Further expansion of the alien seaweed Caulerpa taxifolia var . distichophylla ( Sonder ) Verlaque , Huisman & Procacini ( Ulvophyceae , Bryopsidales ) in the Eastern Mediterranean Sea

Caulerpa taxifolia var. distichophylla (Sonder) Verlaque, Huisman and Procacini is a green alga of Australian origin recently reported as an alien species in the Mediterranean Sea, where it is known from SE Turkey, Sicily, Cyprus and Malta. In the current study we present additional records of this taxon, expanding its known distribution into the Eastern Mediterranean Sea, and provide additional records from Cyprus and the first records from Rhodes Island (Greece). Our specimens were identified through a combination of morphological and molecular methods involving sequencing of ITS and tufA. Locally, C. taxifolia var. distichophylla occurred in high abundances and dominated the benthic community, suggesting that has the potential to become a major pest in the Mediterranean. It was also observed over a very wide depth range, from the sea surface to at least 100 m depth, on a variety of natural soft and hard substrates as well as abandoned fishing nets, suggesting a broad environmental plasticity. One of the findings reported here constitutes the deepest record of an alien Caulerpa in the Mediterranean Sea, even though it remains to be demonstrated that it actually grows at this site and depth rather than being merely a drift specimen.

Recently, an additional Caulerpa taxon has been reported from the Mediterranean Sea, namely Caulerpa taxifolia var.distichophylla (Sonder) Verlaque, Huisman and Procaccini.It was first reported as C. taxifolia from the coasts of South Turkey (Cevik et al. 2007), and later reported from Sicily (Cormaci and Furnari 2009;Meinesz et al. 2010;Jongma et al. 2013;Musco et al. 2014), Cyprus  (Çicek et al. 2013;Tsiamis et al. 2014) and Malta (Schembri et al. 2015).Based on molecular work, it was shown that C. taxifolia var.distichophylla is also an alien species in the Mediterranean Sea, originating from Australia (Jongma et al. 2013).
In the current paper we present data showing further expansion of C. taxifolia var.distichophylla in the Eastern Mediterranean Sea (identified on the basis of both morphological and molecular data) and a survey of different benthic habitats affected by this new arrival.Besides reporting additional records from Cyprus, we report the taxon for the first time from Greece (Rhodes Island).

Sampling
During 1-3 November 2010, benthic sampling was conducted along the coasts of Rhodes Island and a few individuals of Caulerpa taxifolia var.distichophylla were collected at a depth of 20 m with a Smith McIntire grab (0.1 m 2 surface) on crushed shells and sand substrate in Lindos Bay (east of Rhodes Island, SE Greece, Figure 1).These samples were preserved in ethanol.Additional surveys and sampling of the species were conducted on 17 June 2014 by scuba diving on hard substrate at Charaki Bay on the east coast of Rhodes down to 18 m depth.Samples from Charaki Bay were preserved in CTAB (Cetyltrimethylammoniumbromide) buffer (Gachon et al. 2009) for DNA extraction.
On 27 June 2012, while surveying the coastal waters of Cyprus, a few specimens of Caulerpa taxifolia var.distichophylla were collected with a Van Veen grab (0.1 m 2 surface) from 34 m depth, off Sunrise Bay on the east coast of Cyprus.On 5 July 2012, specimens of the same taxon were collected from two depths (22 and 42 m) along a single line transect in Cavo Greco.Additional specimens were collected from the 42 m station on 28 February 2014.
Finally, diving-based surveys and sampling of C. taxifolia var.distichophylla were conducted in Cyprus at Cavo Greco, on the east coast of Cyprus at depths between 36 -40 m, on July 16, 2014.1).The 2012 specimens from Cyprus were preserved in 4% formaldehyde-seawater and deposited in the collection of the Department of Fisheries and Marine Research, Cyprus (HDFMR; Table 1).The 2012 specimens were compared to a few small unclassified Caulerpa specimens deposited in the HDFMR collection since 2008: HDFMR44, Caulerpa sp.Ammochostos district area -SE coast of Cyprus, 2008.

Identification based on morphological features
The following characters were assessed for the morphological identification of specimens: stolon width, the shape, width and length of fronds and pinnules, the number of rhizoidal pillars and pinnules, and the morphology and width of the midrib.

DNA extraction, PCR amplification and sequencing
DNA extractions were performed on material from specimens collected from both Cyprus and Rhodes in 2014 (Table 2) preserved in CTAB extraction buffer (Gachon et al. 2009) using DNeasy Plant Mini Kit (Qiagen, Hilden, Germany).A mortar and pestle were used in order to grind the algal tissue and then 700μL CTAB (preheated to 65ºC) were added to the ground material, followed by disruption with TissueLyser II (Qiagen).The subsequent protocol followed the procedure as in DNeasy Plant Mini Kit.
Polymerase chain reaction (PCR) was performed using specific primer pairs for the tufA gene and ITS region, that had previously been used for Caulerpa spp.(Famà et al. 2002;Jongma et al. 2013;Stam et al. 2006).The chloroplast tufA gene amplified with the primer pair tufAF-tufAR and using PCR conditions slightly modified from those reported by Famà et al. (2002).PCR amplification was performed in a total volume of 25 μL, containing 1.25 units/μL of Taq DNA Polymerase (Promega), 1 × GoTaq™ buffer, 5 mM MgCl 2 , 1.25 mM dNTPs, 1.87 mM of each primer and 1 μL of template DNA (5-50 ng/μl).PCR amplification was carried out with the following profile: one cycle at 94° C for 4 min; 10 cycles at 94° C for 15 s, 1 min at 50° C and 2 min at 72° C; 35 cycles of 15 s at 94° C, 20 s at 50° C and 2 min at 72° C and one cycle at 72° C for 10 min.
For the nuclear ribosomal ITS1+2 region the primer pair JO4-JO6 developed by Stam et al. (2006) was used.PCR reactions were performed in a total volume of 25 μL and used 0.25 units/μL of Taq DNA Polymerase (Promega), 1 × GoTaq™ Buffer, 2.5 mM MgCl 2 , 0.2 mM dNTPs, 0.5 mM of each primer and 1 mL template DNA (5-50 ng/μl).The PCR profile included one cycle of 2 min at 94° C; 10 cycles of 15 s at 94° C, 1 min at 50° C and 2 min at 72° C; 30 cycles of 15 s at 94° C, 20 s at 50° C and 2 min at 72° C; and one cycle of 10 min at 72° C.
PCR products were run on a GelRed™ (Biotium) TBE agarose (1.2 %) gel to check for amplification and correct length.A single reaction was purified using the QIAquick PCR Purification Kit (Qiagen) and sent for sequencing using the Source Bioscience sequencing service.Approximately 50 ng of purified PCR products were sequenced using the original PCR primers.
The alignment of each gene sequence was created with BioEdit Sequence Alignment Editor (Hall 1999) and then the sequences were compared to published data by means of NCBI BLAST searches (Altschul et al. 1997).

Phylogenetic analysis
Publicly available sequences (Jongma et al. 2013) were merged with sequences newly generated in this study and aligned in MEGA6.0 (Tamura et al. 2013) using the ClustalW algorithm.Alignments were refined by eye.Finally Maximum Likelihood (ML) phylogenies were inferred under the Tamura-Nei model of evolution with gaps being treated as pairwise deletions and 1000 bootstrap pseudo-replicates to ensure statistical support of the nodes.

Identification
Based on morphology, the Caulerpa specimens from Cyprus and Rhodes were identified as Caulerpa taxifolia var.distichophylla.They were characterized by a light green thallus, featherlike, delicate, with narrow postrate stolons and erect fronds bearing pinnules (Figure 2A); stolons slender, 0.4-1.0mm thick, with short rhizoidal pillars 1-4 mm long (Figure 2B); about 13 pillars per 10 cm of stolon; erect fronds simple to 3 times branched, 2.5-10 cm high and 1.5-4.2mm wide; erect fronds terete below, compressed towards the apex, bearing distichously and closely arranged (but never overlapping) pinnules in one plane; pinnules compressed, 0.8-2.3mm long and 0.3-0.5 mm wide, up-curved, slightly constricted at their base and gradually tapering into a pointed tip (Figure 2C).No fertile specimens were found.

Field observations
In Cyprus, C. taxifolia var.distichophylla was found both in very shallow waters (Figure 3) and at 42 m depth (Figure 3); however, it was less dominant at the two Cypriot sites than at Charaki in Rhodes.The Cypriot specimens from the Cavo Greco area were collected from sediment comprised of fine biogenic sand: shell fragments/foraminiferans (at 34 m depth), dominated by the non-native seagrass Halophila stipulacea (Forsskål) Ascherson (at 42 m depth) and on muddy substratum dominated by C. racemosa var.cylindracea with scattered specimens of Codium bursa (Olivi) C. Agardh (48 m depth), respectively.They were also found growing on biogenic hard substrate (serpulid tubes

Molecular phylogeny
The cpDNA tufA sequences from specimens collected from Rhodes and Cyprus (Cavo Greco) were 745 bp and 788 bp long, respectively.These sequences provide strong support that the specimens from Rhodes and Cyprus belong to Caulerpa taxifolia var.distichophylla (98/100).
The rDNA ITS1-5.8S-ITS2sequences from Rhodes and Cyprus (Cavo Greco) were 587 bp long and 586 bp, respectively.Likewise, the sequences obtained show strong support that the specimens from Rhodes and Cyprus fall within the Caulerpa taxifolia var.distichophylla (85/100).
All sequences of tufA and ITS1-5.8S-ITS2from both areas have been submitted to EMBL (European Bioinformatics Institute) and accession numbers obtained (Table 1).In Maximum Likelihood trees of both tufA and ITS sequences, C. taxifolia var.distichophylla from both Rhodes and Cyprus clustered with specimens from Italy, Turkey, and Australia and were distinctly different from C. taxifolia from southern France and other localities (Figures 4 and  5).

Discussion
Based on morphological features and molecular analyses, the green alga from Cyprus and Rhodes was confirmed to be Caulerpa taxifolia var.distichophylla.The sequences of tufA and ITS were identical to previously published sequences from specimens of C. taxifolia var.distichophylla from Sicily (Jongma et al. 2013).In general, due to the morphological plasticity that characterizes the genus, identification of Caulerpa species based solely on morphological features can result in high misidentification rates (>12%), and is considered unreliable without supplementary use of molecular methods (Olsen et al. 1998;Stam et al. 2006).Indeed, the morphology of C. taxifolia var.distichophylla is somewhat similar to C. cupressoides (M.Vahl) C. Agardh; however, the latter is currently not known from the Mediterranean.Our specimens do not exhibit the usual morphologies of C. mexicana or C. taxifolia, or the typically robust morphology of the invasive aquarium strain of the latter, but they seem to agree with the description of a C. taxifolia var.distichophylla population found in south eastern Turkey by Cevik et al. (2007, as C. taxifolia).
The tropical green alga C. taxifolia was accidentally introduced to the Mediterranean Sea in 1984 from the Aquarium in Monaco (Meinesz and Hesse 1991;Jousson et al. 1998) and since then it has spread to at least seven Mediterranean countries: Croatia, France, Italy, Monaco, Spain and Tunisia (Meinesz et al. 2001).Its negative effect on benthic systems is significant and has led to concerns, e.g. for the decline of seagrass meadows in southern France and elsewhere in the Mediterranean, even though a causal link could not always be established (Glasby 2013).On the other hand, C. mexicana has been found in the Mediterranean since at least the 1940s (Olsen et al. 1998) and is mostly common in the Eastern Basin, where it has been reported from Israel, Lebanon, Syria and Egypt (Ukabi et al. 2012;Ukabi et al. 2014), with apparently little knowledge about its ecological impact.
This study suggests that C. taxifolia var.distichophylla has recently spread in the Eastern Mediterranean.Originating from South Western Australia where it is endemic (Jongma et al. 2013), C. taxifolia var.distichophylla was originally discovered in Iskenderun Bay, Turkey (approx.200 km from Cyprus and 700 km from Rhodes, respectively) by Cevik et al. (2007), followed by records in South Eastern Sicily (Jongma et al. 2013;Musco et al. 2014), Cyprus (Çicek et al. 2013;Tsiamis et al. 2014) and, most recently, Malta (Schembri et al. 2015).Our records reveal further expansion of the taxon in the Eastern Mediterranean Sea.However, it is impossible to say at present whether this is occurring through natural dispersal or shipping or a combination of both.
Throughout this study, C. taxifolia var.distichophylla was found over a wide depth range, from the surface to at least 100 m depth.To the best of our knowledge, the findings off Lahania (Rhodes) constitute the deepest records not only of this taxon, but of any invasive Caulerpa species in general in the Mediterranean.However, while it seems possible that the species could survive at such a depth at that site, it is important to note that we cannot be sure whether the plants were attached and developing.It would seem essential to confirm this record with in situ photography or a grab sample with substrate still attached.Moreover, a better understanding of its impact on circalittoral habitats in general would also be desirable.Klein and Verlaque (2008) reported C. racemosa from 0 to 70 m depths whereas the other invasive member of this genus, C. taxifolia, has been usually observed at lesser depths, even though the deepest record was made at 99 m at Cap d'Ail, France (Belsher and Meinesz 1995).Indeed, C. racemosa grows better at 30 than at 10 m depth (Cebrian and Ballesteros 2009), but it has never been recorded as deep as the C. taxifolia var.distichophylla reported here.Locally, like at the Charaki site (Rhodes Island, Greece), the taxon can be dominant, and it may be displacing native zooand macrophytobenthos, covering both hard and soft natural, as well as artificial (Figure substrates, thus suggesting a potentially invasive behavior. In contrast, at 38 m depth off Cavo Greco (Cyprus), the stands were much sparser than at Charaki in Rhodes.From a conservation perspective, it should be pointed out that in Rhodes, C. taxifolia var.distichophylla invades and covers Cystoseira communities, which are endangered in much of the Mediterranean due to a variety of anthropogenic causes (Thibaut et al. 2005).Moreover, it is noteworthy that following a two-year monitoring program covering 30 sites along the coast of Sicily, the taxon has been labelled an "invader" because of its impact on benthic system and ongoing spread in the Mediterranean Sea (Musco et al. 2014).
The genus Caulerpa contains several invasive taxa in the Mediterranean and other parts of the world, which correlates with the presence of an efficient wound-healing mechanism in this group (Welling et al. 2009), facilitating vegetative dispersal and establishment of new populations.At present, there is no knowledge about potential grazers of C. taxifolia var.distichophylla in the Mediterranean Sea.In several instances, the snail Bittium reticulatum (da Costa, 1778) was observed on C. taxifolia var.distichophylla, possibly feeding on it.This should be further investigated.In line with Musco et al. (2014), we hypothesize that C. taxifolia var.distichophylla has to be considered potentially invasive, and that the Caulerpa taxifolia species cluster in general seems to possess ecological features (Andreakis and Schaffelke 2012) rendering it particularly adapted to rapidly colonize the shallow-water Mediterranean ecosystem.

Figure 2 .
Figure 2. Morphological features of the Caulerpa specimens (HDFMR91).(A) Part of the thallus: scale bar 1 cm; (B) Details of stolon and rhizoidal pillars: scale bar 1 mm; (C) Details of the upper part of the frond and pinnules: scale bar 1 mm.Photographs by M. Aplikioti (DFMR).

Figure 3 .
Figure 3. Caulerpa taxifolia var.distichophylla in situ off the coast of Rhodes (Charaki, A-I) and Cyprus (Paralimni, J; Cavo Greco, K-L), respectively.A-B: Macrophotographs of C. taxifolia var.distichophylla, showing both individual phylloids and stolons on macrophytobenthos turf on a steeply inclined underwater cliff.C: C. taxifolia var.distichophylla in community with Padina sp. and Acetabularia sp.. D-F: C. taxifolia var.distichophylla on undulating, otherwise flat soft-bottom benthos.E: In several instances, the snail Bittium reticulatum was observed on C. taxifolia var.distichophylla, possibly grazing on it.F: C. taxifolia var.distichophylla invading and overgrowing a Cystoseira community.G: C. taxifolia var.distichophylla colonizing an abandoned fishing net.H: Upward view of a dense community of C. taxifolia var.distichophylla covering a steeply inclined underwater cliff from around 15 m depth.I: At the same site, C. taxifolia var.distichophylla in a community with Acetabularia sp.J: C. taxifolia var.distichophylla in shallow inshore waters in Cyprus, in a community with Padina sp. and Laurencia sp.. K-L: Deep-water community (around 38 m depth) of C. taxifolia var.distichophylla at Cavo Greco, in a community with the native Caulerpa prolifera.Photographs by F.C. Küpper (Aberdeen; A-I), M. Marcou (DFMR; J-L).

Figure 4 .
Figure 4. Maximum likelihood test of phylogeny of the tufA cpDNA gene sequences obtained from the Caulerpa taxifolia var.distichophylla under investigation in this study.The grey box indicates sequences of Caulerpa taxifolia var.distichophylla in this study from Rhodes Island in Greece and Cavo Greco in Cyprus.

Figure 5 .
Figure 5. Maximum likelihood test of phylogeny of the ITS1-5.8S-ITS2rDNA sequences obtained from the Caulerpa taxifolia var.distichophylla under investigation in this study.The grey box indicates sequences of Caulerpa taxifolia var.distichophylla in this study from Rhodes Island in Greece and Cavo Greco in Cyprus.

Table 2 .
Specimens used for DNA sequencing in the present study.
the Natural History Museum, London (BM); the Muséum National d'Histoire Naturelle, Paris (PC); the University of California, Berkeley (UC); and the East Mediterranean Seaweed Herbarium at the National and Kapodistrian University of Athens (ATHU) (Table