e. mating, aggregation and reproduction (e.g. using histology tools) patterns at temporal and spatial scales). These will enable a better understanding of impacts on biodiversity, habitat structure and function. Furthermore, socio-economic studies should aim to estimate economic losses and gains relevant to the impacts of lionfish and its increasing abundance (i.e. fishing industry, tourism industry, diving industry, pharmaceutical industry, jewel industry) as well as to examine the risk of exposure of fishers/divers/tourists to lionfish envenomation, and costs of management techniques. Habitat and climatic niche of the Mediterranean should be further explored with modelling. Dispersal, 108 | P a g e ecophysiology, and genetic studies of P. miles can support better predictions of models (e.g. SDMs) and provide a better understanding on the climatic niche, rate of expansion and potential geographical areas at which the lionfish is likely to establish. Genetics can also be used to better understand lionfish recruitment, evolution, genetic flow along the Mediterranean and monitor of secondary introductions sources.

109 | P a g e REFERENCES:

Ahrenholz DW, Morris JA (2010) Larval duration of the lionfish, Pterois volitans along the Bahamian Archipelago. Environmental Biology of Fishes, 88, 305–309. Akins L (2012) Best practices and strategies for lionfish control. Invasive lionfish: a guide to control and management. Gulf and Fisheries Institute Press, Fort Pierce. Al Mabruk SA, & Rizgalla J (2019) First record of lionfish (Scorpaenidae: Pterois) from Libyan waters. J. Black Sea/Mediterranean Environment, 25, 108- 114. Albins MA (2013) Effects of invasive Pacific red lionfish Pterois volitans versus a native predator on Bahamian coral-reef fish communities. Biological Invasions, 15, 29–43. Albins MA, Hixon MA (2008) Invasive Indo-Pacific lionfish Pterois volitans reduce recruitment of Atlantic coral-reef fishes. Marine Ecology Progress Series, 367, 233–238. Albins MA, Hixon MA (2013) Worst case scenario : Potential long-term effects of invasive predatory lionfish ( Pterois volitans ) on Atlantic and Caribbean coral-reef communities. Environmental Biology of Fishes, 96, 1151–1157. Alemu JB, Schuhmann P, & Agard J (2019) Mixed preferences for lionfish encounters on reefs in Tobago: Results from a choice experiment. Ecological Economics, 164, 106368. Ali, M., Reynaud, C., Capapé, C. (2017). Has a viable population of common lionfish, Pterois miles (Scorpaenidae), established off the Syrian Coast(Eastern Mediterranean)? Suomalainen Tiedeakatemia toimituksia. Sar. A.4: Biologica. 27. 10.19233/ASHN.2017.19. Alshawy F, Lahlah M, Hussein C (2016). First record of the Berber ponyfish Leiognathus berbis Valenciennes, 1835 (Osteichthyes: Leiognathidae) from Syrian marine waters (eastern Mediterranean). Marine Biodiversity Records, 9, 98. Andradi-Brown DA (2019) Invasive lionfish (Pterois volitans and P. miles): distribution, impact, and management. In Mesophotic Coral Ecosystems (pp. 931-941). Springer, Cham. Antoniou C, Kleitou P, Crocetta F, Lorenti M (2019) First record of ectoparasitic isopods on the invasive lionfish Pterois miles ( Bennett , 1828 ). Spixiana, 5–7. Araga C, Tanase, H (1968) Further record of winter fish stranding in the vicinity of Seto. Kyoto University, 16, 207-218 Arias-González JE, González-Gándara C, Luis Cabrera J, Christensen V (2011) Predicted impact of the invasive lionfish Pterois volitans on the food web of a Caribbean coral reef. Environmental Research, 111, 917–925. Ayas D, Agilkaya GŞ, Yaglioglu D (2018) New record of the red lionfish, Pterois volitans (Linnaeus, 1758), in the Northeastern Mediterranean Sea. Düzce University Journal of Science & Technology, 6, 871–877. Azzurro E, Soto S, Garofalo G, Maynou F (2013) Fistularia commersonii in the Mediterranean Sea: invasion history and distribution modeling based on presence-only records. Biological Invasions, 15, 977-990. Azzurro E, Tuset VM, Lombarte A, Maynou F, Simberloff D, Rodríguez‐Pérez A, Solé RV (2014) External morphology explains the success of biological invasions. Ecology letters, 17, 1455-1463. Azzurro E, Bariche M (2017) Local knowledge and awareness on the incipient lionfish invasion in the eastern Mediterranean Sea. Marine and Freshwater 110 | P a g e Research, 1–5. Azzurro E, Stancanelli B, Di Martino V, Bariche M (2017) Range expansion of the common lionfish Pterois miles (Bennett, 1828) in the Mediterranean Sea: an unwanted new guest for Italian waters. BioInvasions Records, 6, 95–98. Ballew NG, Bacheler NM, Kellison GT, Schueller AM (2016) Invasive lionfish reduce native fish abundance on a regional scale. Scientific Reports, 6, 1–7. Barbour AB, Montgomery ML, Adamson AA, Díaz-Ferguson E, Silliman BR (2010) Mangrove use by the invasive lionfish Pterois volitans. Marine Ecology Progress Series, 401, 291–294. Barbour AB, Allen MS, Frazer TK, Sherman KD (2011) Evaluating the Potential Efficacy of Invasive Lionfish ( Pterois volitans ) Removals. Plos ONE, 6, 1–7. Bariche M, Torres M, Azzurro E (2013) The presence of the invasive Lionfish Pterois miles in the Mediterranean Sea. Mediterranean Marine Science, 14, 292–294. Bariche M, Kleitou P, Kalogirou S, Bernardi G (2017) Genetics reveal the identity and origin of the lionfish invasion in the Mediterranean Sea. Scientific Reports, 7, 1–6. Barker BD, Horodysky AZ, Kerstetter DW (2018) Hot or not? Comparative behavioral thermoregulation, critical temperature regimes, and thermal tolerances of the invasive lionfish Pterois sp. versus native western North Atlantic reef fishes. Biological Invasions, 20, 45–58. Bellis B, Carolina C, Christine Z, Hannah H et al.,(2012) Lionfish: Kudzu of the Caribbean. ALS 398 Spring 2012 Benkwitt CE (2015) Non-linear effects of invasive lionfish density on native coral-reef fish communities. Biological Invasions, 17, 1383–1395. Bernal NA, DeAngelis DL, Schofield PJ, & Sealey KS (2015) Predicting spatial and temporal distribution of Indo-Pacific lionfish (Pterois volitans) in Biscayne Bay through habitat suitability modeling. Biological Invasions, 17, 1603-1614.Bernardi G, Golani D, Azzurro E (2010) The genetics of Lessepsian bioinvasions. Fish invasions of the Mediterranean Sea: change and renewal, 71-84. Bernardi G, Azzurro E, Golani D, Miller MR (2016) Genomic signatures of rapid adaptive evolution in the bluespotted cornetfish, a Mediterranean Lessepsian invader. Molecular ecology, 25, 3384-3396. Betancur-R. R, Hines A, Arturo AP, Ortí G, Wilbur AE, Freshwater DW (2011) Reconstructing the lionfish invasion: Insights into Greater Caribbean biogeography. Journal of Biogeography, 38, 1281–1293. Bethoux JP, Gentili B (1996) The Mediterranean Sea, coastal and deep-sea signatures of climatic and environmental changes. Journal of Marine Systems, 7, 383–394. Biggs CR, Olden JD (2011) Multi-scale habitat occupancy of invasive lionfish (Pterois volitans) in coral reef environments of Roatan, Honduras. Aquatic Invasions, 6, 347–353. Biondo M V. (2017) Quantifying the trade in marine ornamental fishes into Switzerland and an estimation of imports from the European Union. Global Ecology and Conservation, 11, 95–105. Bos AR, Sanad AM, Elsayed K (2017) Gymnothorax spp. ( Muraenidae ) as natural predators of the lionfish Pterois miles in its native biogeographical range. Environ Biol Fish, 100, 745–748. Botnen H, Gollasch S (1999) Results of the 2nd ocean-going workshop on the oil tanker NORDIC TORINITA on the voyage from Cork (Ireland) to Sture (Norway). In Final report of the EU concerted action testing monitoring Systems for Risk Assessment of harmful introductions by ships to European waters. Attachment VIII, 26 pp.

111 | P a g e Brown JH, Gillooly JF, Allen AP, Savage VM, West GB (2004) Toward a metabolic theory of ecology. Ecology, 85, 1771–1789. Burke L, Maidens J (2004) Reefs at Risk in the Caribbean. World Resources Institute, Washington, DC, 80 pp. Byrnes, JE, Reynolds PL, Stachowicz JJ (2007) Invasions and extinctions reshape coastal marine food webs. PloS one, 2, e295. Carlsson NO, Bustamante H, Strayer DL and Pace ML (2011) Biotic resistance on the increase: native predators structure invasive zebra mussel populations. Freshwater Biology; 56, 1630-7. Carlton JT (1996) Marine Bioinvasions: The Alteration of Marine Ecosystems by Nonindigenous Species. Oceanography, 9, 36–43. Chainho P, Fernandes A, Amorim A et al.,(2015) Non-indigenous species in Portuguese coastal areas, coastal lagoons, estuaries and islands. Estuarine, Coastal and Shelf Science, 167, 199–211. Chapman JK, Anderson LG, Gough CLA, Harris AR (2016) Working up an appetite for lionfish: A market-based approach to manage the invasion of Pterois volitans in Belize. Marine Policy, 73, 256–262. CIESM (2008) Climate warming and related changes in Mediterranean marine biota. N° 35 in CIESM Workshop Monographs [F. Briand, Ed.] Monaco, 152 pp. http: //www.ciesm. org/online/monographs/Helgoland.html Clavel J, Julliard R, Devictor V (2011) Worldwide decline of specialist species: Toward a global functional homogenization? Frontiers in Ecology and the Environment, 9, 222–228. Claydon JAB, Calosso MC, Traiger SB (2012) Progression of invasive lionfish in seagrass, mangrove and reef habitats. Marine Ecology Progress Series, 448, 119–129. Coates KA, Fourqurean JW, Kenworthy WJ, Logan A, Manuel SA, Smith SR (2013) Introduction to Bermuda: geology, oceanography and climate. In Coral Reefs of the United Kingdom Overseas Territories (pp. 115-133). Springer, Dordrecht. Collins M, Knutti R, Arblaster J, Dufresne JL, Fichefet T, Friedlingstein P, ... & Shongwe M (2013) Long-term climate change: projections, commitments and irreversibility. In Climate Change 2013-The Physical Science Basis: Contribution of Working Group I to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change (pp. 1029-1136). Cambridge University Press. Corrales X, Coll M, Ofir E, Heymans JJ, Steenbeek J, Goren M, ... & Gal G (2018) Future scenarios of marine resources and ecosystem conditions in the Eastern Mediterranean under the impacts of fishing, alien species and sea warming. Scientific reports, 8, 14284. Côté IM, Green SJ (2012) Potential effects of climate change on a marine invasion: The importance of current context. Current Zoology, 58, 1–8. Côté IM, Maljković A (2010) Predation rates of indo-pacific lionfish on bahamian coral reefs. Marine Ecology Progress Series, 404, 219–225. Côté IM, Smith NS (2018) The lionfish Pterois sp. invasion: Has the worst-case scenario come to pass? Journal of Fish Biology, 92, 660–689. Côté IM, Green SJ, Hixon MA (2013a) Predatory fish invaders: Insights from Indo-Pacific lionfish in the western Atlantic and Caribbean. Biological Conservation, 164, 50–61. Côté IM, Green SJ, Morris JA, Akins JL, Steinke D (2013b) Diet richness of invasive Indo-Pacific lionfish revealed by DNA barcoding. Marine Ecology Progress Series, 472, 249–256. Côté IM, Darling ES, Malpica-Cruz L, Smith NS, Green SJ, Curtis-Quick J, Layman C (2014a) What doesn't kill you makes you wary? Effect of repeated culling on the behaviour of an invasive predator. PLoS One, 9, e94248. Côté IM, Akins L, Underwood E, Curtis-Quick J, Green SJ (2014b) Setting the record straight on invasive lionfish control: Culling works (No. e398v1). PeerJ PrePrints.

112 | P a g e Coutts a DM, Moore KM, Hewitt CL (2003) Ships’ sea chest: an overlooked transfer mechanism for non-indigenous marine species? Marine Pollution Bulletin, 46, 1504–1515. Crocetta F, Agius D, Balistreri P, Bariche M, Bayhan YK, Çakir M, Ciriaco S (2015) New Mediterranean Biodiversity Records ( October 2015 ). Mediterranean Marine Science, 16, 682–702. Chagaris, D., Binion-Rock, S., Bogdanoff, A., Dahl, K., Granneman, J., Harris, H., Mohan, J., Rudd, M.B., Swenarton, M.K., Ahrens, R., 2017. An ecosystem-based approach to evaluating impacts and management of invasive lionfish. Fisheries 42, 421-431. https://doi.org/10.1080/03632415.2017.1340273. Curtis-Quick J, Underwood E, Green S, Akins LAD, Harborne A, Côté I (2013) Interactions Between the Caribbean Spiny Lobster , Panulirus argus , and Invasive Lionfish , Pterois volitans : Who Displaces Whom ? The 66th Gulf and Caribbean Fisheries Institute. Dabruzzi TF, Bennett WA, Fangue NA (2017) Thermal ecology of red lionfish Pterois volitans from Southeast Sulawesi, Indonesia, with comparisons to other Scorpaenidae. Aquatic Biology, 26, 1–14. Dahl KA, Patterson WF (2014) Habitat-specific density and diet of rapidly expanding invasive red lionfish, Pterois volitans, populations in the northern Gulf of Mexico. PLoS ONE, 9. Dailanis et al. (2016) New Mediterranean Biodiversity Records (July 2016). Mediterranean Marine Science, 17, 608–626. Darling ES, Green SJ, O’Leary JK, Côté IM (2011) Indo-Pacific lionfish are larger and more abundant on invaded reefs: A comparison of Kenyan and Bahamian lionfish populations. Biological Invasions, 13, 2045–2051. D’Amen M, & Azzurro E (2019) Lessepsian fish invasion in Mediterranean marine protected areas: a risk assessment under climate change scenarios. ICES Journal of Marine Science. De León R, Vane K, Bertuol P, Chamberland VC, Simal F, Imms E, Vermeij MJ (2013) Effectiveness of lionfish removal efforts in the southern Caribbean. Endangered Species Research, 22, 175-182. DeRivera CE, Ruiz GM, Hines AH, Jivoff P (2005). Biotic resistance to invasion: native predator limits abundance and distribution of an introduced crab. Ecology 86, 3364-3376. 10.1890/05-0479. Diamant A, Whipps CM, Kent ML (2004) A new species of Sphaeromyxa (Myxosporea: Sphaeromyxina: Sphaeromyxidae) in devil firefish, Pterois miles (Socrpaenidae) from the nortnern Red Sea: Morphology, ultrastructure, and phylogeny. Journal of Parasitology, 90, 1434–1442. Diaz JH (2015) Marine Scorpaenidae Envenomation in Travelers: Epidemiology, Management, and Prevention. Journal of Travel Medicine, 22, 251–258. Diffenbaugh NS, Pal JS, Giorgi F, Gao X (2007) Heat stress intensification in the Mediterranean climate change hotspot. Geophysical Research Letters, 34, 1–6. Dimitriou A, Chartosia N, Hall-Spencer MJ, Kleitou P, Jimenez C, Antoniou C, Hadjioannou L, Kletou D, Sfenthourakis S (2019) Genetic data suggest multiple introductions of the lionfish (Pterois miles) into the Mediterranean Sea following various pathways. Diversity. Special issue Biological Invasions 2020 Horizon. DeRivera CE, Ruiz GM, Hines AH, Jivoff P (2005) Biotic resistance to invasion: native predator limits abundance and distribution of an introduced crab. Ecology, 86, 3364-3376. Eddy C, Pitt J, Oliveira K, Morris JA, Potts J, Bernal D (2019) The life history characteristics of invasive lionfish (Pterois volitans and P. miles) in Bermuda. Environmental Biology of Fishes, 102, 887-900.

113 | P a g e El-Serehy HA, Abdallah HS, Al-Misned FA, Irshad R, Al-Farraj SA, Almalki ES (2018) Aquatic ecosystem health and trophic status classification of the Bitter Lakes along the main connecting link between the Red Sea and the Mediterranean. Saudi Journal of Biological Sciences, 25, 204–212. FAO (2018) The State of Mediterranean and Black Sea Fisheries. General Fisheries Commission for the Mediterranean. Rome. 172 pp. Farquhar SD. (2017) Back to the source: lionfish imported into the United States via the ornamental aquarium trade. J. Environ. Ecol, 8, 23-31. Filiz H, Tarkan AS, Bilge G, Yapıcı S (2017) Assessment of invasiveness potential of Pterois miles by the Aquatic Species Invasiveness Screening Kit. Journal of Black Sea/Mediterranean Environment, 23, 17-37. Fishbase (2013) Fish Identification: Pterois [Online]. Available at: https://www.fishbase.de/identification/SpeciesList.php?genus=Pterois [Accessed: 31 August 2018]. Fishbase (2013) Fish Identification: Pterois [Online]. Available at: https://www.fishbase.de/identification/SpeciesList.php?genus=Pterois [Accessed: 31 August 2018]. Fishelson L (1975) Ethology and reproduction of pteroid fishes found in the gulf of Aqaba (Red Sea), especially Dendrochirus brachypterus (Cuvier),(Pteroidae, Teleostei).[Conference paper]. Pubblicazioni della Stazione Zoologica, Napoli. Frazer TK, Jacoby CA, Edwards MA, Barry SC, Manfrino CM (2012) Coping with the lionfish invasion: can targeted removals yield beneficial effects?. Reviews in Fisheries Science, 20, 185-191. Freshwater DW, Hines A, Parham S, Wilbur A, Sabaoun M, Woodhead J, ... & Paris CB (2009) Mitochondrial control region sequence analyses indicate dispersal from the US East Coast as the source of the invasive Indo-Pacific lionfish Pterois volitans in the Bahamas. Marine Biology, 156(6), 1213- 1221. Galanidi M, Zenetos A, Bacher S (2018) Assessing the socio-economic impacts of priority marine invasive fishes in the Mediterranean with the newly proposed SEICAT methodology. Mediterranean Marine Science, 19, 107-123. Galil BS (2006) Shipwrecked – Shipping Impacts on the Biota of the Mediterranean Sea. In: The E cology of Transportation: Managing Mobility for the Environment, 1st edn, Vol. 10 (eds Davenport J, Davenport JL), pp. 39–69. Springer Netherlands. Galil B, Marchini A, Occhipinti-Ambrogi A, Ojaveer H (2017) The enlargement of the Suez Canal—Erythraean introductions and management challenges. Management of Biological Invasions, 8(2), 141-152. Gardner PG, Frazer TK, Jacoby CA, Yanong RPE (2015) Reproductive biology of invasive lionfish (Pterois spp.). Frontiers in Marine Science, 2, 1–10. Garyfallou GT, Madden JF (1996) Lionfish envenomation. Annals of Emergency Medicine, 28, 456–457. Gerin R, Poulain PM, Taupier-Letage I, Millot C, Ismail SB, Sammari C (2009) Surface circulation in the Eastern Mediterranean using drifters (2005-2007). Ocean Science, 5, 559-574. Giakoumi S, Pey A, Di Franco A, Francour P, Kizilkaya Z, Arda Y, ... & Guidetti P (2019a). Exploring the relationships between marine protected areas and invasive fish in the world's most invaded sea. Ecological Applications, 29, e01809. Giakoumi S, Katsanevakis S, Albano PG, Azzurro E, Cardoso AC, Cebrian E, ... & Mačić V (2019b) Management priorities for marine invasive species. Science of The Total Environment, 688, 976-982. Giakoumi, S, Pey A, Thiriet P, Francour P, & Guidetti P (2019c) Patterns of predation on native and invasive alien fish in Mediterranean protected and unprotected areas. Marine environmental research, 150, 104792. Giovos I, Batjakas I, Doumpas N, Kampouris TE, Poursanidis D, Paravas V (2019). The current status of lionfish invasion in Greece and future steps towards

114 | P a g e control and mitigation, In: Lionfish Invasion and its Management in the Mediterranean Sea, (eds Hüseyinoğlu MF, Öztürk B), pp. 88-100. Turkish Marine Research Foundation (TUDAV), Turkey. Giovos I, Bernardi G, Romanidis-Kyriakidis G, Marmara D, Kleitou P (2018). First records of the fish Abudefduf sexfasciatus (Lacepède, 1801) and Acanthurus sohal (Forsskål, 1775) in the Mediterranean Sea. BioInvasions Records, 7. Goodbody-Gringley G, Eddy C, Pitt JM, Chequer AD, Smith SR (2019). Ecological Drivers of Invasive Lionfish (Pterois volitans and Pterois miles) Distribution Across Mesophotic Reefs in Bermuda. Frontiers in Marine Science, 6, 258. Gökoğlu M, Teker S, Julian D. (2017) Westward extension of the lionfish Pterois volitans Linnaeus, 1758 along the Mediterranean coast of Turkey. Natural and Engineering Sciences 2(2), 67-72. Golani D, Sonin O (1992) New Records of the Red Sea Fishes, Pterois miles (Scorpaenidae) and Pteragogus pelycus (Labridae) from the Eastern Mediterranean Sea. Japanese Journal of Icthyology, 39, 38–40. Golani D (2004) First record of the muzzled blenny (Osteichthyes: Blenniidae: Omobranchus punctatus) from the Mediterranean, with remarks on ship- mediated fish introduction. Journal of the Marine Biological Association of the United Kingdom, 84(4), 851-852. Golani D, Azzurro E, Corsini-Foka M, Falautano M, Andaloro F, Bernardi G (2007) Genetic bottlenecks and successful biological invasions: the case of a recent Lessepsian migrant. Biology letters, 3, 541-545.Gollasch S, Lenz J, Dammer M, Andres HG (2000) Survival of tropical ballast water organisms during a cruise from the Indian Ocean to the North Sea. Journal of Plankton Research, 22(5), 923–937. Gollasch S, Macdonald E, Belson S, Botnen H, Christensen J, Hamer J, et al.,(2002) Life in ballast tanks. In E. Leppäkoski, S. Gollasch, & S. Olenin (Eds.), Invasive aquatic species of Europe: Distribution, impacts and management (pp. 217–231). Dordrecht, The Netherlands: KLUWER Academic Publishers. Gollasch S, David M (2019) Ballast Water: Problems and Management. In World Seas: an Environmental Evaluation (pp. 237-250). Academic Press. Goren M, Gayer K, Lazarus N (2009) First record of the Far East chameleon goby Tridentiger trigonocephalus (Gill, 1859) in the Mediterranean Sea. Aquatic Invasions, 4, 413-415. Goren M, Galil BS (2008) Additional records of Bregmaceros atlanticus in the eastern Mediterranean—an invasion through the Suez Canal or in ballast water?. Marine Biodiversity Records, 1. Green SJ, Côté IM (2009) Record densities of Indo-Pacific lionfish on Bahamian coral reefs. Coral Reefs, 28, 107. Green SJ, Akins JL, Maljkovic A, Côté IM (2012) Invasive lionfish drive Atlantic coral reef fish declines. Plos o, 7, e32596. Green SJ, Dulvy NK, Brooks AM, Akins JL, Cooper AB, Miller S, Côté IM (2014) Linking removal targets to the ecological effects of invaders: a predictive model and field test. Ecological Applications, 24, 1311-1322. Gress E, Andradi-Brown DA, Woodall L, Schofield PJ, Stanley K, Rogers AD (2017) Lionfish ( Pterois spp.) invade the upper-bathyal zone in the western Atlantic. PeerJ, 5, e3683. Gülenç, 2019 - Marine aquariums and lionfish trade in Turkey. In: Lionfish Invasion and its Management in the Mediterranean Sea, (eds Hüseyinoğlu MF, Öztürk B), pp. 88-100. Turkish Marine Research Foundation (TUDAV), Turkey. Gurlek M, Erguden D, Uyan A, Dogdu S, Yaglıoglu D, Ozturk B, Turan C (2016) First record of red lionfish, Pterois volitans (Linnaeus, 1785) in the Mediterranean Sea. Natural and Engineering Sciences, 1, 27–32. Guzmán-Méndez, I. A., Rivera-Madrid, R., Díaz-Jaimes, P., García-Rivas, M. D. C., Aguilar-Espinosa, M., & Arias-González, J. E. (2017). First genetically

115 | P a g e confirmed record of the invasive devil firefish Pterois miles (Bennett. 1828) in the Mexican Caribbean. BioInvasions Rec, 6, 99-103. Hackerott S, Valdivia A, Green SJ, Côté IM, Cox CE, Akins L, ... & Bruno JF (2013) Native predators do not influence invasion success of Pacific lionfish on Caribbean reefs. PLoS one, 8, e68259. Haddad V, Stolf HO, Risk JY, França FOS, Cardoso JLC (2015) Report of 15 injuries caused by lionfish (Pterois volitans) in aquarists in Brazil: A critical assessment of the severity of envenomations. Journal of Venomous Animals and Toxins Including Tropical Diseases, 21. Haines LJ, Côté IM (2019) Homing decisions reveal lack of risk perception by Caribbean damselfish of invasive lionfish. Biological Invasions, 21, 1657- 1668. Hamner, R. M., Freshwater, D. W., & Whitfield, P. E. (2007). Mitochondrial cytochrome b analysis reveals two invasive lionfish species with strong founder effects in the western Atlantic. Journal of Fish Biology, 71, 214-222. Han W (2001) Modeling salinity distributions in the Indian Ocean. Journal of Geophysical Research, 106, 859–877. Hardison DR, Holland WC, Darius HT et al.,(2018) Investigation of ciguatoxins in invasive lionfish from the greater caribbean region : Implications for fishery development. 1, 1–15. Hare JA, & Whitfield PE (2003) An integrated assessment of the introduction of lionfish (Pterois volitans/miles complex) to the western Atlantic Ocean. Harms-Tuohy CA, Appeldoorn RS, Craig MT (2018). The effectiveness of small-scale lionfish removals as a management strategy: effort, impacts and the response of native prey and piscivores. Manag. Biol. Invasions, 9, 149-162. Hunt CL, Kelly GR, Windmill H, Curtis-Quick J, Conlon H, Bodmer MD, ... Exton DA (2019). Aggregating behaviour in invasive Caribbean lionfish is driven by habitat complexity. Scientific reports, 9, 783. Imamura H, Yabe M (1996) Larval record of a red firefish, Pterois volitans, from northwestern Australia (Pisces: Scorpaeniformes). 北海道大學水産學部研究彙報= BULLETIN OF THE FACULTY OF FISHERIES HOKKAIDO UNIVERSITY, 47(2-3), 41-46. Insacco G, Zava B (2017). Chlorurus rhakoura Randall & Anderson, 1997 (Perciformes, Scaridae), an Indo-Pacific fish new for the Mediterranean Sea. Mediterranean Marine Science, 18, 285-291. Jensen A, Collins K, Lockwood AP (Eds.) (2012) Artificial reefs in European seas. Springer Science & Business Media. Jimenez C, Andreou V, Hadjioannou L, Petrou A, Alhaija RA, Patsalou P (2017) Not everyone’s cup of tea: Public perception of culling invasive lionfish in Cyprus. Journal of the Black Sea/Mediterranean Environment, 23, 38–47. Jimenez C, Andreou V, Huseyinoglu F, Hadjioannou L, Patsalou P, Petrou A (2018). Of natural predation and gastronomy: Who's eating the invasive lionfish (Pterois miles) in Cyprus (Levantine Sea) p 138. In Mees, J.; Seys, J. (Eds.) Book of abstracts – 53rd European Marine Biology Symposium. Oostende, Belgium, 17-21 September 2018. VLIZ Special Publication, 82. Vlaams Instituut voor de Zee - Flanders Marine Institute (VLIZ): Oostende. 199 pp. Available at www.vliz.be/en/event/embs53. Accessed on 18 August 2019 Jiménez-Muñoz L, Quintanilla M, Filomena A (2019). Managing the lionfish: influence of high intensity ultrasound and binders on textural and sensory properties of lionfish (Pterois volitans) surimi patties. Journal of food science and technology, 56, 2167-2174. Johnston MW, Purkis SJ (2014) Are lionfish set for a Mediterranean invasion? Modelling explains why this is unlikely to occur. Marine pollution bulletin, 88, 138-147. Johnston, M. W., Purkis, S. J., & Dodge, R. E. (2015). Measuring Bahamian lionfish impacts to marine ecological services using habitat equivalency analysis. Marine biology, 162(12), 2501-2512. 116 | P a g e Johnson EG, Swenarton MK (2016) Age, growth and population structure of invasive lionfish ( Pterois volitans/miles ) in northeast Florida using a length- based, age-structured population model. PeerJ, 4, e2730. Johnston MW, Bernard AM, Shivji MS (2017) Forecasting lionfish sources and sinks in the Atlantic: are Gulf of Mexico reef fisheries at risk? Coral Reefs, 36, 169–181. Jørgensen, C., Peck, M. A., Antognarelli, F., Azzurro, E., Burrows, M. T., Cheung, W. W., ... & McKenzie, D. (2012). Conservation physiology of marine fishes: advancing the predictive capacity of models.Jud ZR, Layman CA, Lee JA, Arrington DA (2011) Recent invasion of a Florida (USA) estuarine system by lionfish Pterois volitans/P. miles. Aquatic Biology, 13, 21–26. Jud ZR, Nichols PK, Layman CA (2015) Broad salinity tolerance in the invasive lionfish Pterois spp. may facilitate estuarine colonization. Environmental Biology of Fishes, 98, 135–143. Katsanevakis S, Zenetos A, Belchior C, Cardoso AC (2013) Invading European Seas: Assessing pathways of introduction of marine aliens. Ocean and Coastal Management, 76, 64–74. Kimball ME, Miller JM, Whitfield PE, Hare JA (2004) Thermal tolerance and potential distribution of invasive lionfish ( Pterois volitans / miles complex ) on the east coast of the United States. Marine Ecology Progress Series, 283, 269–278. Kimbro DL, Cheng BS, & Grosholz ED (2013) Biotic resistance in marine environments. Ecology Letters, 16(6), 821-833. Kindinger TL, Albins MA (2017) Consumptive and non-consumptive effects of an invasive marine predator on native coral-reef herbivores. Biological Invasions, 19, 131–146. Kirtman B, Power SB, Adedoyin AJ, Boer GJ, Bojariu R, Camilloni I, ... & Prather M (2013) Near-term climate change: projections and predictability. In Climate Change 2013-The Physical Science Basis: Contribution of Working Group I to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change (pp. 953-1028). Cambridge University Press. Kletou D, Hall-Spencer JM, Kleitou P (2016) A lionfish (Pterois miles) invasion has begun in the Mediterranean Sea. Marine Biodiversity Records, 9, 1–7. Kleitou P, Savva I, Kletou D, Hall-Spencer JM, Antoniou C, Christodoulides Y, …, Rees S (2019) Invasive lionfish in the Mediterranean: Low public awareness yet high stakeholder concerns. Marine Policy, 104, 66-74. Kochzius M, Blohm D (2005) Genetic population structure of the lionfish Pterois miles (Scorpaenidae, Pteroinae) in the Gulf of Aqaba and northern Red Sea. Gene, 347, 295–301. Kochzius M, Söller R, Khalaf MA, Blohm D (2003) Molecular phylogeny of the lionfish genera Dendrochirus and Pterois (Scorpaenidae, Pteroinae) based on mitochondrial DNA sequences. Molecular Phylogenetics and Evolution, 28, 396–403. Kousteni V, Bakiu R, Benhmida A, Crocetta F, Di Martino V, Dogrammatzi A, ... & Huseyinoglu MF (2019) New Mediterranean Biodiversity Records (April, 2019). Mediterranean Marine Science, 20, 230-247. Kulbicki M, Beets J, Chabanet P et al.,(2012) Distributions of Indo-Pacific lionfishes Pterois spp. in their native ranges: Implications for the Atlantic invasion. Marine Ecology Progress Series, 446, 189–205. Layman CA, Jud ZR, Nichols P (2014) Lionfish alter benthic invertebrate assemblages in patch habitats of a subtropical estuary. Marine biology, 161, 2179- 2182. Lee S, Buddo DS a, Aiken K a (2011) Habitat Preference in the Invasive Lionfish (Pterois volitans / miles) in Discovery Bay, Jamaica: Use of GIS in Management Strategies. Proceedings of the 64th Gulf and Caribbean Fisheries Institute, 10.

117 | P a g e Lejeusne C, Chevaldonné P, Pergent-Martini C, Boudouresque CF, Pérez T (2010) Climate change effects on a miniature ocean: the highly diverse, highly impacted Mediterranean Sea. Trends in Ecology and Evolution, 25, 250–260. Lesser MP, Slattery M (2011) Phase shift to algal dominated communities at mesophotic depths associated with lionfish ( Pterois volitans ) invasion on a Bahamian coral reef. Biological Invasions, 13, 1855–1868. Lyons TJ, Tuckett QM, Hill JE (2019) Characterizing the US trade in lionfishes. PloS one, 14(8). Lyons TJ (2018) Invasion Risk of the Lionfishes Pterois, Dendrochirus, and ParaPterois. Master's ( M.S.). University of Florida, United States of America Maclsaac HJ, DeRoy EM, Leung B, Grgicak-Mannion, Ruiz GM (2016) Possible ballast water transfer of lionfish to the Eastern Pacific Ocean. PLoS ONE, 11, 1–12. Maljković A, Van Leeuwen TE, Cove SN (2008) Predation on the invasive red lionfish, Pterois volitans (Pisces: Scorpaenidae), by native groupers in the Bahamas. Coral Reefs, 27, 501–501. Malpica-Cruz L, Haider W, Smith NS, Fernández-Lozada S, & Côté IM (2017) Heterogeneous Attitudes of Tourists toward Lionfish in the Mexican Caribbean: Implications for Invasive Species Management. Frontiers in Marine Science, 4, 138. Marba N, Duarte C (1997) Interannual changes in seagrass (Posidonia oceanica) growth and environmental change in the Spanish Mediterranean littoral zone. Limnology and Oceanography, 42, 800–810. Marbà N, Jordà G, Agustí S, Girard C, Duarte CM (2015) Footprints of climate change on Mediterranean Sea biota. Frontiers in Marine Science, 2, 1–11. Marras S, Cucco A, Antognarelli F, Azzurro E, Milazzo M, Bariche M, ... & Sinerchia M (2015) Predicting future thermal habitat suitability of competing native and invasive fish species: from metabolic scope to oceanographic modelling. Conservation physiology, 3(1). Metaxas D a, Bartzokas a, Vitsas a (1991) Temperature-Fluctuations in the Mediterranean Area During the Last 120 Years. International Journal of Climatology, 11, 897–908. Michailidis N, Corrales X, Karachle PK, Chartosia N, Katsanevakis S, Sfenthourakis S (2019) Modelling the role of alien species and fisheries in an Eastern Mediterranean insular shelf ecosystem. Ocean & coastal management, 175, 152-171. Mito S (1956) On the egg development and hatched larvae of a scorpaenoid fish, Pterois lunulata Temminck et Schlegel. Sci Bull Fac Agric Kyushyu Univ, 16, 381-385. Morris JA, Breen N (2011) Nutritional properties of the invasive lionfish: A delicious and nutritious approach for controlling the invasion. Aquaculture, Aquariums, Conservation & Legislation, 5, 99–102. Morris JA, Whitfield PE (2009) Biology, Ecology, Control and Management of the Invasive Indo-Pacific Lionfish: An Updated Integrated Assessment. 57 pp. Morris JA, Akins JL, Barse A, Cerino D, Freshwater DW (2008) Biology and Ecology of the Invasive Lionfishes, Pterois miles and Pterois volitans. Marine Ecology, 29, 1–6. Morris JA, Sullivan C V., Govoni JJ (2011) Oogenesis and spawn formation in the invasive lionfish, Pterois miles and Pterois volitans. Scientia Marina, 75, 147–154. Morris JA, Shertzer KW, Rice JA (2011) A stage-based matrix population model of invasive lionfish with implications for control. Biological Invasions, 13, 7-12. Morris JA (2012) Invasive lionfish: a guide to control and management. Gulf and Caribbean Fisheries Institute, 113. Mumby PJ, Harborne AR, Brumbaugh DR (2011) Grouper as a natural biocontrol of invasive Lionfish. PLoS ONE, 6, 2–5.

118 | P a g e NCCOS (2017) he Role of Temperature and Depth in Fish Community Distribution off North Carolina with Implications for Invasive Lionfish Distribution [Online]. Available at: https://coastalscience.noaa.gov/project/temperature-depth-invasive-lionfish-distribution/[Accessed: 31 August 2018] Oray IK, Sinay E, Saadet Karakulak F, Yildiz T (2015) An expected marine alien fish caught at the coast of Northern Cyprus: Pterois miles (Bennett, 1828). Journal of Applied Ichthyology, 31, 733–735. Orejas C, Jiménez C, Gori A, Rivera J, Iacono CL, Aurelle D, Hadjioannou L, Petrou A, Achilleos K (2019) Corals of Aphrodite: Dendrophyllia ramea Populations of Cyprus. In Orejas C, Jiménez C (eds.), Mediterranean Cold-Water Corals: Past, Present and Future, Coral Reefs of the World 9, https://doi.org/10.1007/978-3-319-91608-8_12 pp. 257-260. Özbek EÖ, Mavruk S, Saygu İ, Öztürk B (2017) Lionfish distribution in the eastern Mediterranean coast of Turkey. Journa of Black Sea/Mediterranean Environment, 23, 1–16. PADI, 2019. 2019 Worldwide Corporate Statistics, Data for 2013-2018. PADI. Padilla DK, Williams SL (2004) Beyond ballast water: Aquarium and ornamental trades as sources of invasive species in aquatic ecosystems. Frontiers in Ecology and the Environment, 2, 131–138. Papacostas KJ, Freestone AL (2019) Stronger predation in a subtropical community dampens an invasive species-induced trophic cascade. Biological invasions, 21, 203-15. Parravicini V, Azzurro E, Kulbicki M, & Belmaker J (2015) Niche shift can impair the ability to predict invasion risk in the marine realm: an illustration using Mediterranean fish invaders. Ecology Letters, 18 (3), 246-253. Paxton AB, Peterson CH, Taylor JC, Adler AM, Pickering EA, Silliman BR (2019). Artificial reefs facilitate tropical fish at their range edge. Communications biology, 2, 168. Peake J, Bogdanoff AK, Layman CA et al.,(2018) Feeding ecology of invasive lionfish (Pterois volitans and Pterois miles) in the temperate and tropical western Atlantic. Biological Invasions. Penning M, Reid G, Koldewey H, Dick G, Andrews B, Arai K, ... Tonge S (2009) Turning the tide: a global aquarium strategy for conservation and sustainability. Bern: World Association of Zoos and Aquariums Executive Office. Potts JC, Berrane D, Morris, Jr. JA (2010) Age and growth of lionfish from the Western North Atlantic. Proceedings of the 63rd Gulf and Caribbean Fisheries Institute. Poursanidis D (2015) Ecological Niche Modeling of the the invasive lionfish Pterois miles (Bennett, 1828) in the Mediterranean Sea. Eleventh Panhellenic Symposium on Oceanography and Fisheries, Mytilene, Lesvos island, Greece, 13–15 May 2015. Anavyssos Attiki Greece; Hellenic Center for Marine Research, pp. 621–624. Poursanidis D, Kalogirou S, Azzurro E, Parravicini V, Bariche M, Dohna H (2019) Niche analysis predicts a rapid spread of invasive lionfish in the Mediterranean Sea. Under Review. Ramos-Ascherl Z, Williams EH, Bunkley-Williams L, Tuttle LJ, Sikkel PC, Hixon MA (2015) Parasitism in Pterois volitans (Scorpaenidae) from Coastal Waters of Puerto Rico, the Cayman Islands, and the Bahamas. Journal of Parasitology, 101, 50–56. Raymond WW, Albins MA, Pusack TJ (2015) Competitive interactions for shelter between invasive Pacific red lionfish and native Nassau grouper. Environmental Biology of Fishes, 98, 57–65. Rensch G, Murphy-Lavoie HM (2018) Lionfish, Scorpionfish, And Stonefish Toxicity. In StatPearls [Internet]. StatPearls Publishing.

119 | P a g e Rilov G, Galil B (2009) Marine Bioinvasions in the Mediterranean Sea – History, Distribution and Ecology. In: Biological Invasions in Marine Ecosystems. Ecological, Management, and Geographic Perspectives, Vol. 204 (eds Rilov G, Crooks JA), pp. 549–575. Springer-Verlag Berlin Heidelberg. Roberts M, Cresswell W, Hanley N (2018) Prioritising invasive species control actions: evaluating effectiveness, costs, willingness to pay and social acceptance. Ecological Economics 152, 1-8. https://doi.org/10.1016/j.ecolecon.2018.05.027. Rocha LA, Rocha CR, Baldwin CC, Weigt LA, McField M (2015) Invasive lionfish preying on critically endangered reef fish. Coral Reefs, 34, 803–806. Roy, H.E., Adriaens, T., Aldridge, D.C., Bacher, S., Bishop, J.D.D., Blackburn, T.M., Branquart, E., Brodie, J., Carboneras, C., Cook, E.J., Copp, G.H., Dean, H.J., Eilenberg, J., Essl, F., Gallardo, B., Garcia, M., García-Berthou, E., Genovesi, P., Hulme, P.E., Kenis, M., Kerckhof, F., Kettunen, M., Minchin, D., Nentwig, W., Nieto, A., Pergl, J., Pescott, O., Peyton, J., Preda, C., Rabitsch, W., Roques, A., Rorke, S., Scalera, R., Schindler, S., Schönrogge, K., Sewell, J., Solarz, W., Stewart, A., Tricarico, E., Vanderhoeven, S., van der Velde, G., Vilà, M., Wood, C.A., Zenetos, A. (2015) Invasive Alien Species - Prioritising prevention efforts through horizon scanning ENV.B.2/ETU/2014/0016. European Commission Samuel-Rhoads Y, Iona S, Zodiatis G, Stylianou S, Hayes D, Georgiou G (2010) Sea surface temperature and salinity rise in the Levantine Basin. In: Cοmmision Internationale pour l’exploration scientifique de la mer Méditerranée, Vol. 39, p. 177. Venise, Italy. Samuel-Rhoads Y, Hayes D, Konnaris G, Georgiou G (2013) Climate change impacts on sea surface temperature in the Eastern Mediterranean, Levantine Basin. In: Commision Internationale pour l´exploration scientifique de la Mer Méditerranée, Vol. 40, p. 204. Marseille, France. Sandel V, Martínez-Fernández D, Wangpraseurt D, Sierra L (2015) Ecology and management of the invasive lionfish Pterois volitans/miles complex (Perciformes: Scorpaenidae ) in Southern Costa Rica. Revista de Biología Tropical, 63, 213–221. Sala E, & Giakoumi S (2017) No-take marine reserves are the most effective protected areas in the ocean. ICES Journal of Marine Science, 75, 1166-1168. Saunder PR, Taylor PB (1959) Venom of the lionfish Pterois volitans. The American journal of physiology, 197, 437–440. Schofield PJ (2010) Update on geographic spread of invasive lionfishes (Pterois volitans [Linnaeus, 1758] and P. miles [Bennett, 1828]) in the Western North Atlantic Ocean, Caribbean Sea and Gulf of Mexico. Aquatic Invasions, 5, 117–122. Schult RF, Acquisto NM, Stair CK, Wiegand TJ (2017) Case Report A Case of Lionfish Envenomation Presenting to an Inland Emergency Department. Case Reports in Emergency Medicine, 3–6. Scyphers SB, Powers SP, Akins JL, Drymon JM, Martin CW, Schobernd ZH, ... Switzer TS (2015) The role of citizens in detecting and responding to a rapid marine invasion. Conservation Letters, 8, 242-250. Seatemperature (2018) Cape Hatteras average January sea temperature [Online] Available at: https://www.seatemperature.org/north-america/united- states/cape-hatteras-january.htm [Accessed: 10 August 2018]. Semmens BX, Buhle ER, Salomon AK, Pattengill-Semmens C V. (2004) A hotspot of non-native marine fishes: Evidence for the aquarium trade as an invasion pathway. Marine Ecology Progress Series, 266, 239–244. Sisma-Ventura G, Yam R, Shemesh A (2014) Recent unprecedented warming and oligotrophy of the eastern Mediterranean Sea within the last millennium. Geophysical Research Letters, 41, 5158–5166. Skliris N, Sofianos S, Gkanasos A, Mantziafou A, Vervatis V, Axaopoulos P, Lascaratos A (2012) Decadal scale variability of sea surface temperature in the Mediterranean Sea in relation to atmospheric variability. Ocean Dynamics, 62, 13–30. Sommeng AN, Arya RMY, Ginting MJ, Pratami DK, Hermansyah H, Sahlan M, Wijanarko A (2019). Antiretroviral activity of Pterois volitans (Red Lionfish) venom in the early development of human immunodeficiency virus/acquired immunodeficiency syndrome antiretroviral alternative source,

120 | P a g e Veterinary World, 12, 309-315.. Sri Balasubashini M, Karthigayan S, Somasundaram ST, Balasubramanian T, Viswanathan V, Raveendran P, Menon VP (2006) Fish venom (Pterios volitans) peptide reduces tumor burden and ameliorates oxidative stress in Ehrlich’s ascites carcinoma xenografted mice. Bioorganic and Medicinal Chemistry Letters, 16, 6219–6225. Stern N, Jimenez C, Huseyinoglu MF et al.,(2018) Constructing the genetic population demography of the invasive lionfish Pterois miles in the Levant Basin , Eastern Mediterranean. Mitochondrial DNA Part A, 1–7. Stevens JL, Jackson RL, Olson JB (2016) Bacteria associated with lionfish ( Pterois volitans / miles complex ) exhibit antibacterial activity against known fish pathogens. Marine Ecology Progress Series, 558, 167–180. Turan C, Öztürk B (2015) First record of the lionfish Pterois miles (Bennett 1828) from the Aegean Sea. Journal of the Black Sea/Mediterranean Environment, 21, 334–338. Turan C, Ergüden D, Gürlek M, Yağlıoğlu D, Uyan A, Uygur N (2014) First record of the Indo-Pacific lionfish Pterois miles (Bennett, 1828) (Osteichthyes: Scorpaenidae) for the Turkish marine waters. J. Black Sea/Mediterranean Environment, 20, 158–163. Turan C, Uygur N, İğde M (2017) Lionfishes Pterois miles and Pterois volitans in the North-eastern Mediterranean Sea: Distribution, Habitation, Predation and Predators. NESciences, 2, 35–43. Turan C (2019) Impacts of climate change and species distribution modelling: Future scenarios to invasive alien lionfish distribution in the Mediterranean Sea. Biodiversity Information Science and Standards, 3, e37363. Tuttle L (2017) Direct and indirect effects of invasive lionfish on coral-reef cleaning mutualists. Marine Ecology Progress Series, 569, 163–172. Ulbrich U, May W, Li L, Lionello P, Pinto JG, Somot S (2006) The Mediterranean Climate Change under Global Warming. Mediterranean Climate Variability, 4, 399–415. USFWS (2014) Pterois miles Ecological Risk Screening Summary U.S. Fish and Wildlife Service – Web Version – 7/28/2014. Accessed from: https://www.fws.gov/fisheries/ans/erss/highrisk/Pterois-miles-WEB-7-28-2014.pdf. Vargas-Yáñez M, Jesús García M, Salat J, García-Martínez MC, Pascual J, Moya F (2008) Warming trends and decadal variability in the Western Mediterranean shelf. Global and Planetary Change, 63, 177–184. Vargas-Yáñez M, Zunino P, Benali A et al.,(2010a) How much is the western Mediterranean really warming and salting? Journal of Geophysical Research: Oceans, 115, 1–12. Vargas-Yáñez M, Moya F, García-Martínez MC et al.,(2010b) Climate change in the Western Mediterranean Sea 1900-2008. Journal of Marine Systems, 82, 171–176. Vásquez-Yeomans L, Carrillo L, Morales S, Malca E, Morris JA, Schultz T, Lamkin JT (2011) First larval record of Pterois volitans (Pisces: Scorpaenidae) collected from the ichthyoplankton in the Atlantic. Biological Invasions, 13, 2635–2640. Vergés A, Tomas F, Cebrian E et al.,(2014) Tropical rabbitfish and the deforestation of a warming temperate sea. Journal of Ecology, 102, 1518–1527. Vetrano SJ, Lebowitz JB, Marcus S (2002) Lionfish envenomation. The Journal of Emergency Medicine, 23, 379–382. Whitfield PE, Hare JA, David AW, Harter SL, Muñoz RC, Addison CM (2007) Abundance estimates of the Indo-Pacific lionfish Pterois volitans/miles complex in the Western North Atlantic. Biological Invasions, 9, 53–64. Whitfield PE, Muñoz RC, Buckel CA, Degan BP, Freshwater DW, Hare JA 2014. Native fish community structure and Indo-Pacific lionfish Pterois volitans

121 | P a g e densities along a depth-temperature gradient in Onslow Bay, North Carolina, USA. Marine Ecology Progress Series, 509, 241-254. Wiedenmann J, Baumstark A, Pillen TL, Meinesz A, Vogel W (2001) DNA fingerprints of Caulerpa taxifolia provide evidence for the introduction of an aquarium strain into the Mediterranean Sea and its close relationship to an Australian population. Marine Biology, 138, 229–234. Wilcox CL (2014) Molecular investigations of the Pteroinae : insights into the invasive lionfishes from the native range. University of Hawaii, 111 pp. Wilcox CL, Motomura H, Matsunuma M, Bowen BW (2017) Phylogeography of Lionfishes (Pterois) Indicate Taxonomic Over Splitting and Hybrid Origin of the Invasive Pterois volitans. Journal of Heredity, 162–175. Wonham MJ, Carlton JT, Ruiz GM, Smith LD (2000) Fish and ships: Relating dispersal frequency to success in biological invasions. Marine Biology, 136, 1111–1121. Yokes MB, Andreou V, Bakiu R, Bonanomi S, Camps J, Christidis G, ... Karhan SÜ 2018. New Mediterranean Biodiversity Records (November 2018). Mediterranean Marine Science, 19, 673-689. Zannaki, K., Corsini-Foka, M., Kampouris, T. E., & Batjakas, I. E. (2019). First results on the diet of the invasive Pterois miles (Actinopterygii: Scorpaeniformes: Scorpaenidae) in the Hellenic waters. Acta Ichthyologica et Piscatoria, 49(3). Zenetos A, Çinar ME, Crocetta F, Golani D, Rosso A, Servello G, ... & Verlaque M (2017) Uncertainties and validation of alien species catalogues: The Mediterranean as an example. Estuarine, Coastal and Shelf Science, 191, 171-187.

122 | P a g e ANNEX Scoring of Likelihoods of Events (taken from UK Non-native Organism Risk Assessment Scheme User Manual, Version 3.3, 28.02.2005)

Score Description Frequency Very unlikely This sort of event is theoretically possible, but is never known to have 1 in 10,000 years occurred and is not expected to occur Unlikely This sort of event has not occurred anywhere in living memory 1 in 1,000 years Possible This sort of event has occurred somewhere at least once in recent years, but 1 in 100 years not locally Likely This sort of event has happened on several occasions elsewhere, or on at 1 in 10 years least one occasion locally in recent years Very likely This sort of event happens continually and would be expected to occur Once a year

123 | P a g e ANNEX Scoring of Magnitude of Impacts (modified from UK Non-native Organism Risk Assessment Scheme User Manual, Version 3.3, 28.02.2005)

Score Biodiversity and Ecosystem Services impact Economic impact (Monetary loss Social and human health impact ecosystem impact and response costs per year) Question 2.18-22 Question 2.23-25 Question 2.26-30 Question 2.31-32 Minimal Local, short-term No services affected1 Up to 10,000 Euro No social disruption. Local, mild, population loss, no short-term reversible effects to significant ecosystem individuals. effect Minor Some ecosystem Local and temporary, 10,000-100,000 Euro Significant concern expressed at impact, reversible reversible effects to one or local level. Mild short-term changes, localised few services reversible effects to identifiable groups, localised. Moderate Measureable long-term Measureable, temporary, local 100,000-1,000,000 Euro Temporary changes to normal damage to populations and reversible effects on one activities at local level. Minor and ecosystem, but or several services irreversible effects and/or larger little spread, no numbers covered by reversible extinction effects, localised. Major Long-term irreversible Local and irreversible or 1,000,000-10,000,000 Euro Some permanent change of ecosystem change, widespread and reversible activity locally, concern expressed spreading beyond local effects on one / several over wider area. Significant area services irreversible effects locally or reversible effects over large area. Massive Widespread, long-term Widespread and irreversible Above 10,000,000 Euro Long-term social change, population loss or effects on one / several significant loss of employment, extinction, affecting services migration from affected area. several species with Widespread, severe, long-term, serious ecosystem irreversible health effects. effects

1 Not to be confused with „no impact“. 124 | P a g e ANNEX Scoring of Confidence Levels (modified from Bacher et al.,2017)

Confidence level Description Low There is no direct observational evidence to support the assessment, e.g. only inferred data have been used as supporting evidence and/or Impacts are recorded at a spatial scale which is unlikely to be relevant to the assessment area and/or Evidence is poor and difficult to interpret, e.g. because it is strongly ambiguous and/or The information sources are considered to be of low quality or contain information that is unreliable. Medium There is some direct observational evidence to support the assessment, but some information is inferred and/or Impacts are recorded at a small spatial scale, but rescaling of the data to relevant scales of the assessment area is considered reliable, or to embrace little uncertainty and/or The interpretation of the data is to some extent ambiguous or contradictory. High There is direct relevant observational evidence to support the assessment (including causality) and Impacts are recorded at a comparable scale and/or There are reliable/good quality data sources on impacts of the taxa and The interpretation of data/information is straightforward and/or Data/information are not controversial or contradictory. Very high There is direct relevant observational evidence to support the assessment (including causality) from the risk assessment area and Impacts are recorded at a comparable scale and There are reliable/good quality data sources on impacts of the taxa and The interpretation of data/information is straightforward and Data/information are not controversial or contradictory.

125 | P a g e ANNEX Ecosystem services classification (CICES V4.3) and examples

For the purposes of this risk analysis, please feel free to use what seems as the most appropriate category / level of impact (Division – Group – Class), reflecting information available.

Section Division Group Class Examples Provisioning Nutrition Biomass Cultivated crops Cereals (e.g. wheat, rye, barely), vegetables, fruits etc. Reared animals and their outputs Meat, dairy products (milk, cheese, yoghurt), honey etc. Wild plants, algae and their outputs Wild berries, fruits, mushrooms, water cress, salicornia (saltwort or samphire); seaweed (e.g. Palmaria palmata = dulse, dillisk) for food Wild animals and their outputs Game, freshwater fish (trout, eel etc.), marine fish (plaice, sea bass etc.) and shellfish (i.e. crustaceans, molluscs), as well as equinoderms or honey harvested from wild populations; Includes commercial and subsistence fishing and hunting for food Plants and algae from in-situ aquaculture In situ seaweed farming Animals from in-situ aquaculture In-situ farming of freshwater (e.g. trout) and marine fish (e.g. salmon, tuna) also in floating cages; shellfish aquaculture (e.g. oysters or crustaceans) in e.g. poles Water Surface water for drinking Collected precipitation, abstracted surface water from rivers, lakes and other open water bodies for drinking Ground water for drinking Freshwater abstracted from (non-fossil) groundwater layers or via ground water desalination for drinking Materials Biomass Fibres and other materials from plants, algae Fibres, wood, timber, flowers, skin, bones, sponges and other products, which and animals for direct use or processing are not further processed; material for production e.g. industrial products such as cellulose for paper, cotton for clothes, packaging material; chemicals extracted or synthesised from algae, plants and animals such as turpentine, rubber, flax, oil, wax, resin, natural remedies and medicines (e.g. chondritin from sharks), dyes and colours, ambergris (from sperm whales used in perfumes); Includes consumptive ornamental uses. Materials from plants, algae and animals for Plant, algae and animal material (e.g. grass) for fodder and fertilizer in agricultural use agriculture and aquaculture Genetic materials from all biota Genetic material from wild plants, algae and animals for biochemical industrial and pharmaceutical processes e.g. medicines, fermentation, detoxification; bio-prospecting activities e.g. wild species used in breeding programmes etc.

126 | P a g e Water Surface water for non-drinking purposes Collected precipitation, abstracted surface water from rivers, lakes and other open water bodies for domestic use (washing, cleaning and other non- drinking use), irrigation, livestock consumption, industrial use (consumption and cooling) etc. Ground water for non-drinking purposes Freshwater abstracted from (non-fossil) groundwater layers or via ground water desalination for domestic use (washing, cleaning and other non- drinking use), irrigation, livestock consumption, industrial use (consumption and cooling) etc. Energy Biomass-based Plant-based resources Wood fuel, straw, energy plants, crops and algae for burning and energy energy sources production Animal-based resources Dung, fat, oils, cadavers from land, water and marine animals for burning and energy production Mechanical Animal-based energy Physical labour provided by animals (horses, elephants etc.) energy Regulation & Mediation of Mediation by Bio-remediation by micro-organisms, algae, Bio-chemical detoxification/decomposition/mineralisation in land/soil, Maintenance waste, toxics biota plants, and animals freshwater and marine systems including sediments; and other decomposition/detoxification of waste and toxic materials e.g. waste water nuisances cleaning, degrading oil spills by marine bacteria, (phyto)degradation, (rhizo)degradation etc. Filtration/sequestration/storage/accumulation Biological filtration/sequestration/storage/accumulation of pollutants in by micro-organisms, algae, plants, and animals land/soil, freshwater and marine biota, adsorption and binding of heavy metals and organic compounds in biota Mediation by Filtration/sequestration/storage/accumulation Bio-physicochemical filtration/sequestration/storage/accumulation of ecosystems by ecosystems pollutants in land/soil, freshwater and marine ecosystems, including sediments; adsorption and binding of heavy metals and organic compounds in ecosystems (combination of biotic and abiotic factors) Dilution by atmosphere, freshwater and marine Bio-physico-chemical dilution of gases, fluids and solid waste, wastewater in ecosystems atmosphere, lakes, rivers, sea and sediments Mediation of smell/noise/visual impacts Visual screening of transport corridors e.g. by trees; Green infrastructure to reduce noise and smells Mediation of Mass flows Mass stabilisation and control of erosion rates Erosion / landslide / gravity flow protection; vegetation cover flows protecting/stabilising terrestrial, coastal and marine ecosystems, coastal wetlands, dunes; vegetation on slopes also preventing avalanches (snow, rock), erosion protection of coasts and sediments by mangroves, sea grass, macroalgae, etc. Buffering and attenuation of mass flows Transport and storage of sediment by rivers, lakes, sea Liquid flows Hydrological cycle and water flow Capacity of maintaining baseline flows for water supply and discharge; e.g. maintenance fostering groundwater; recharge by appropriate land coverage that captures effective rainfall; includes drought and water scarcity aspects.

127 | P a g e Flood protection Flood protection by appropriate land coverage; coastal flood prevention by mangroves, sea grass, macroalgae, etc. (supplementary to coastal protection by wetlands, dunes) Gaseous / air Storm protection Natural or planted vegetation that serves as shelter belts flows Ventilation and transpiration Natural or planted vegetation that enables air ventilation Maintenance of Lifecycle Pollination and seed dispersal Pollination by bees and other insects; seed dispersal by insects, birds and physical, maintenance, other animals chemical, habitat and biological gene pool conditions protection Maintaining nursery populations and habitats Habitats for plant and animal nursery and reproduction e.g. seagrasses, microstructures of rivers etc. Pest and Pest control Pest and disease control including invasive alien species disease control Disease control In cultivated and natural ecosystems and human populations Soil formation Weathering processes Maintenance of bio-geochemical conditions of soils including fertility, and nutrient storage, or soil structure; includes biological, chemical, physical composition weathering and pedogenesis Decomposition and fixing processes Maintenance of bio-geochemical conditions of soils by decomposition/mineralisation of dead organic material, nitrification, denitrification etc.), N-fixing and other bio-geochemical processes; Water Chemical condition of freshwaters Maintenance / buffering of chemical composition of freshwater column and conditions sediment to ensure favourable living conditions for biota e.g. by denitrification, re-mobilisation/re-mineralisation of phosphorous, etc. Chemical condition of salt waters Maintenance / buffering of chemical composition of seawater column and sediment to ensure favourable living conditions for biota e.g. by denitrification, re-mobilisation/re-mineralisation of phosphorous, etc. Atmospheric Global climate regulation by reduction of Global climate regulation by greenhouse gas/carbon sequestration by composition greenhouse gas concentrations terrestrial ecosystems, water columns and sediments and their biota; transport and climate of carbon into oceans (DOCs) etc. regulation Micro and regional climate regulation Modifying temperature, humidity, wind fields; maintenance of rural and urban climate and air quality and regional precipitation/temperature patterns

Cultural Physical and Physical and Experiential use of plants, animals and land- In-situ whale and bird watching, snorkelling, diving etc. intellectual experiential /seascapes in different environmental settings interactions interactions with biota, 128 | P a g e ecosystems, and land- /seascapes [environmental settings] Physical use of land-/seascapes in different Walking, hiking, climbing, boating, leisure fishing (angling) and leisure environmental settings hunting Intellectual and Scientific Subject matter for research both on location and via other media representative interactions Educational Subject matter of education both on location and via other media Heritage, cultural Historic records, cultural heritage e.g. preserved in water bodies and soils Entertainment Ex-situ viewing/experience of natural world through different media Aesthetic Sense of place, artistic representations of nature Spiritual, Spiritual and/or Symbolic Emblematic plants and animals e.g. national symbols such as American eagle, symbolic and emblematic British rose, Welsh daffodil other interactions with biota, ecosystems, and land- /seascapes [environmental settings] Sacred and/or religious Spiritual, ritual identity e.g. 'dream paths' of native Australians, holy places; sacred plants and animals and their parts Other cultural Existence Enjoyment provided by wild species, wilderness, ecosystems, land-/seascapes outputs Bequest Willingness to preserve plants, animals, ecoystems, land-/seascapes for the experience and use of future generations; moral/ethical perspective or belief

129 | P a g e ANNEX EU Biogeographic Regions and MSFD Subregions See https://www.eea.europa.eu/data-and-maps/figures/biogeographical-regions-in-europe-2 and https://www.eea.europa.eu/data-and-maps/data/msfd-regions-and-subregions/technical-document/pdf

130 | P a g e