Present distribution and possible vectors of introductions of the alga Heterosiphonia japonica (Ceramiales, Rhodophyta) in Europe

In order to trace the introduction history of the western Pacific species Heterosiphonia japonica to and within Europe, information on distribution pattern of the species in Europe, oyster import and shipping activity has been compiled and analyzed. Additionally survival of fragments of H. japonica during a simulated journey in ballast water from Asia (Korea) to Europe (France) has been observed. The species was first recorded in Europe near an oyster culture area in Brittany (France) in 1984. Since then, it has spread quickly in Europe. The first introduction into Europe and secondary dispersal in southern Europe has most likely been trough movement of the Pacific oyster Crassostrea gigas. However, survival of H. japonica in temperatures varying from 14 to 28 degrees during a simulated 28-days journey in ballast water was demonstrated, and an introduction by ships from the Pacific Ocean may thus be considered as possible. Spreading of the species in the North Sea area and Scandinavia is most likely due to transport by ships, e.g. as attached to ships hulls, as fragments in ballast water or entangled e.g. in fishing gear.


Introduction
Spreading of marine species across natural barriers by human-mediated means is an increasing problem.An overview published in 2006 reported a total of 664 non-indigenous marine species (NIMS), including brackish water species, in European and adjacent waters (Gollasch 2006).The highest rate of arrival of NIMS to European and adjacent waters is seen after 1950 (Gollasch 2006).Reise et al. (1999) sorted the introductions of NIMS to the North Sea according to the most likely vector for introduction.Before 1940 more than half of the introductions to the North Sea probably were by ship, while more than half of the introductions most likely have been through aquaculture after 1940 (Reise et al. 1999).According to Gollasch (2006) the main vectors for introductions of marine species to European and adjacent waters have been ships (400 species), the Suez Canal (253 species) and aquaculture (251 species).Ships have carried boring organisms (wooden ships), organisms attached to their hull or associated with ballast sand or rock, or in the ballast water.Many NIMS have been intentionally introduced in order to cultivate the species, which has subsequently resulted in establishment of viable populations outside the cultivation areas.A great number of NIMS have also come to Europe associated with introduced species for cultivation.In particular, the extensive import of Pacific oysters has resulted in many NIMS in the North Sea (Reise et al. 1999;Maggs and Stegenga 1999).
Macroalgae constitute an important group of NIMS.According to Hewitt et al. (2007), the highest numbers of accidentally introduced macroalgae are found in the Mediterranean and in Australia and New Zealand waters (90 and 99, respectively), followed by the northeast Atlantic (38).Ballast water and aquaculture are considered to be important ways for introduction of macroalgae, in addition to hull fouling (Hewitt et al. 2007).
The first European observation of the Pacific red alga Heterosiphonia japonica Yendo was in France (Brittany) in 1984 (Verlaque and Cabioch, pers. comm.)(Annex 1, Figure 1).It is now widespread in Europe.Yendo (1920) described two varieties of the species from Japan; Heterosiphonia japonica f. nipponica with four pericentral cells and Heterosiphonia japonica f. pacifica which has four or five pericentral cells.According to Choi (pers. comm.) the former variety is the species present in Korea and Europe.Recent genetic studies show that H. japonica sensu Choi et al. (2002) most likely is misplaced in the genus Heterosiphonia (Choi et al. 2002).Spanish, French (Atlantic and Mediterranean) and Norwegian material of H. japonica has been shown to be genetically similar to H. japonica sensu Choi et al. (2002) from Korea (Bjaerke 2004).
The vector for the introduction of H. japonica to Europe from Asia remains unknown, but imports of oysters from The Pacific to Europe has been suggested (Maggs and Stegenga 1999).H. japonica may e.g. have grown on oyster shells of half grown or adult oysters, or on empty shells on which oyster spat have been set.Here, two possible ways by which H. japonica may have spread to Europe from the West Pacific will be considered; import of oyster from the Pacific and transportation in ballast water.In addition, an attempt will be made to trace the subsequent spreading pattern and possible vectors for secondary spreading inside Europe.

Geographical survey of Heterosiphonia japonica
In order to trace the establishment of H. japonica in Europe information from different sources has been compiled: publications, responses to a request made through the ALGAE-L e-mail list, which reaches a great number of European phycologists (see http://www.seaweed.ie/),and information from other sources.Care was taken to ensure that all non-published information about distribution of H. japonica is correct; e.g. by only including verified registrations of H. japonica, or collecting preserved samples or pictures showing characteristic features of H. japonica from the information sources.Based on the collected information the time of first recordings and spreading of H. japonica in various countries in Europe have been established.

Assessment of introduction vectors of Heterosiphonia japonica
In order to examine oyster import from the Pacific as a possible vector for introduction of H. japonica to Europe, information about time periods and places for imports of Pacific Oysters (Crassostrea gigas) has been compiled.Also, transport of oysters within Europe as a possible vector for secondary introductions has been examined.The overview has been based on information from available publications.
Boat traffic to and within European waters was assessed as a possible alternative vector for the introduction of H. japonica to Europe, and for subsequent spreading within Europe.H. japonica may be transferred by boats in different ways, e.g. by ballast water, growing on the hull or e.g.ropes, or entangled in fishing nets.Ships are most likely to function as vector for introduction along busy traffic lines.In order to get an overview of boat traffic to and within European waters the web site Sailwx (http://www.sailwx.info/) was used, which collects weather reports from ships and stores positions.The collection of ship positions by this web site will account for about 2-3 % of the total boat traffic according to the web master of the web site.Positions may be under-reported close to harbours or along busy shipping areas.
In order to test survival of H. japonica under conditions resembling those in ballast water a laboratory experiment was conducted, simulating a journey from South Korea to Le Havre, France, through the Suez Canal.Fragments of uniseriate small side branches (pseudolaterals) of H. japonica, 1-2 mm long, were put in two round glass beakers (6 cm high and 10 cm wide) filled with natural seawater, with 25 fragments in each.In addition, two 5-7 cm long shoots of H. japonica were kept in seawater in two separate beakers.The shoots and fragments were kept in total darkness in a temperature-regulated room for 28 days, which would be a normal duration for a bulk boat from Korea to Le Havre (according to the APL Asia-Europe timetable for the CEX China Europe Express).The temperature in the room was gradually increased   1971-1976 (1971-1975) Spat (cultchless or set on shells) 5)

Distribution of H. japonica in Europe
A survey of information on the first registrations of Heterosiphonia japonica in various countries in Europe is shown in Annex 1 and Figure 1.Some years after the first registration in Europe in 1984 (Bretagne, France) the species was found in Galicia (NW Spain, 1988) H. japonica has spread very rapidly around the Norwegian south coast since it was first recorded on the southwest coast (Figure 1).The species has spread along the coast of Galicia after 1988, and in France it has been found along the coast of Brittany (Annex 1, Figure 1).Gunnarsson, pers. comm.) or in south Spain (C.Pena-Martín, pers.comm.), and it is not mentioned in a recent overview of introduced marine species in Belgium (Kerckhof et al. 2007).Gametophytic specimens are readily obtained in culture (Bjaerke and Rueness 2004), but seem to be very rare in nature.Examinations of H. japonica samples in Norway have only shown vegetative or tetrasporophytic specimens (Husa and Sjøtun 2006).Gametophytes have been found in Roscoff, France (1984), Galicia, Spain (2004, 2005and 2008) and in The Netherlands (2004) (Annex 1; M. Verlaque and J. Cabioch, pers. comm., H. Stegenga, pers. comm.).Gametophytic specimens from Galicia are placed in the SANT-Algae herbarium, University of Sanitiago de Compostela.

Oyster import as vector
Many of the sites of the first observation of Heterosiphonia japonica in Europe are situated near areas with oyster farming (Annex 1).In order to examine if the import of Pacific Oysters is a possible vector for introduction of H. japonica into Europe, information from Ruesink et al. (2005) and others about main introductions and import events were summarized (Table 1).The first introduction of Crassostrea into Europe may have taken place from Taiwan, as a fouling organism on ships's hull before 1819 (Wolff and Reise 2002).In more recent time, imports of Pacific Oysters from the west coast of the USA and Canada to Portugal, the UK, The Netherlands, Denmark and Norway have taken place between 1926 and 1986 (Table 1).In 1966 Pacific Oysters were imported from Japan to The Netherlands, and between 1971 and 1977 an extensive import of Crassostrea gigas took place from the Pacific to France.During 1971-1975 adult oysters were imported from British Columbia, Canada, and were directly planted in the sea at several places in France (Grizel and Héral 1991).During 1971During -1977 large amounts of spat were imported from Japan to various places in France.The spat was partly cultchless and partly set on oysters or scallop shells (Grizel and Héral 1991).The most extensive import took place to Marennes-Oléron during 1971-1975, but much was also imported to other areas in the Bay of Biscay, to Brittany, and to the Mediterranean (Grizel and Héral 1991).In addition, there were plans to import Ostrea denselamellosa to France from Korea in 1982 (Mann 1983), but only one record of such an import from Japan to Brittany in 1982 has been found (le Borgne and Le Pennec 1983).In 1982 the import of oysters to France from the Pacific stopped due to the discovery of a parasite in Japan (Wolff and Reise 2002).
An extensive transport of oysters for cultivation has also taken place within Europe (Ruesink et al. 2005).In order to investigate if such transport may have caused secondary introductions of H. japonica reports of oyster transfers within Europe from the 1970s and onwards were taken into consideration, since the first record of H. japonica in Europe is from 1984.Norway imported seed oysters and spat from Scotland (the UK) during 1976-1986(Strand and Vølstad 1997), and Denmark has imported seed oysters from the UK, The Netherlands and France during the last 30 years of the last century (Nehring 2006).According to the overview and information summarized by Ruesink et al. (2005), Denmark also imported oysters from Germany in 1980, Ireland from France in 1993, The Netherlands from France during 1964-1981 and Portugal from France during 1990-1992.Spain has imported Pacific oysters from France since the 1980s (Ruesink et al. 2005;Iglesias et al. 2005).The import of oysters from France to The Netherlands was stopped in 1981 due to the discovery of a disease (Drinkwaard 1999).

Ships as vector
Transfer of H. japonica by ships, e.g. in ballast water, is an alternative to transport by oyster imports.When completing the experiment with survival of thallus parts in darkness and under temperature stress 86 % of the uniseriate pseudolateral fragments of H. japonica had normal pigmentation and were recorded as alive in one beaker, and 63 % in the other.One of the two main shoots was without normal pigmentation in any part, while the other had normal pigmentation in several side shoots when completing the experiment (Figure 2).The viability was confirmed by a resumption of growth in fragments after two weeks in culture conditions (Figure 2).

Introduction of H. japonica to Europe
In order to assess possible vectors involved in the introduction of a species it is crucial to know the donor region of the species in question.Outside Europe H. japonica is reported from Korea (Choi 2001), China, Japan, Russia, Alaska and California (according to references in the database AlgaeBase, Guiry and Guiry 2008).However, according to S. Lindstrom (pers.comm.),H. japonica is not present in north-west America.The north-west American H. densiuscula was called H. japonica for a period since Abbott (1972) considered these two to be conspecific (S.Lindstrom, pers.comm.).H. japonica and H. densiuscula separate clearly from each other with regard to morphology (Choi 2001), andChoi (2001) regards these as two separate species.Since H. japonica is not present in British Columbia (Canada), the introduction to Europe of this species cannot have followed from the import of oysters from British Columbia to European countries, and the donor region of the European H. japonica must be the western Pacific (Korea, China or Japan).While European material of H. japonica was genetically similar to Korean material when analysed by using cox2-3 spacer (Bjaerke 2004), no comparisons have been made between European and Chinese or Japanese material of H. japonica.
In order to assess the most likely vector for an introduction it is equally important to localize the first site of arrival.It is not known to what extent the reports of H. japonica from various parts of Europe actually reflect the time of establishment of this species in each area.Some areas have probably been more searched than others, and e.g. in France and South Europe there are several red algae with a similar habit as H. japonica, which may have made it difficult to spot the new species (Peña and Bárbara 2006).However, it is likely that the now reported first site of observation, Brittany in France, actually is the first site where H. japonica became established in Europe.Brittany was one of the main areas for import of oyster spat from Japan to Europe in the 1970s (Grizel and Héral 1991), and this import seems to be a very likely vector for the first introduction of H. japonica to Europe.There are several ways in which algae, like H. japonica, may have hitchhiked with imported oysters.Algal material may have been used as cover during transport.The oyster spat was transported to Europe partly set on shells, and a number of species may have survived transport growing on these shells.Some measures were taken to prevent introduction of new species to France, like immersion of the spat in fresh water for some time (Grizel and Héral 1991), but it is not known how efficient this was or how well this treatment was carried out.H. japonica was not registered in Brittany until several years after the main period of oyster import from Japan had stopped, but in some cases it takes time before introduced species build up large enough populations to become discovered.
Even though imports of oyster spat from Japan to France is a very likely vector for the introduction of H. japonica to Europe, we cannot rule out that the European H. japonica may have another donor region than Japan, or that there may have been multiple introductions of H. japonica from the western Pacific to Europe.The invasive kelp Undaria pinnatifida is an example of a species which may have spread with several vectors to different parts of the world (Voisin et al. 2006).In addition to its arrival in the Mediterranean with oysters (Voisin et al. 2006), U. pinnatifida has also probably spread to other parts of the world by ships (Wallentinus 2007).H. japonica may have spread in a similar way, by both oyster transfers and ships.The result of the experiment with survival during a simulated journey in ballast water shows that thallus parts and fragments of uniseriate pseudolaterals of H. japonica are capable of surviving in total darkness and under temperature stress for a considerable amount of time, at least as long as a journey by a bulk carrier takes from Korea to Le Havre.A high survival of H. japonica sporelings after 40 days in darkness was also observed by Bjaerke and Rueness (2004).Survival of fragments and small thallus parts is sufficient for successful establishment of H. japonica at a site of arrival, since uniseriate pseudolateral branches of this species show great ability to regenerate at a variety of temperatures (Husa and Sjøtun 2006).
However, the experiment simulating a journey in ballast water only takes temperature stress and light deficiency for algal fragments in ballast water into consideration.Other types of stress, e.g.burial of algal fragments in sediments, variation in salinity or hypoxic or even anoxic conditions, may be more important than temperature and light in limiting transportation success of live algae by ballast water.On the other hand, Flagella et al. (2007) did not find the physical conditions in ballast tanks they sampled to be lethal for macroalgal species, and David et al. (2007) identified as many as 120 early macroalgal stages per litre ballast water in ships in a Mediterranean harbour.

Spreading of H. japonica in Europe
About 25 years after the first observation of H. japonica in Europe it is widespread in both south and north Europe.H. japonica is found in a variety of marine habitats, but does not tolerate much wave-exposure (Husa et al. 2004) and grows poorly at 15 psu or below (Bjaerke and Rueness 2004).It grows abundantly as an epiphyte, on any type of solid substratum, or it can grow loose on soft substratum (Husa et al. 2004).Thus, there are few stretches along the European coasts where the physical conditions would be unfavourable for establishment, once the species has arrived.
H. japonica may spread naturally, by spores or fragments carried by currents, or it may spread by growing attached to loose buoys or other drifting items.However, H. japonica has spread over large areas in relatively short time in Europe.It has also spread countercurrent in some places, e.g.southwards and eastwards along the Norwegian and southwards along the Swedish west coast.This suggests that some direct human-mediated secondary spread of the species is most likely in these cases.
After the import stop of Pacific Oysters from Japan in 1982, oysters have frequently been transferred between many countries within Europe.H. japonica may have spread to Galicia (NW Spain) during the 1980s by ships or oyster transfers.Galicia is one of the main oyster cultivation areas in Spain, and Spain has been importing oysters from France from the 1980s onwards (Iglesias et al. 2005).The establishment of H. japonica in Ireland may possibly have been a result of oyster imports from France during the 1990s, since the site where it was found is close to an oyster farm, and in addition relatively little boat traffic connects Ireland with the English Channel and Spain.The oyster cultivation in the Mediterranean is likely to have caused the spreading of H. japonica between the Atlantic and the Mediterranean coasts, since the process of oyster cultivation in France involves frequent transfers of oysters between the Mediterranean and the Atlantic coast (Mineur et al. 2007b).In addition, one cannot rule out that H. japonica may have spread into the Mediterranean as a result of transport of other bivalves than oysters for cultivation (see e.g.Breber 1985).
H. japonica is reported to grow well in oyster beds in The Netherlands and in the Mediterranean (Haydar and Wolff 2003;Mineur et al. 2005).The fact that oyster beds represent a good habitat for H. japonica does not necessarily mean that oysters are the vector for introduction to the area, even though it cannot be ruled out in the cases where H. japonica has first been recorded in an area close to an oyster cultivation site.The first record of H. japonica in The Netherlands was in 1994 in an empty oyster farm, and it was suggested that oyster transfer was the vector of introduction (Maggs and Stegenga 1999).However, the import stop of oysters from France to The Netherlands as early as in 1981 (Drinkwaard 1999) makes oyster imports a less likely vector for the introduction of H. japonica to The Netherlands.H. japonica can also have spread naturally northwards along the coast from Brittany, but the fact that it is not recorded in Belgium suggests that there has been a vector involved for the spreading to the North Sea.
An alternative to transport with oysters in the North Sea is transport of H. japonica by other types of shellfish or by ships.The coasts around the North Sea, specially the south and southeast part including the western part of the Baltic Sea have busy shipping lines.The fact that H. japonica is not yet recorded in the Baltic Sea, other than along parts of the Swedish west coast, is probably due to a lethal salinity limit of 10 psu to the species (Bjaerke 2004).The spreading of H. japonica along the Norwegian southwest coast and along the Swedish west coast is most likely facilitated by boat traffic, since this is a busy shipping area of cargo, fishing and leisure boats.Along the Norwegian coast H. japonica has been observed to spread very rapidly (Husa et al. 2004).The high rate and pattern of spreading within Norway suggest that the local spread is most likely due to boat traffic along the coast (Husa et al. 2004).Bjaerke (2004) analysed samples of H. japonica from several places along its area of distribution in Norway by the AFLP fingerprint method.She found different groupings and high similarity between the northernmost and the southernmost samples, suggesting both multiple secondary introductions into Norway and spreading from site to site along the coast.
H. japonica may spread short distances by ship's traffic in a number of ways, e.g.attached to the hull of ships or growing on ropes, or other types of equipment transported by ships, as well as on buoys, as fragmented thalli in ballast water or other water bodies transported by ships e.g. for transportation of fish, or entangled in different types of fishing gear or equipment used in aquaculture.Under stressed conditions H. japonica is easily fragmented, and the fragments have a high rate of survival and regeneration after exposure to high environmental stress (Husa and Sjøtun 2006).However, since H. japonica has a relatively fragile thallus and is e.g.not found in waveexposed areas, it is not very likely to have spread over long distances attached to the hull of ships.On the other hand, in a study of benthic algae growing on hulls of ships arriving in Mediterranean harbours a number of small and delicate species in the order Ceramiales were observed (Mineur et al. 2007a).
H. japonica may also have spread by ballast water within Europe.The first recording of H. japonica in North Europe was from a site close to Bergen (Lein 1999), which is one of the main ports in Norway.A study of origin of ballast water from ships arriving at the oil terminals around Bergen showed that the main part of the ballast water came from ports between North France and Germany (Dragsund et al. 2007).However, not far from the locality where H. japonica was first observed in Norway, an oyster producer was keeping imported oysters from south Europe in tanks during the 1990s, with water running from the tanks directly to the sea (S.Mortensen, pers. comm.).If H. japonica were growing on the oysters, fragments or spores of H. japonica would easily escape to the surrounding areas.Thus, we cannot rule out that oyster import was the vector for the first introduction to Scandinavia.This demonstrates how difficult it is to determine vectors for introductions, and also how difficult it is to limit secondary introductions.

Figure 1 .
Figure 1.Sites of first observations of Heterosiphonia japonica in different countries of Europe.Symbols show year or time periods during which observations were made.The site of the first observation in Europe is shown by a star.Dotted lines show coast lines where more or less continuous distribution has been observed.A detailed description of localities is provided in Annex 1.

Figure 2 .
Figure 2. Left: photo of Heterosiphonia japonica after 28 days in darkness and under temperature stress.Right: the development of surviving uniseriate pseudolateral fragments of H. japonica two weeks after the experiment (Photograph by V. Husa).

Figure 3
Figure 3 shows reported positions of ships in the Mediterranean and eastern parts of North Atlantic during random months in 2005 and 2006, according to the Sailwx database.The patterns of the main traffic are similar during all months.A main shipping line extends from the Suez Canal and the Mediterranean, around Portugal and into the English Channel.The coast from Brittany and northwards through the English Channel is also a very busy shipping route up to Germany and Denmark.The coast of south Norway also represents a main shipping line, and the North Sea is in general busy, specially the south and southeast part.Some areas with relatively little traffic seem to be the Bay of Biscay, the waters around Ireland and the western and northern part of the UK.In the Mediterranean the busiest shipping lines appear to be on the Mediterranean coast of France and Spain.

Figure 3 .
Figure 3. Data from the web page Sailwx showing positions of ships in European waters (see Figure 1 for names) during 4 random months in 2005-2006.
Annex 1. Recordings of Heterosiphonia japonica in Europe.The year of first observation in various countries is given.Information is based on personal communication, unpublished data (V.Peña and I. Bárbara) and publications.Available information about reproductive status of Heterosiphonia japonica in samples is included.* shows year of first recording in a country, ** indicates an oyster cultivation farm nearby.