Chrysymenia wrightii ( Rhodymeniales , Rhodophyta )-a new non-native species for the European Atlantic Coast

Chrysymenia wrightii, originally described from Japan, was found for the first time from the European Atlantic coast. It was collected in several subtidal rocky bottom habitats (9-14 meters depth) of the Ría de Arousa (Galicia, NW Spain). In this work, a description of Galician gametophytic and tetrasporophytic plants is provided. They are similar to the Mediterranean and Japanese plants. DNA sequence data of materials from Galicia and Korea were investigated using nuclear SSU and ITS1-5.8S-ITS2 rDNA and plastid rbcL sequences. No genetic variation was observed in the SSU, and only one substitution was detected in ITS and rbcL data between Galician and Korean samples, respectively. Our molecular data indicate that the Galician populations of C. wrightii are probably due to a recent introduction event from the northwest Pacific. Despite the fact that C. wrightii was formerly recorded as a new non-native species from a Mediterranean hotspot (Thau Lagoon, 1987-1989), it was never reported for the European Atlantic coast. After 30 years of its first report in the Mediterranean Sea, the simultaneous occurrence of C. wrightii in four subtidal localities of NW Spain suggests that this species may have colonized the region unnoticed for several years which may be related to its subtidal habitat and short life cycle. Similarly to the Mediterranean Sea, the introduction of C. wrightii on the European Atlantic coasts could be enhanced by the intensive aquaculture widely spread along the Galician coast. Curiously, C. wrightii is apparently restricted to both areas, the Ría de Arousa and the Thau Lagoon. In addition, many non-native species markedly represented in Galicia since the 1980’s were firstly reported in the Ría de Arousa. Consequently, the Ría de Arousa should be considered an outstanding European Atlantic hotspot of introduced marine species similar to the Solent region (South of England) or the Thau Lagoon.


Introduction
The introduction of non-native species is mainly caused by vessels and aquaculture (Streftaris et al. 2005).In the Galician region (NW Spain), fishing and aquaculture industry have a great development.
Although Chrysymenia wrightii (Harvey) Yamada was reported as non-native seaweed for the European coast (Thau Lagoon, Mediterranean Sea) (Ben Maïz et al. 1987), it has not been detected on the European Atlantic coast.In the NW of Iberian Peninsula, no Chrysymenia species are known.Solely, C. ventricosa was reported in the Cantabric Sea (Gorostiaga et al. 2004;Llera González and Álvarez Raboso 2007) whereas the rest of the Iberian records for this warm-temperate species are known from the South of Spain (Conde et al. 1996) and the Mediterranean Sea (Ballesteros 1989).During current surveys in subtidal maërl beds in the Galician rías, several specimens of the northwest Pacific species C. wrightii were collected from rocky bottom areas.The aim of this work is to add C. wrightii as a new non-native species to the European Atlantic seaweeds catalogue and describe its morphology, habitat, current distribution and extent of its populations, as well as the genetic variation of the Atlantic and Pacific populations.

Materials and methods
Plants were first collected (October 2005) by SCUBA diving in a subtidal rocky bottom habitat covered with mud (9-12 meters depth) of the Ría de Arousa during the explorations of the Galician maërl beds.From a new subtidal exploration along this ría (September and October 2007) we found C. wrightii in the original site examined in 2005 and in three new localities, Cambados, Islote Galiñeiro and Tragove (Figure 1, Annex 1) which are located 11 km from the first locality.Samples were preserved in 4% formalin seawater at 4°C in darkness.In order to study reproductive structures, sterile plants collected in Cabo Cruz (September 2007) were cultured in sterile seawater (31-32 PSU) at 16ºC and 4:20 light:dark photoperiod.Plants were dissected by hand with a stainless steel razor blade.Herbarium specimens were deposited at the herbarium of the University of Santiago de Compostela (SANT-Algae).
In order to study the genetic variation between Galician and Korean individuals of C. wrightii, several silica-dried specimens from both areas (Annex 2) were compared at the Polar Biodiversity Laboratory, Korea Polar Research Institute (KOPRI).Genomic DNA from samples was extracted using DNeasy ® Plant Mini Kit (Qiagen, Hilden, Germany).After DNA extraction, the rest of silica-dried samples were deposited at the KOPRI herbarium.The nuclear SSU and ITS rDNA, and plastid rbcL were amplified from total genomic DNA using polymerase chain reaction (PCR) and the primer combinations of Saunders andKraft (1994, 1996) for SSU, Tai et al. (2001) for ITS1-5.8S-ITS2,and Freshwater and Rueness (1994) for rbcL.Agarose gelpurification or direct purification with High Pure TM PCR Product Purification Kit (Roche Diagnostics, Indianapolis, IN, USA) or LaboPass PCR Purification Kit (COSMO Genetech, Seoul, Korea) was used to clean PCR products.DNA purified using this method was sequenced using the BigDye TM terminator cycle sequencing ready reaction kit (PE Applied Biosytems [ABI], Foster City, CA, USA).Sequence data were collected using an ABI PRISM 3730 DNA Analyzer, and were edited using the SeqEd DNA sequence editor (ABI) software package.The edited sequences were aligned relative to one another using the SeqPup multiple alignment program (Gilbert 1995) and MacClade 4 program (Maddison and Maddison 2003).The final alignment for SSU, rbcL and ITS1-5.8S-ITS2consisted of three, three and seven taxa (Annex 2), respectively.The sequences of the 1771, 1526 and 703 aligned nucleotide positions of SSU, rbcL and ITS1-5.8S-ITS2data were edited to remove the 5' and 3' PCR primer regions (Saunders and Kraft 1994;Freshwater and Rueness 1994;Tai et al. 2001), as well as ambiguously aligned regions, to yield 1723, 1448 and 658 base pairs for phylogenetic inference, respectively.Pairwise distance for SSU, rbcL and ITS1-5.8S-ITS2data were performed usuing PAUP* 4.0b10 for Macintosh (Swofford 2002).

Results and discussion
Galician plants of Chrysymenia wrightii are similar to the ones described for the Mediterranean coast (Ben Maïz et al. 1987) and the Japanese coast (Yamada 1932;Lee 1978).They are 15-40 cm high, red-brownish in colour, terete and hollow (Figure 2).The thallus is gelatinous and tender, attached by a discoid holdfast and cylindrical axes, 3-4 mm at the base to 1-2 mm at the apex.Plants grow monopodially, 2-4 times branched (alternate, opposite or irregular) with branches constricted at the base and tapering at the tips.The cortical layer consists of 2-3 small cells (5-8×6-12µm) and the subcortical layer has 3-5 large cells and inner irregular rizoidal cells (Figure 3).The medullary layer of 3-5 cells is loosely and irregularly arranged.Medullary cells are hyaline, elongated (130-400×60-90µm).Gland cells (up to 70 µm diameter) and hyphaelike filaments (2-5 cells) are developed from innermost medullary cells, solitary or in aggregation (Figure 3) as it is commented by Yamada (1932).
Cystocarps match with the descriptions of Lee (1978) and Yamada (1932).Whereas, Ben Maïz et al. (1987) described cystocarps (500-800 µm in diam.)without ostiole for the Mediterra-nean plants.In Galicia, this feature is observed only in immature cystocarps.Male plants were not observed.Tetrasporangia divided cruciately (28-42×25-30 µm) scattered in the cortical layer and originate from inner cortical cells (Figure 4).Galician plants show a morphological variability in size and diameter of axes.Plants are profuse in branching, second order branches being abundant.In general, gametophytic plants are more irregular in form and branching compared to the tetrasporophytic plants, because adventitious short branches (similar in form to apical branches) grow from the pericarp of senescent cystocarps.This growth process is quite peculiar since reproductive structures are usually the last growth stadium, but this species apparently have a high cell activity.Profusion in branching is a good strategy for non-native and invasive species providing lots of vegetative propagules by fragmentation.Although we have not observed new individuals of C. wrightii growing from the cystocarpic branches, they could provide an excellent way of spreading along the Atlantic coast of Europe as it was pointed out for other non-native species with effective dispersal by vegetative propagation (Eno et al. 1997;Bjaerke and Rueness 2004;Husa and Sjötun 2006).
Three SSU rDNA, three rbcL and seven ITS1-5.8S-ITS2 rDNA sequences were completed from seven samples of two different localities in Galicia and three different sites in Korea and were deposited in GenBank (Annex 2).No ambiguities were observed in the sequence data.Comparisons with sequences retrieved from GenBank showed that the SSU rDNA sequences were identical with that of C. wrightii from Japan (Saunders et al. 1999;AF117129).All sequence data for two samples (CH1407 and CH1536) from Galicia were identical to each other.No genetic variation was seen in the SSU, and only one substitution was observed in rbcL (T↔C in position 914; data not shown) and ITS data (A↔G in position 423; Figure 5) between Galician and all Korean samples, respectively.In addition a deletion (T in position 169) and an insertion (T in position 538) were also observed in the ITS1 and ITS2 region, respectively between Galician and four Korean ones (CH1727, CH1728, CH1729 and CH1774; Figure 5).Only one deletion (T in position 169) was observed between the sample from Samchuk (CH1770) and the other four samples from Korea (Figure 5).Our molecular data indicate that the C. wrightii populations in Galicia are probably due to a recent introduction event from somewhere of the northwest Pacific near Korean coast.
In the Mediterranean Sea the vector of introduction for Chrysymenia wrightii is likely to be mollusc culture (Verlaque 2001;Cormaci et al. 2004).Taking into account that intensive aquaculture is widely spread on the Galician coast, the introduction of C. wrightii was likely enhanced by this vector.In the NW Spain, aquaculture has played an important role in the introduction of other non-native species especiallly since the 1980´s.Examples include Sargassum muticum, Undaria pinnatifida, Lomentaria hakodatensis, Grateloupia turuturu, G. subpectinata, Gracilaria vermiculophylla, Heterosiphonia japonica, Dasya sessilis and Ulva pertusa.
Chrysymenia wrightii was added as a new non-native species for the European coast (Thau Lagoon, Mediterranean Sea), based on collections from 1978 to 1985 (Ben Maïz et al. 1987).Subsequently, the species is regularly found in the same area (which is a hotspot of introduced species) ( (Verlaque 2001).In Galicia, Grateloupia subpectinata Holmes is an example of a non-native species which has not expanded along the coast.Hitherto, it was only known in 6 localities of the Ría de Arousa (López Rodríguez et al. 1991;Bárbara et al. 2002) and since the 1990's it has not colonized new localities in other Galician rías.At the present, C. wrightii is restricted to both the Mediterranean coast (Thau Lagoon) and the Atlantic coast (4 localities in the Ría de Arousa).Taking into account that the Ría de Arousa provides the new record of C. wrightii on the Atlantic coasts, as it happened with other nonnative species which are now widely distributed, we conclude that the Ría de Arousa should be considered as an interesting European Atlantic hotspot for the introduction of marine species such as the Solent region in England (Farnham 1980) and the Thau Lagoon in the Mediterranean France (Verlaque 2001).
The present record of Chrysymenia wrightii in the Ría de Arousa occurred 30 years after its detection in the Mediterranean.The subtidal habitat (9-14 meters depth) where the Galician populations were observed could have favoured an unnoticed occurrence for several years.In contrast in the Mediterranean Sea it could have been detected easier because it occurs at shallow localities, from 0.5 to 6 m depth (Ben Maïz et al. 1987).The short life cycle of C. wrightii could further complicate its detection.On the Japanese coasts it is described as an annual species (Lee 1978) which appears for a short period, May to October, whereas in the Mediterranean Sea it was found from January to November (Ben Maïz et al. 1987).However, in Galicia it was only collected in September and October despite many subtidal explorations (2006)(2007) carried out along the Ría de Arousa in different months, habitats and water depths.Based on both features that complicate the detection of C. wrightii in Galicia (subtidal habitat and short life cycle), it would be possible that C. wrightii has been introduced earlier in the Ría de Arousa, 1-2 decades ago or more, but it has been unnoticed.Arguably, C. wrightii could also occur unnoticed nowadays along other Atlantic coasts which may only be proven with further subtidal surveys.New studies could also verify if the present record is an isolated case of introduction that will not thrive along the Atlantic Iberian Peninsula or if it has already become an established member of the Galician non-native seaweed flora or even if C. wrightii could be considered a future invasive species.Currently C. wrightii occurs in two areas of the Ría de Arousa: (a) locality 1 (1 ha surface) and (b) localities 2-4 (100 ha potential surface).Both are located in the vicinity of aquaculture sites where non-native species like Undaria pinnatifida or Grateloupia turuturu were firstly recorded for the Spanish coast.
Historical reports and our molecular data indicate that the C. wrightii populations in Galicia were recently introduced and originate from somewhere in the northwest Pacific and may have used the Mediterranean coast as a stepping stone.To complete the information about this non-native species, extensive samplings of plants and more variable genetic markers could more clearly locate the source populations and further identify the most likely dispersal vector(s).

Figure 3 .
Figure 3. Chrysymenia wrightii in Galicia, vegetative structure: A -transverse section of an apical branch showing the hollow thallus; B-C -cortical, subcortical and hyphaelike filaments in transverse section; D -cortical cells in surface view, outer part; E -hyphaelike filaments and gland cells in surface view, inner part; E -gland cells; F -hyphaelike filaments.Scale bar = 200 µm (Photographs by Ignacio Bárbara).