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Distribution, abundance and colony size of the invasive coral Oculina patagonica de Angelis, 1908 (Cnidaria, Scleractinia) in Malta

The zooxanthellate, scleractinian coral Oculina patagonica is known from various locations in both west and east basins of the Mediterranean Sea, but there are only three casual records of this cryptogenic species from the Central-Ionian area, all from Malta in 2017. Surveys at 28 sites around the Maltese coasts undertaken during the present work revealed 43 colonies spread across ten sites. The coral occurred primarily on artificial substrata in harbour areas at depths shallower than 6 m, but a few colonies occurred on natural rocky bottoms and in non-harbour sites. The largest colonies reached a mean diameter of up to 95 cm and occurred on artificial structures that are only a few decades old, while the majority of the colonies were much smaller (< 40 cm diameter). This, together with the clustering of records in harbour areas, suggests that O. patagonica was likely introduced in Maltese waters in recent decades via maritime transport. The present results also confirm that this species is established in the Central Mediterranean area.


Introduction
The zooxanthellate, scleractinian coral Oculina patagonica de Angelis, 1908, was first recorded from the Mediterranean Sea in 1966, based on a large colony found off Savona Harbour, Italy (Zibrowius 1974). The original description of this species was based on fossil specimens from the south-west Atlantic, and it was therefore deemed to be non-indigenous to the Mediterranean and probably introduced through shipping (Zibrowius 1974). Subsequently, O. patagonica was recorded in high abundances from the southeastern coast of Spain, and later, in various countries throughout the Mediterranean basin including Spain, France, Algeria, Tunisia, Croatia, Greece, Turkey, Egypt, Lebanon and Israel (Cvitković et al. 2013;Salomidi et al. 2013;Rubio-Portillo et al. 2014a, and references therein;Terrón-Sigler et al. 2015). However, this species has never been recorded alive outside the Mediterranean, and it has recently been argued that O. patagonica may originate from the eastern Atlantic but remained undetected until it began expanding its distribution in the Mediterranean (Leydet and Hellberg 2015). Given the uncertainty around its native or alien status, it is currently considered a cryptogenic species (Serrano et al. 2018).
Live colonies of O. patagonica have been reported on both natural and artificial substrata, but in much higher densities and cover on the latter (Salomidi et al. 2013;Serrano et al. 2013Serrano et al. , 2018. Most records of this species are from shallow water, with peak abundances within the 2-6 m depth range and rare occurrences below a depth of 10 m (Fine et al. 2001;Coma et al. 2011;Salomidi et al. 2013;Rubio-Portillo et al. 2014a), although a few colonies have been observed at depths down to 28 m (Serrano et al. 2013). The successful, rapid expansion of this species in the Mediterranean in recent years has been attributed to the availability of artificial structures (Salomidi et al. 2013;Serrano et al. 2013Serrano et al. , 2018Rubio-Portillo et al. 2014a), while a phase-shift in dominance from macroalgae to O. patagonica on natural substrata has also been reported (Serrano et al. 2012).
Although O. patagonica is widespread in the Mediterranean, it has only recently been reported from the Central-Ionian area. In 2017, a report on the occurrence of O. patagonica in Maltese waters was carried in local news media based on two colonies discovered by one of us (JE) in Sliema Creek (35.9079°N; 14.5009°N) (The Sunday Times of Malta, 12 Nov 2017), but was not published in the scientific literature. A second report from a different locality in Malta (Birzebbuga; 35.8254°N; 14.5342°E) was subsequently made during the same year (Chartosia et al. 2018), suggesting that the species is established in Maltese waters. A third Maltese record, this time from Marsaxlokk (35.8361°N; 14.5479°E), was recently reported in Katsanevakis et al. (2020); this record is dated 19 July 2017 and therefore represents the first finding of this species from the Central Mediterranean. In the present work, we assessed the status of O. patagonica along Maltese coasts by mapping its distribution and estimating abundance; we also measured the size of colonies of the coral in an attempt to gain insight into when the species was first introduced.

Materials and methods
Twenty-eight sites along Maltese coasts were surveyed noting the presence or absence of Oculina patagonica, for a total coastal length of 7.2 km (see Supplementary material Table S1). At each site, surveys employing SCUBA diving were made along shore-parallel belt transects located at depths of 0 m down to a maximum of 20 m. Transect lengths varied based on the availability of suitable hard substrata, but mostly ranged between 80 m and 500 m. Of the sites studied, 14 where characterised by natural substrata, eight were artificial shorelines, while the rest included a mixture of both natural rocky bottoms and artificial structures.
Colonies of O. patagonica were identified based on the following combination of characters, which also enabled distinguishing them from the native colonial scleratinian Cladocora caespitosa (Linnaeus, 1767), and from the non-indigenous Oulastrea crispata (Lamarck, 1816) that has been recently reported from Corsica and Catalonia (Hoeksema and Ocaña Vicente 2014;Mariani et al. 2018): small polyp calices (< 3 mm diameter); plocoid growth form; calices separated by a wide coenosteum covered by a very evident coenosarc; living tissue light brown in colour; encrusting colonies covering substratum as a thin lamina.
Whenever a colony of O. patagonica was found, its location (GPS coordinates), depth, and substratum type were recorded. When possible, standardised photographs of the colonies were also taken, together with a scale, enabling the length, width and area covered, and the fraction of the colony having living tissue, to be estimated using the image analysis software ImageJ (Schneider et al. 2012). To quantify the proportion of living tissue, a grid was overlaid on the image and the number of grid cells with visible living tissue was counted; those parts of the colony that either appeared white (i.e. with denuded skeleton) or were overgrown by living algal tissue were considered as dead.

Results
A total of 43 colonies of Oculina patagonica were recorded from 10 sites, while no colonies of this species were noted at any of the other 18 sites visited (see Figure 1; Tables S1, S2; the two previously unpublished records made in 2017 from Sliema Creek are included). Only two of the sites hosting O. patagonica (Marsascala and Zonqor; Sites 6 and 7 in Figure 1) are located outside harbour environments. The highest abundance was of 12 colonies at Birzebbuga -Pretty Bay (Site 3 in Figure 1), which translates to 0.15 colonies per square metre. The colonies were mostly found on artificial substrata (34 colonies) but were also recorded on a natural substratum (9 colonies), always on vertical or steeply-sloping bottoms. Most occurred at depths between 0.5 m and 2.0 m; the shallowest was found at a depth of 0.1 m, while the deepest record throughout this study was at 6.0 m. Over four-fifths of the colonies appeared to be in good health, with more than 75% of the colony covered by living tissue, while only three colonies were less than 50% alive (see Table S2). In most cases, the dead parts were overgrown by algae; however, bleaching and denuded skeletons were also observed in some instances ( Figure 2; see also Table S2).

Discussion
Until recently, the only three records of Oculina patagonica in the Central Mediterranean were those from Malta in 2017. The present findings show that this species is in fact well-established in Maltese waters, given the large number of healthy colonies recorded from multiple sites. This study has therefore helped fill in gaps regarding the distribution of O. patagonica in the Mediterranean Sea, which confirms that the species is established in the Central Mediterranean. The majority of the colonies recorded from Maltese shores were found on artificial structures such as quays and wharves constructed within the last century. The affinity for artificial habitats is often considered an indication of non-native origin (Chapman and Carlton 1991) and suggests that the Maltese population of O. patagonica probably results from a human-mediated introduction. Given that most of the colonies were sited within busy harbours, transport via maritime vessels is the most likely mode of introduction to Maltese waters. Shipping is also thought to be the main introduction pathway in several other Mediterranean countries (Zibrowius 1974;Fine et al. 2001;Sartoretto et al. 2008;Cvitković et al. 2013).
The sizes of colonies found in Malta also suggest a recent introduction of this species within the last few decades. The largest two colonies, which had a mean diameter of 95 cm and 87 cm, occurred in Marsaskala and Sliema Creek (Sites 6 and 14 in Figure 1). Both these colonies were found growing on artificial substrata; attempts were made to ascertain the date of construction of the artificial structures on which they occurred. The Marsaskala "breakwater" surveyed has been in place since 1947 (Marsaskala Local Council, personal communication, 25 th November 2019) while the structures in Sliema Creek have been re-built several times, with the latest occasion being in the 1980s (Sandro Lanfranco, personal communication, 27 th November 2019). This suggests that the largest colonies of O. patagonica found in Maltese waters cannot be older than around 40-70 years. The majority of the colonies measured in the present work were much smaller (mean diameter < 40 cm) and hence unlikely to be more than two to three decades old, based on published growth rate values (e.g. Fine et al. 2001;Serrano et al. 2017). However, it should be noted that the growth rate of this species is very variable and depends on the season, temperature, light intensity, bleaching events, and also on the size of the colony (since growth rate may change as the colony ages), such that coral size alone cannot be used to accurately calculate the establishment time of a colony (Rubio-Portillo et al. 2014b). In addition, asexual reproduction can produce clones of genetically identical colonies found in close proximity, which can then fuse when in contact, resulting in a single large colony (Sartoretto et al. 2008). Indeed, this could potentially account for the two large colonies recorded from Marsaskala and Sliema Creek.
Although the present results suggest that O. patagonica may have been introduced in Malta no more than a few decades ago, it is still somewhat surprising that this conspicuous species remained overlooked until 2017. This may be attributed to several reasons. Firstly, being an encrusting coral, this species cannot be detected by sea users such as fishers; it is only divers and snorkelers who could potentially come across it. Secondly, most of the recorded colonies are sited within busy harbours, which are not popular with recreational SCUBA divers or snorkelers. Only two of the sites hosting this species lie outside harbours, Marsaskala and Zonqor (Sites 6 and 7 in Figure 1), and in both cases the colonies of O. patagonica are not in areas commonly frequented by divers or snorkelers. Thirdly, to the untrained eye, this species can be mistaken for the native C. caespitosa, especially when small. This combination of factors makes it difficult for this species to be detected and recognised as a new invader by non-scientists. In fact, the three field observations of this species in 2017 were all made by scientists.
The present results, including colonies as large as 0.7 m 2 , show that O. patagonica is a strong competitor for space with other sessile biota, especially on vertical substrata, as has also been noted elsewhere in the Mediterranean (Zibrowius 1974;Zibrowius and Ramos 1983;Fine and Loya 2003;Sartoretto et al. 2008;Armoza-Zvuloni et al. 2012). This species has been described as an "opportunistic dominant settler" that can overgrow other calcareous organisms and eliminate algae and soft faunal species at its growing edge (Sartoretto et al. 2008). It can therefore have an impact on native Maltese benthic communities, particularly if it continues to invade natural rocky bottoms, such as at the two sites Marsaxlokk and Zonqor (Sites 5 and 7 in Figure 1) surveyed in the present study. Most of the sites where O. patagonica has been recorded in Malta are also hotspots for the Mediterranean endemic stony coral C. caespitosa, an endangered species (Casado de Amezua et al. 2015) that is protected under regional (Annex 2 of the Barcelona Convention) and Maltese legislation. Thus O. patagonica can have detrimental effects on C. caespitosa, since the latter is the weaker competitor and can be overgrown by the invader (Sartoretto et al. 2008).
The biological features of O. patagonica (high growth rate, early reproduction, ability to reproduce both sexually and asexually, wide environmental tolerance and ability to survive low temperatures and in polluted areas), together with an increase in seawater temperature that extends the growing period, and the increased availability of open space provided by artificial habitats, facilitate its proliferation (Fine et al. 2001;Serrano et al. 2013;Salomidi et al. 2013). Conditions in Maltese waters, where the sea temperature ranges 14-28 °C, seem ideal for this species, whose optimal temperature range for growth is 16-26 °C (Rubio-Portillo et al. 2014b;Serrano et al. 2017). Therefore, proliferation may also be expected along Maltese rocky coasts, particularly on artificial substrata in harbour environments, but may also extend to natural rocky bottoms in more pristine sites. Given its potential impact on native communities, monitoring changes in the distribution and abundance of O. patagonica is advisable. Furthermore, since the highest abundances at present are found in busy commercial harbours, further spread of this species to other locations within the Central Mediterranean via maritime transport seems inevitable. Geo-referenced locations of the 28 study sites with details on site type (harbour vs non-harbour), substratum type (artificial vs natural) and orientation, length / depth / area of surveyed site, and number of colonies and standardised abundance of Oculina patagonica recorded from each site