Blitzkrieg in a Marine Invasion: Caulerpa racemosa var. cylindracea (Bryopsidales, Chlorophyta) Reaches the Canary Islands (North-East Atlantic)

On the basis of morphological and genetic studies (rDNA ITS1, 5.8S, ITS2, and a 18S rDNA intron), we confirm here that Caulerpa racemosa var. cylindracea (Sonder) Verlaque, Huisman et Boudouresque, a southwestern Australian taxon recently introduced into the Mediterranean Sea also occurs in the Canary Islands. This is the first report of C. racemosa var. cylindracea in the Atlantic. It was observed for the first time in the Canary Archipelago in 1997–1998. The speed and regional scale of expansion (north Atlantic Ocean and the Mediterranean Sea) of this invasive species appear to be among the most dramatic ever recorded. The possible outcome of this introduction in the Atlantic is discussed.     

Genetic evidence for the identity of Caulerpa racemosa (Forsskål) J. Agardh (Caulerpales,Chlorophyta) in the Adriatic Sea

Three taxa of Caulerpa racemosa that differ morphologically and genetically have been reported in the Mediterranean Sea. The ‘invasive variety’ was recorded for the first time in the early 1990s in Libya. In less than 10 years, it was found in almost all parts of the Mediterranean. The first record of C. racemosa in the Croatian part of the Adriatic Sea was in 2000 and as many as 35 different localities were established by the end of 2004. In terms of morphology, the specimens from the Adriatic Sea resemble the ‘invasive variety’. To confirm this, we analysed populations from two different localities (the island of Mljet and the peninsula of Pelje?ac) using the ITS1-5.8S-ITS2 region of 18S-5.8S-26S rDNA unit as a molecular marker. The ITS1-5.8S-ITS2 region from nine individual plants was amplified by PCR, cloned in a pCR 2.1 vector and sequenced. Phylogenetic comparison of sequences from specimens found in the Adriatic Sea with specimens of the same and similar taxa, found both inside and outside the Mediterranean, provided genetic evidence that C. racemosa populating the Adriatic Sea corresponds to the Mediterranean C. racemosa var. cylindracea (Sonder) Verlaque, Huisman?&?Boudouresque, i.e. to the ‘invasive variety’.     

MEDITERRANEAN CAULERPA TAXIFOLIA AND C. MEXICANA (CHLOROPHYTA) ARE NOT CONSPECIFIC

In 1984, Caulerpa taxifolia (Vahl) C. Agardh was reported along the coast of Monaco. Over the past decade it has spread along 60 km of the Mediterranean coastline and presently represents a potential risk to biodiversity. Several explanations have been advanced regarding the presence of C. taxifolia in the Mediterranean. One hypothesis maintains that the alga was introduced accidentally into the sea at Monaco, where it has been used as a decorative alga in aquaria. Caulerpa taxifolia has not been reported in earlier marine floras of the Mediterranean, and its sudden appearance has suggested that it may be a recent introduction. Another hypothesis proposes that C. taxifolia and Caulerpa mexicana Sonder ex Kützing are morphological variants of one another and hence conspecific taxa. Caulerpa mexicana has been found in the eastern Mediterranean since at least 1941. In order to establish the taxonomic identities of these taxa, individuals from five populations of C. taxifolia and four populations of C. mexicana were collected from within and outside of the Mediterranean. Comparative DNA sequence analysis of the nuclear ribosomal cistron, including the 3′-end of the 18S, ITS1, 5.8S, and ITS2 regions, show clear phylogenetic separation of the two taxa using parsimony and maximum likelihood analyses. Separation is maintained whether the analyses are based on just the more conserved 18S data or just the fast- evolving spacers. The two species are thus not conspecific. For specimens of uncertain identity (i.e. taxifolia–mexicana intermediates), a PCR diagnostic amplification can easily be performed because the ITS1 in C. taxifolia is 36 nucleotides shorter than the ITS1 in C. mexicana. Whether or not C. taxifolia has been present for a longer period of time in the marine flora, either as a cryptic endemic species or as the result of one or more introductions, represents an additional hypothesis that will require identification of biogeographic populations from throughout the world, as well as a population-level study of the Mediterranean region.     

<Emphasis Type="Italic">Caulerpa racemosa</Emphasis> var. <Emphasis Type="Italic">cylindracea</Emphasis> as a potential source of organic matter for benthic consumers: evidences from a stable isotope analysis

Caulerpa racemosa var. cylindracea has been invading various types of substrates in wide areas throughout the Mediterranean Sea. However, the effects of the distribution of this alga on zoobenthos are scanty. The aim of this study was to evaluate the effects of C. racemosa invasion on the feeding habits of some zoobenthic taxa inhabiting the upper infralittoral rocky shores. This was done by identifying the isotopic N and C ratios of several potential food sources and testing differences in isotope composition among the taxa collected from areas invaded and not-invaded areas by C. racemosa. Results suggest that C. racemosa detritus was a significant food source for the polychaete Syllis prolifera, the gammarid Corophium sextonae and the gastropods Cerithium rupestre and Pisinna glabrata. They would also suggest that stable carbon and nitrogen isotope ratios deserve further attention as a possible valuable approach to understand the overall effect of C. racemosa spread on the trophic interactions among the species.     

A FORENSIC AND PHYLOGENETIC SURVEY OF CAULERPA SPECIES (CAULERPALES,CHLOROPHYTA) FROM THE FLORIDA COAST,LOCAL AQUARIUM SHOPS,AND E‐COMMERCE: ESTABLISHING A PROACTIVE BASELINE FOR EARLY DETECTION1

Baseline genotypes were established for 256 individuals of Caulerpa collected from 27 field locations in Florida (including the Keys), the Bahamas, US Virgin Islands, and Honduras, nearly doubling the number of available GenBank sequences. On the basis of sequences from the nuclear rDNA‐ITS 1+2 and the chloroplast tufA regions, the phylogeny of Caulerpa was reassessed and the presence of invasive strains was determined. Surveys in central Florida and southern California of >100 saltwater aquarium shops and 90 internet sites revealed that >50% sold Caulerpa. Of the 14 Caulerpa species encountered, Caulerpa racemosa was the most common, followed by Caulerpa sertularioides, Caulerpa prolifera, Caulerpa mexicana, and Caulerpa serrulata. None of the >180 field‐collected individuals (representing 13 species) was the invasive strain of Caulerpa taxifolia or C. racemosa. With one exception (a sample of C. racemosa from a shop in southern California belonged to the invasive Clade III strain), no invasive strains were found in saltwater aquarium stores in Florida or on any of the internet sites. Although these results are encouraging, we recommend a ban on the sale of all Caulerpa species (including “live rock”) because: morphological identification of Caulerpa species is unreliable (>12% misidentification rate) and invasive strains can only be identified by their aligned DNA sequences, and because the potential capacity for invasive behavior in other Caulerpa species is far from clear. The addition of the Florida region to the genetic data base for Caulerpa provides a valuable proactive resource for invasion biologists as well as researchers interested in the evolution and speciation of Caulerpa.     

Invasion of alien macroalgae in different Mediterranean habitats

The study evaluated different macroalgal invasions in the main Mediterranean coastal habitats on hard bottom. Biodiversity, species composition and structure of macroalgal assemblages were compared among non-invaded areas and areas invaded by the Chlorophyta Caulerpa racemosa var. cylindracea and by the turf-forming Rhodophyta Womersleyella setacea in three different habitats: shallow rocky bottom, deep rocky bottom and dead matte of the seagrass Posidonia oceanica. Results showed that alien macroalgae constituted a relevant component of benthic assemblages in invaded areas of the Mediterranean Sea. Assemblages invaded by Womersleyella setacea and Caulerpa racemosa showed lower values of diversity and large differences in the structure and species composition related to non-nvaded assemblages. The species that mostly suffered from invasion were erect species reproducing sexually; moreover, the dominance of W. setacea led to low abundance of native filamentous algae, while C. racemosa colonization seemed particularly threatening for encrusting algae. All the studied habitats appeared highly invasible by alien macroalgae, even if W. setacea appeared more invasive in deeper habitats, while colonization of C. racemosa seemed more serious in shallower habitats; the dead matte of P. oceanica represented a suitable substrate for the spread of both species. Differences among assemblages in different habitats were reduced in invaded areas.     

Caprellid assemblages (Crustacea: Amphipoda) in shallow waters invaded by <Emphasis Type="Italic">Caulerpa racemosa</Emphasis> var. <Emphasis Type="Italic">cylindracea</Emphasis> from southeastern Spain

Growth of the invasive algae Caulerpa racemosa var. cylindracea in shallow habitats may influence the faunal assemblage composition. We studied its effects on caprellid assemblages associated with shallow-water habitats of hard and soft bottoms from the SE Iberian Peninsula (native rocky-bottom algae, C. racemosa from hard and soft bottoms, and Caulerpa prolifera, Cymodocea nodosa and Posidonia oceanica from soft bottoms). Samples were taken in two different sampling periods (September 2004 and March 2005). A total of seven caprellid species were identified, with important differences in their distribution in different habitats. Total abundance of caprellids was very high in March on native algae on hard bottoms, and on C. racemosa on both soft and hard bottoms. On both hard and soft bottoms, abundances of Caprella hirsuta recorded from C. racemosa were low. On the other hand, a higher abundance of other species, namely C. acanthifera, C. santosrosai, Phtisica marina and Pseudoprotella phasma, was recorded from C. racemosa. The results indicate that C. racemosa may have a positive influence on some caprellid species, while seasonal changes are also evident. It is concluded that introduced C. racemosa may serve as a new habitat, promoting and maintaining caprellid populations in shallow Mediterranean habitats.     

Dinucleotide microsatellite markers in the genus <Emphasis Type="Italic">Caulerpa</Emphasis>

Caulerpa spp. are clonal green marine algae which often act as invasive species when growing outside their native biogeographical borders. Over the two past decades, Caulerpa taxifolia has spread along the Mediterranean coast, presently occurring at 70 sites and covering nearly 3,000 ha of subtidal area. New genetic markers (microsatellites) have been developed to assess clonal structure and genetic diversity of recently established populations of the invasive species C. taxifolia and Caulerpa racemosa in comparison with populations of the native Caulerpa prolifera in the Mediterranean. Our results show that nine polymorphic markers have been developed for C. prolifera, seven for C. taxifolia, and three for C. racemosa. Genetic diversity in Caulerpa was assessed in two geographical scales: one at a population scale where 40 thalli units were collected from C. prolifera in Cala d’Or, Mallorca, Spain, and another at a species scale, where 30 sample units were analyzed for C. prolifera, 24 for C. taxifolia, and 24 for C. racemosa from different sites in the Mediterranean, Atlantic, and Pacific Ocean. Number of alleles, expected heterozygosity, and marker amplification success are provided in each case.     

Invasive alga <Emphasis Type="Italic">Caulerpa racemosa</Emphasis> var. <Emphasis Type="Italic">cylindracea</Emphasis> makes a strong impact on the Mediterranean sponge <Emphasis Type="Italic">Sarcotragus spinosulus</Emphasis>

Here we present the first observation of the impact of the invasive Caulerpa racemosa var. cylindracea on native photophilic sponge species in the Adriatic Sea, with special focus on Sarcotragus spinosulus. Caulerpa racemosa var. cylindracea is able to completely overgrow the sponge, developing an exceptionally thick canopy with a maximum measured density of 1,887 m of stolons m⁻² and 40,561 fronds m⁻². Necrosis of the sponge surface was significantly correlated with the algal dry biomass, frond number and stolon length. Dense algal canopy, penetration of the algal stolon and rhizoids into the sponge oscula and covering of the ostiae probably diminishes the seawater circulation through the sponge and consequently results in its smothering and even death. We suggest that chemotropism is the reason why C. racemosa penetrates the sponge oscula and establishes such dense canopy on the sponge.     

A comparative study: Assessment of the antioxidant system in the invasive green alga Caulerpa racemosa and some macrophytes from the Mediterranean

Reactive oxygen species (ROS) are of great importance in plant metabolism. However, uncontrolled activation of ROS might have deleterious effects in cells. Eleven Mediterranean countries are still under threat of an introduced taxon of Caulerpa racemosa var. cylindracea. In the present study, it has been aimed to compare the antioxidant status of this highly invasive alga with some Mediterranean macrophytes collected in the same habitat. For this purpose, such antioxidant enzyme activities as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and lipid peroxidation (LPO) as well, levels have been determined in C. racemosa, Cystoseira barbata C. Agardh, Padina pavonica (Linnaeus) Thivy and Enteromorpha sp. The highest SOD, CAT, GSH-Px enzyme activities and the lowest LPO level have been measured in the invasive C. racemosa. In conclusion, C. racemosa var. cylindracea seems more tolerant in warring with ROS than the Mediterranean species tested do. These results could partly explain the amazing success of C. racemosa var. cylindracea introduced in the Mediterranean.     

Genetic polymorphism in Caulerpa taxifolia (Ulvophyceae) chloroplast DNA revealed by a PCR‐based assay of the invasive Mediterranean strain

Abstract An invasive, cold‐tolerant strain of the tropical green alga Caulerpa taxifolia was introduced recently in the Mediterranean Sea and along the Californian coast. We screened 50 aquarium and open‐sea C. taxifolia specimens for the presence/absence of an intron located in the rbcL gene of chloroplast DNA. We also reanalysed a total of 229 sequences of the Internal Transcribed Spacer (ITS) of ribosomal DNA, combining previously published sequences from different studies with 68 new sequences to complement rbcL data. The introduced Mediterranean strain was found to be characterized by the absence of the rbcL intron and by the occurrence of a particular monomorphic ITS type. A PCR assay based on rbcL gene was developed to detect new introductions of the invasive strain of C. taxifolia. This rapid and inexpensive test could be useful to assist environment managers in the preservation of coastal marine ecosystems.     

Tracking the origin of the invading Caulerpa (Caulerpales, Chlorophyta) with Geographic Profiling, a criminological technique for a killer alga

The application of the Geographic Profiling technique (with the “Rossmo formula”) proved to be effective in assessing the spreading origin of invading species of Caulerpa in the Mediterranean. Geoprofiling is a technique more frequently used in criminology. We applied this method to an algal invasion for the first time. The method was calibrated with the distribution data of Caulerpa taxifolia, whose spreading in the Mediterranean Sea started from the aquarium of Monaco. This is the first time that Geographic Profiling is calibrated on a data set of sites of presence of a biological invader, of which the spreading origin is known. The application on Caulerpa racemosa var. cylindracea showed that the probable spreading center of the alga should be located in Western Sicily for the Mediterranean and in the southern part of Tenerife for the Canary Islands (Las Galletas, a fishing village). The spreading centers correspond to areas with ports, indicating that the spreading of this alga should be related to (fishing) boats. This result confirmed the opinion of other authors. The parameters used in the Rossmo Formula, obtained through calibration with the known origin of C. taxifolia invasion for identifying the origin of the spread of C. racemosa var. cylindracea may be extended to other algae with similar propagation mode and similar habitat requirements.     

A study of Sarcocornia A.J. Scott (Chenopodiaceae) from Western Mediterranean Europe

The latest publications on Sarcocornia taxonomy and phylogeny recognize six taxa in this genus on the Iberian Peninsula: S. perennis, S. fruticosa, S. alpini, S. alpini subsp. carinata, S. hispanica, and S. pruinosa. The present study represents a comprehensive revision of the different taxa in the Sarcocornia genus present in Western Mediterranean Europe by means of morphological, micromorphological and phylogenetic internal transcribed spacer (ITS) analysis. Morphological and micromorphological data were studied from Sarcocornia samples from 113 populations in coastal salt marshes and inland salt pans in Portugal, France, Spain and Italy. Sixteen new ITS sequences were obtained from Mediterranean Sarcocornia species and analysed together with previous reported data. Published karyological, ecological and biogeographical data from Western Mediterranean Europe were also reviewed. The results indicate the presence of a new species, S. lagascae, found growing in coastal Mediterranean areas of the Iberian Peninsula. The species S. fruticosa was found to be absent from the Iberian territories.     

Phytotoxic activity of caulerpenyne from the Mediterranean invasive variety of Caulerpa racemosa: a potential allelochemical

Allelopathy has been postulated as a factor in the colonisation success of Caulerpa racemosa var. cylindracea, an introduced chlorophyte alga which has become invasive in the Mediterranean Sea. In order to reveal their possible phytotoxic activity, secondary metabolites were isolated from a population growing in the Gulf of Naples (Italy) and tested on leaf tissue of the native seagrass Cymodocea nodosa, which often co-occurs with Caulerpa in nature. The seaweed metabolites were identified by ¹H and ¹³C NMR analysis and leaf portions of C. nodosa were exposed to crude diethyl ether extract and purified compounds in the laboratory, under controlled conditions of irradiance and temperature for a period of 144 h. Eventual changes in the photosynthetic performance of the seagrass tissue following the treatment were tested by monitoring its optimal quantum yield with a pulse amplitude modulated (PAM) fluorometer. One of the identified compounds, caulerpenyne, showed a phytotoxic effect, while the other purified metabolites did not reveal any effect at the assayed concentrations. Thus, our results seem to suggest a possible allelopathic activity of caulerpenyne, which may play a role in the successful competition of the invasive C. racemosa var. cylindracea with native macrophytes, such as seagrasses.     

Molecular genetic delineation of Phaeocystis species (Prymnesiophyceae) using coding and non-coding regions of nuclear and plastid genomes

Sequence variation among 22 isolates representing a global distribution of the prymnesiophyte genus Phaeocystis has been compared using nuclear-encoded 18S rRNA genes and two non-coding regions: the ribosomal DNA internal transcribed spacer 1 (ITS1) separating the 18S rRNA and 5.8S rRNA genes and the plastid ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO) spacer flanked by short stretches of the adjacent large and small subunits (rbcL and rbcS). 18S rRNA can only resolve major species complexes. The analysis suggests that an undescribed unicellular Phaeocystis sp. (isolate PLY 559) is a sister taxon to the Mediterranean unicellular Phaeocystis jahnii; this clade branched prior to the divergence of all other Phaeocystis species, including the colonial ones. Little divergence was seen among the multiple isolates sequenced from each colonial species complex. RUBISCO spacer regions are even more highly conserved among closely related colonial Phaeocystis species and are identical in Phaeocystis antarctica, Phaeocystis pouchetii and two warm-temperate strains of Phaeocystis globosa, with a single base substitution in two cold-temperate strains of P. globosa. The RUBISCO spacer sequences from two predominantly unicellular Phaeocystis isolates from the Mediterranean Sea and PLY 559 were clearly different from other Phaeocystis strains. In contrast, ITS1 exhibited substantial inter- and intraspecific sequence divergence and showed more resolution among the taxa. Distinctly different copies of the ITS1 region were found in P. globosa, even among cloned DNA from a single strain, suggesting that it is a species complex and making this region unsuitable for phylogenetic analysis in this species. However, among nine P. antarctica strains, four ITS1 haplotypes could be separated. Using the branching order in the ITS1 tree we have attempted to trace the biogeographic history of the dispersal of strains in Antarctic coastal waters.     

An investigation on the antioxidant status of the invasive alga Caulerpa racemosa var. cylindracea (Sonder) Verlaque, Huisman, et Boudouresque (Caulerpales, Chlorophyta)

Caulerpa racemosa var. cylindracea is a potential invader of the Mediterranean Sea and 11 Mediterranean countries are under threat from this alga. In the present study, in order to investigate seasonal changes in the antioxidant status of C. racemosa var. cylindracea, antioxidant enzyme activities such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and lipid peroxidation (LPO) levels were determined in C. racemosa var. cylindracea samples, which were collected in May 2003, September 2003, December 2003, and March 2004 from Turkish coastlines. SOD and CAT activities showed similar trends. These enzymes increased from May to December, then sharp decreases were observed. On the other hand, GSH-Px enzyme activities showed a fluctuation between May 2003 and March 2004. In contrast to increases in SOD and CAT activities up to December 2003, LPO level decreased in this period. No significant correlation was observed between antioxidant status and solar radiation. In conclusion, the antioxidant status of C. racemosa var. cylindracea is strictly not affected by both solar radiation and seawater temperature; however, the growth of epiphytes on fronds may change antioxidant status. Further investigations are strongly warranted to understand the contributions of non-enzyme-based antioxidants such as glutathione, vitamin E, and vitamin C.     

The rDNA ITS Region in the Lessepsian Marine Angiosperm <Emphasis Type="BoldItalic">Halophila stipulacea</Emphasis> (Forssk.) Aschers. (Hydrocharitaceae): Intragenomic Variability and Putative Pseudogenic Sequences

Halophila stipulacea is a dioecious marine angiosperm, widely distributed along the western coasts of the Indian Ocean and the Red Sea. This species is thought to be a Lessepsian immigrant that entered the Mediterranean Sea from the Red Sea after the opening of the Suez Canal (1869). Previous studies have revealed both high phenotypic and genetic variability in Halophila stipulacea populations from the western Mediterranean basin. In order to test the hypothesis of a Lessepsian introduction, we compare genetic polymorphism between putative native (Red Sea) and introduced (Mediterranean) populations through rDNA ITS region (ITS1-5.8S-ITS2) sequence analysis. A high degree of intraindividual variability of ITS sequences was found. Most of the intragenomic polymorphism was due to pseudogenic sequences, present in almost all individuals. Features of ITS functional sequences and pseudogenes are described. Possible causes for the lack of homogenization of ITS paralogues within individuals are discussed.     

Rhinomonas nottbecki n. sp. (Cryptomonadales) and Molecular Phylogeny of the Family Pyrenomonadaceae

The cryptomonad Rhinomonas nottbecki n. sp., isolated from the Baltic Sea, is described from live and fixed cells studied by light, scanning, and transmission electron microscopy together with sequences of the partial nucleus‐ and nucleomorph‐encoded 18S rRNA genes as well as the nucleus‐encoded ITS1, 5.8S, ITS2, and the 5′‐end of the 28S rRNA gene regions. The sequence analyses include comparison with 43 strains from the family Pyrenomonadaceae. Rhinomonas nottbecki cells are dorsoventrally flattened, obloid in shape; 10.0–17.2 μm long, 5.5–8.1 μm thick, and 4.4–8.8 μm wide. The inner periplast has roughly hexagonal plates. Rhinomonas nottbecki cells resemble those of Rhinomonas reticulata, but the nucleomorph 18S rRNA gene of R. nottbecki differs by 2% from that of R. reticulata, while the ITS region by 11%. The intraspecific variability in the ITS region of R. nottbecki is 5%. In addition, the predicted ITS2 secondary structures are different in R. nottbecki and R. reticulata. The family Pyrenomonadaceae includes three clades: Clade A, Clade B, and Clade C. All Rhinomonas sequences branched within the Clade C, while the genus Rhodomonas is paraphyletic. The analyses suggest that the genus Storeatula is an alternating morphotype of the genera Rhinomonas and Rhodomonas and that the family Pyrenomonadaceae includes some species that were described multiple times, as well as novel species.     

Effects of <Emphasis Type="Italic">Caulerpa racemosa</Emphasis> invasion on soft-bottom assemblages in the Western Mediterranean Sea

The present study aimed at evaluating the effects of Caulerpa racemosa var. cylindracea spread on Mediterranean soft-bottom assemblages. The benthic assemblages colonized by C. racemosa were compared with non invaded assemblages at multiple spatial scales. In addition, a manipulative experiment has been conducted over a one-year period in order to compare the structure of native assemblages invaded by the alga with others where the alga has been manually removed and others that were not invaded. Results of both studies showed that Mediterranean soft-bottom assemblages invaded by C. racemosa differed from non invaded ones in terms of species composition, abundance and patterns of spatial variability. Moreover, in areas cleaned out from the alga, the benthic assemblages begin to recover their structure and after one year they get more similar to the assemblages observed in non invaded areas. The present paper, taking into account different aspects of C. racemosa invasion in Mediterranean soft bottoms, highlighted that the observed increase in alpha diversity did not correspond to an increase in the overall diversity of the studied system. Infact, the loss of beta diversity in invaded sites, together with the spread of species typical of vegetated habitats, may contribute to a homogenization of the Mediterranean coastal system. Furthermore, a possible role of ecosystem engineer for C. racemosa can be hypothesized.