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.     

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 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.     

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.     

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.     

New features in the vegetative and reproductive morphology of Cystoclonium purpureum (Cystocloniaceae,Rhodophyta) from the North Atlantic Ocean

The aim of this study was to evaluate whether the success of invasive Caulerpa cylindracea (Caulerpa racemosa var. cylindracea) in Mediterranean habitats may be linked to particular nutritional requirements and/or nutrient accumulation/reallocation dynamics within the algal thallus. To this end, a manipulative field experiment was carried out transplanting C. cylindracea and supplying nutrients to subtidal rocky macroalgal assemblages. Morphological features of C. cylindracea were measured, while percentage cover and tissue nitrogen, carbon and phosphorus were determined for C. cylindracea and two co-occurring species (the introduced Laurencia chondrioides and native Flabellia petiolata) in nutrient-enriched and unenriched conditions. Growth of C. cylindracea was greater in the nutrient-enriched plots while no effect of nutrient enrichment was detected in co-occurring macroalgae. A particularly high requirement for nitrogen has been observed in C. cylindracea, but without severe N-limitation, while a general nutritional state of P-limitation was detected in both invasive and co-occurring macroalgae. The absence of inhibition in hypertrophic conditions and under high levels of ammonia together with a strong uptake and quick reallocation of nutrients within the coenocytic thallus probably represent the main advantages over the other co-occurring species which could explain part of the success of C. cylindracea in the Mediterranean Sea.     

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.     

Antiproliferative and newly attributed apoptotic activities from an invasive marine alga: Caulerpa racemosa var. cylindracea

Caulerpa racemosa (Forsskål) is a green marine alga which spreads from tropical to warm-water regions. Due to having invasive capacity C. racemosa var. cylindracea is a well-known biological pollution in Mediterranean Sea. One of the most important secondary metabolites of C. racemosa is Caulerpenyne (CPN). In the present study, antiproliferative and apoptotic effects of C. racemosa var. cylindracea extract and purified CPN on two well-known neuroblastoma cell lines, SHSY5Y and Kelly, are investigated. The antiproliferative and, additionally, newly attributed apoptotic effects of both C. racemosa var. cylindracea extract and purified CPN on SHSY5Y and Kelly cell lines have been shown in the present study. IC₅₀ values are 0.59 ± 0.06; 1.06 ± 0.23 g wet alga/methanol and 5.64 ± 0.09; 6.02 ± 0.09 μM CPN for C. racemosa var. cylindracea extract and purified CPN on SHSY5Y and Kelly cell lines, respectively. Percentages of apoptotic cells of SHSY5Y and Kelly in 0, 0.1 and 1 μM CPN conditions were 1.00 ± 0.71, 3.00 ± 0.71 and 49.40 ± 3.78, 39.60 ± 6.19 and 78.00 ± 2.74, 69.40 ± 3.78, respectively. In conclusion, the present study shows the antiproliferative effect of C. racemosa var. cylindracea extract and newly attributed apoptotic effects of C. racemosa var. cylindracea this extract. Compared to other alkylating anticancer drugs, CPN and also C. racemosa var. cylindracea extract might be considered as an alternative native source of antitumor drugs. Inasmuch as both C. racemosa extract and CPN have shown both antiproliferative and apoptotic effects on SHSY5Y and Kelly cell lines, the CPN and CPN derivatives might be considered as multifunctional agents in cell metabolism.     

Comparative study of the growth of the two co-occurring introduced green algae Caulerpa taxifolia and Caulerpa racemosa along the Tuscan coast (Italy,western Mediterranean)

Caulerpa taxifolia and C. racemosa are tropical green algae now found in the Mediterranean, where the former was accidentally introduced and the latter presumably also introduced; both species have spread rapidly and invaded a variety of habitats. Since 1996, the two algae have co-inhabited a region along the Tuscan coast near Livorno. The present study has been carried out to evaluate the invasive capacity of the two species. After one year, the increase in surface area was 67.5 % for C. taxifolia and 284.8 % for C. racemosa, while the increase in patch number was 11.4 % and 121.4 % respectively. The stolon growth of the two algae followed a similar trend, as is showing the same temporal variations with the maximum growth rate between August and October. At the end of the study period, the increase in stolon length and number was significantly higher in C. racemosa than in C. taxifolia. The results of the present study show a higher invasive potential in C. racemosa than in C. taxifolia, although further studies are necessary to evaluate the competition between the two algae and the effect of their simultaneous presence on the benthic community.     

Molecular Phylogeny and Barcoding of Caulerpa (Bryopsidales) Based on the tufA,rbcL, 18S rDNA and ITS rDNA Genes

The biodiversity assessment of different taxa of the genus Caulerpa is of interest from the context of morphological plasticity, invasive potential of some species and biotechnological and pharmacological applications. The present study investigated the identification and molecular phylogeny of different species of Caulerpa occurring along the Indian coast inferred from tufA, rbcL, 18S rDNA and ITS rDNA nucleotide sequences. Molecular data confirmed the identification of 10 distinct Caulerpa species: C. veravalensis, C. verticillata, C. racemosa, C. microphysa, C. taxifolia, C. sertularioides, C. scalpelliformis, C. serrulata, C. peltata and C. mexicana. All datasets significantly supported the sister relationship between C. veravalensis and C. racemosa var. cylindracea. It was also concluded from the results that the specimen identified previously as C. microphysa and C. lentillifera could not be considered as separate species. The molecular data revealed the presence of multiple lineages for C. racemosa which can be resolved into separate species. All four markers were used to ascertain their utility for DNA barcoding. The tufA gene proved a better marker with monophyletic association as the main criteria for identification at the species level. The results also support the use of 18S rDNA insertion sequences to delineate the Caulerpa species through character-based barcoding. The ITS rDNA (5.8S-ITS2) phylogenetic analysis also served as another supporting tool. Further, more sequences from additional Caulerpa specimens will need to be analysed in order to support the role of these two markers (ITS rDNA and 18S insertion sequence) in identification of Caulerpa species. The present study revealed the phylogeny of Caulerpa as complete as possible using the currently available data, which is the first comprehensive report illustrating the molecular phylogeny and barcoding of the genus Caulerpa from Indian waters.     

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.     

Improving Transferability of Introduced Species’ Distribution Models: New Tools to Forecast the Spread of a Highly Invasive Seaweed

The utility of species distribution models for applications in invasion and global change biology is critically dependent on their transferability between regions or points in time, respectively. We introduce two methods that aim to improve the transferability of presence-only models: density-based occurrence thinning and performance-based predictor selection. We evaluate the effect of these methods along with the impact of the choice of model complexity and geographic background on the transferability of a species distribution model between geographic regions. Our multifactorial experiment focuses on the notorious invasive seaweed Caulerpacylindracea (previously Caulerpa racemosa var. cylindracea ) and uses Maxent, a commonly used presence-only modeling technique. We show that model transferability is markedly improved by appropriate predictor selection, with occurrence thinning, model complexity and background choice having relatively minor effects. The data shows that, if available, occurrence records from the native and invaded regions should be combined as this leads to models with high predictive power while reducing the sensitivity to choices made in the modeling process. The inferred distribution model of Caulerpacylindracea shows the potential for this species to further spread along the coasts of Western Europe, western Africa and the south coast of Australia.     

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.     

GENOMIC DNA ISOLATION FROM GREEN AND BROWN ALGAE (CAULERPALES AND FUCALES) FOR MICROSATELLITE LIBRARY CONSTRUCTION1

A method for isolating high‐quality DNA is presented for the green algae Caulerpa sp. (C. racemosa, C. prolifera, and C. taxifolia) and the brown alga Sargassum muticum. These are introduced, and invasive species in Europe, except for the native C. prolifera. Previous methods of extraction, using cetyl trimethyl ammonium bromide or various commercial kits, were used to isolate genomic DNA but either no DNA or DNA of very low quality was obtained. Genomic libraries were attempted with Caulerpa sp. on three occasions and either the restriction enzyme, the Taq polymerase, or the T4 ligase was inhibited, probably by the large amount of polysaccharides in these algae. The method presented here consists of the rapid isolation of stable nuclei, followed by DNA extraction. Yields of 6–10 μ g genomic DNA from 1 g fresh blades were obtained. After genomic DNA was isolated from fresh material, the quality was checked by agarose gel. Quantification of DNA concentration was performed using UV spectrophotometric measurement of the A ₂₆₀/ A ₂₈₀ ratio. The DNA was suitable for PCR, cloning, and hybridization. The DNA isolated using this method allowed successful construction of microsatellite libraries for Caulerpa species and S. muticum . The technique is inexpensive and appropriate for the isolation of multiple samples of DNA from a small amount of fresh material.     

The Sesquiterpene Caulerpenyne from Caulerpa spp. is a Lipoxygenase Inhibitor

Many algae contain secondary metabolites with the potential to gain importance as pharmaceutically active secretions. Caulerpa racemosa var. cylindracea and Caulerpa prolifera are very abundant on the Mediterranean coastlines. The methanolic extracts of C. racemosa and C. prolifera were tested for inhibitory effects on soybean lipoxygenase. The extract of C. prolifera showed potent inhibitory effect in a lipoxygenase enzyme activity assay. HPLC comparison revealed that C. racemosa extract contained less caulerpenyne, the major secondary metabolite of both algae. In accordance with these findings, purified caulerpenyne inhibited lipoxygenase with an IC₅₀ of 5.1 μM. The enzyme kinetic studies indicated that both K _M and V _max decreased from 0.041 to 0.019 mM and 312.5 to 151.5 U mL⁻¹ in the presence of 5 μM caulerpenyne, revealing an un-competitive type of inhibition kinetics. The major secondary metabolite of Caulerpa species, caulerpenyne, is thus a novel lipoxygenase inhibitor that can be easily obtained in high quantities from the abundant algae.     

<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.     

Native species behaviour mitigates the impact of habitat-forming invasive seaweed

Habitat-forming invasive species cause large, novel changes to the abiotic environment. These changes may elicit important behavioural responses in native fauna, yet little is known about mechanisms driving this behaviour and how such trait-mediated responses influence the fitness of native species. Low dissolved oxygen is a key abiotic change created by the habitat-forming invasive seaweed, Caulerpa taxifolia, which influences an important behavioural response (burrowing depth) in the native infaunal bivalve Anadara trapezia. In Caulerpa-colonised areas, Anadara often emerged completely from the sediment, and we experimentally demonstrate that water column hypoxia beneath the Caulerpa canopy is the mechanism instigating this “pop-up” behaviour. Importantly, pop-up in Caulerpa allowed similar survivorship to that in unvegetated sediment; however, when we prevented Anadara from popping-up, they suffered >50% mortality in just 1 month. Our findings not only highlight the substantial environmental alteration by Caulerpa, but also an important role for the behaviour of native species in mitigating the effects of habitat-forming invasive species.     

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.     

Antioxidant status of Lobiger serradifalci and Oxynoe olivacea (Opisthobranchia, Mollusca)

Invasion of the Mediterranean Sea by the two world-wide famous exotic algae species, Caulerpa taxifolia and Caulerpa racemosa, is still a problem and has adverse effects on the Mediterranean sublittoral ecosystem. Biological control studies revealed that the two native Sacoglossans, Oxynoe olivacea and Lobiger serradifalci, may have an effect on the expansion of invasive Caulerpa spp. in the Mediterranean. In the framework of this study, antioxidant enzyme activities, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), lipid peroxidation (LPO) and oxidized glutathione (GSSG) levels, as oxidative stress markers in L. serradifalci and O. olivacea were determined at two different temperature conditions (20 and 27 °C). In both species, SOD, CAT and GSH-Px activities were found to be positively correlated with temperature. The SOD activities in L. serradifalci were higher than those in O. olivacea at both temperatures, whereas the CAT and GSH-Px activities were significantly (p<0.05) higher in O. olivacea, compared to L. serradifalci. As expected, both species showed decreased LPO levels at 27 °C compared to 20 °C. GSSG level at 27 °C in O.olivacea was significantly (p<0.05) higher than that of 20 °C. On the other hand, no statistical (p>0.05) difference in L.serradifalci existed between GSSG levels at two temperatures. But, despite the variations in the antioxidant enzyme activities, there was no significant difference in LPO levels between the species, suggesting that the oxidative consequences of a given environmental condition may vary among different species. Inasmuch as the GSSG levels were in accordance with antioxidant enzyme activities, GSH might have acted as a cofactor of GSH-Px and an individual antioxidant in these sea slugs.