First record of Caulerpa cylindracea ( Caulerpaceae , Chlorophyta ) in Andalusia ( Southern Spain )

Altamirano, M., Andreakis, N., Souza-Egipsy, V., Zanolla, M. & De la Rosa, J. 2014. First record of Caulerpa cylindracea (Caulerpaceae, Chlorophyta) in Andalusia (Southern Spain). Anales Jard. Bot. Madrid 71(2): e007 Three different species of Caulerpa (Caulerpaceae, Chlorophyta) cooccur in the Mediterranean Sea: two of them are found at the centraleastern basin and are typically considered non aggressive components of the Lessepsian flora [(C. chemnitzia (Esper) J.V. Lamouroux and C. racemosa var. lamourouxii (Turner) Weber-van Bosse f. requienii (Montagne) Weber van Bosse)]; a third taxon, C. cylindracea Sonder has aggressively expanded its range since its first observation in 1990, and it is nowadays reported from nearly all the Mediterranean countries. We report a population of C. cylindracea from Almería (Andalusia, Southern Iberian Peninsula) at −30 m depth as to be the westernmost record of the invasive variety on the Mediterranean European coast. Therefore, we made use of morphological description and molecular phylogenetics to provide a complete identification of this invasive seaweed in Southern Spain. Our findings are discussed in light of the composition of the receptor communities, such as maërl bed, edges of Posidonia oceanica (Linnaeus) Delile, and their ecology. Our results confirmed the suggested directionality of the invasive pathway to be westward to the Strait of Gibraltar, mainly supported by sea currents and vectors of anthropogenic origin.


INTRODUCTION
Populations of an invasive Caulerpa taxon have been systematically reported from nearly all Mediterranean countries since the early nineties. (Nizamuddin, 1991;Klein &Verlaque, 2008, Rivera-Ingraham & al., 2010, genetically related to populations of southwestern Australia (Verlaque & al., 2003). Formerly this taxon was identified as C. racemosa var. occidentalis (J. Agardh) Børgesen (Verlaque & al., 2000), to be renamed few years later as C. racemosa var. cylindracea (Sonder) Verlaque, Huisman & Boudouresque, i.e. the 'invasive variety' (Verlaque & al., 2003). Two other varieties of C. racemosa were accepted to co-occur with the invasive one in the Mediterranean Sea, C. racemosa var. turbinata (J. Agardh) Eubank and C. racemosa var. lamourouxii (Turner) Weber-van Bosse f. requienii (Montagne) Weber-van Bosse), both confined at the central-eastern Mediterranean Sea and considered as typical seaweed components of the Lessepsian flora, with a non-aggressive character (Verlaque & al., 2000). All these varieties were accepted on the basis of morphological identification and molecular phylogenetic analyses (Famà & al., 2000;Durand & al., 2002;Verlaque & al., 2000Verlaque & al., , 2003. However, recent molecular studies of the Caulerpa racemosa-peltata complex have revealed the existence of at least eleven distinct species-level entities in the complex (Sauvage & al., 2013;Belton & al., 2014), together with a confirmed high phenotypic plasticity of certain lineages/species that morphologically partial overlap with some others (Belton & al., 2014). The latter revision of this complex drove to reinstate earlier-described species for subspecies, varieties and forms of C. racemosa and C. peltata, among them the invasive variety of C. racemosa, for which the earlier name C. cylindracea Sonder was reinstated (Belton & al., 2014).
Caulerpa cylindracea seems to show a westward directionality of expansion in the Mediterranean, since the first report of the species in Libya in 1990 (Nizamuddin, 1991;Klein & Verlaque, 2008;Rivera-Ingraham & al., 2010). For this fast expansion potential and the vast impact on local biota (e.g. on maërl beds, edges of Posidonia oceanica, photophilic algae communities), homogenizing the sea bottom independently of species richness of the recipient marine communities (revised by Klein & Verlaque, 2008), the taxon is considered to be one amongst the ten most invasive seaweed species in the Mediterranean Sea EEA, 2007).
Both morphological and molecular analyses are necessary to discriminate among Caulerpa taxonomical units, especially to precisely identify the invasive one C. cylindracea. The large number of the nuclear rDNA ITS1-5.8S-ITS2 region sequences obtained from specimens collected worldwide can be used to distinguish among Caulerpa taxa (Famà & al., 2000;Durand & al., 2002;Verlaque & al., 2000Verlaque & al., , 2003Nuber & al., 2007;Sauvage & al., 2013;Belton & al., 2014). Yet, to identify the "invasive taxon", a combination of morphological identikit and molecular data has been applied in solely 5 amongst the 13 Mediterranean countries in which C. racemosa sensu lato has been reported so far (France, Italy, Greece, Croatia and Cyprus) (reviewed in Klein & Verlaque, 2008), since the first report from Libya in 1990 (Nizamuddin, 1991) (see Fig. 1 for an updated distribution map).
In this study we make use of both morphological differences and molecular evidences to assess the identity and origins of C. cylindracea, collected for first time in Andalusia region (Southern Spain), corresponding up till now to the westernmost identified population of the European Mediterranean coasts. Our results are discussed following the course of the invasive process and the present distribution of the remaining Caulerpa species and varieties encountered in the central and eastern parts of the basin.

Specimen's preliminary identification & collection
An introduced population of Caulerpa cylindracea was localized by SCUBA diving at −30 m of depth at Villaricos coast (Almería, SE Iberian Peninsula, 37°17′17′′N 1°41′04′′O, Fig. 1  * ) in October 2009; several thalli were collected by M. Altamirano and J. de la Rosa from four areas within the population separated at least 5 m from one another. Selected samples were either preserved in 4% formalin in seawater for morphological identification or carefully cleaned from epiphytes and desiccated in silica gel for genetic analysis. Herbarium sheets were prepared and deposited in the Herbarium of the University of Málaga (MGCPhyc 5087).

Morphological identification
The following morphological characters from different parts of the thalli (Fig. 2) were analyzed: height, width and attachment to stolons of the fronds; height, diameter, shape and arrangement of the ramuli; diameter of the stolons; length, width, spacing and morphology of the rhizoids.
To evaluate the partition of individual sequence polymorphisms, genealogical relationships among ITS1-5.8S-ITS2 copies within individuals were calculated using the Median Joining algorithm (ε=0, equally weighted characters) implemented in the software Network v4.5.1.6 (http://www. fluxus-technology.com). The method identifies groups of closely related sequences and uses "median vectors" to connect sequences into a tree or network. Median vectors can be interpreted biologically as extinct individuals or haplotypes that have not been sampled yet (Bandelt & al., 1999).

DISCUSSION
Morphological identification coupled with molecular phylogenetic analysis provides neat evidence that isolates of Caulerpa collected from Almería (Andalusia, Southern Spain) correspond to the invasive species C. cylindracea. The isolates found in Almería represent the most westerly population of this taxon in the Mediterranean European coast and are genetically related to the group of genotypes encountered in the central and eastern Mediterranean Sea. Population was encountered in the same depth as the populations reported from the east Spanish coasts corresponding to circalittoral sites (Ruíz & al., 2007;Cebrián & Ballesteros, 2009;Guillén & al., 2010). Collected thalli exhibited similar morphological and ecological characteristics with short fronds and not dense arranged mats. This study represents the first complete identification of C. cylindracea in the Alboran Sea (Western Mediterranean), confirming the previously suggested directionality of the invasive pathway to be towards west, to the Strait of Gibraltar.
At this moment the westernmost populations of C. cylindracea in the world are located in Canary Islands (Western Atlantic Ocean), where the species has been observed since the late 1990s, and whose origin was confirmed to be from Mediterranean populations (Verlaque & al., 2004). The nearest known population from Canary Islands is in Ceuta (North coast of Africa), where the species was observed for first time in 2007 (Rivera- Ingraham & al., 2010). For these western populations, long distance anthropogenic mediated transport should be suspected, as the nearest populations by the time of the first observations, were very far away. However, for the new reported population of C. cylindracea in Almería (Southern Spain), a short-distance transport from near populations in Murcia province (Ruíz & al., 2007) might be accepted, mainly due to shipping and diving activities at the zone. Up to now, no new populations have been reported in Southern Spain, but this could be due to the severe limit that depth over 30 m poses in locating new introductions.
Two amongst the three Caulerpa taxa encountered in the Mediterranean Sea (Verlaque, 2000(Verlaque, , 2003, namely, C. chemnitzia (Esper) J.V. Lamouroux (formerly C. racemosa var. turbinata (J. Agardh) Eubank) and C. racemosa var. lamourouxii f. requienii occur only in the central and eastern part of the basin. In comparison, the third taxon, C. cylindracea, following initial observation in 1990 (Nizamuddin, 1991), expanded its distribution range in the last 20 years by sea currents and vectors of anthropogenic origin (Klein & Verlaque, 2008). Several eco-physiological characteristics may account for the distinctive distribution patterns encountered between the invasive and the non-invasive Caulerpa species in the Mediterranean Sea. For instance, it has been stated that the non invasive taxa do not occur in locations where winter isotherms drop off below 15 °C (Verlaque & al., 2000). The invasive species on the other hand, has been recorded even below 10°C during colder winters (Verlaque & al., 2000). Furthermore, differences in ecological behavior (i.e. bathymetric distribution, type of substratum and light adaptation) are remarkable between invasive and non-invasive taxa thus reflecting differences in colonization capabilities and survival rates of newly introduced propagules.
The invasive species has been reported from shallow waters down to 60 m depth, from sciaphilic to photophilic conditions and on both soft and hard substrata, being able of competing against a wider range of communities (Klein & Verlaque, 2008). Taken together, these features represent an advantage in challenging local communities when compared with the other two non-invasive forms. However, differences in minimum lethal temperature boundaries among varieties, may explain their current geographical distribution patterns in the Mediterranean Sea: C. chemnitzia and C. racemosa var. lamourouxii are confined to the central and eastern part of the basin; the invasive species occurs also in colder waters. This behavior is additionally suggested by the relative growth rates of the invasive taxon during the winter period, which remains positive down to 10 °C in laboratory conditions (Flagella & al., 2008). The present report and that for  Table 1; circles represent distinct ITS1-5.8S-ITS2 copies; circle size is proportional to the frequency of that copy; triangles indicate large substitutions denoted by the number; bars across lines connecting clone sequences indicate bp substitutions.
Ceuta (Rivera-Ingraham & al., 2010) validate geographical distribution niche models, developed for this invasive species based on surface temperature distribution occurring in the Mediterranean and European coastlines (Verbruggen & al., 2009).
High intra-individual levels of sequence polymorphisms are typically encountered in all isolates belonging to the Mediterranean C. cylindracea and this is believed to be the result of incomplete sequence homogenization by concerted evolution, differences in ploidy levels and multinucleate coenocytic thalli, all responsible of promoting intra-individual polymorphisms in ITS1-5.8S-ITS2 copies (Famà & al., 2000;Kapraun, 2005). Given our data, we cannot clarify on the contribution of sexual reproduction versus clonal propagation to the extreme levels of ITS sequence variation observed at the intra-individual level. It is highly probable however that both, the introduction of positively selected genetic variants and the elevated population density encountered locally, contribute to the recent establishment and remarkable expansion of C. cylindracea population on the Mediterranean Spanish coast.