Distribution of the invasive tunicate Styela clava in Long Island Sound, New England, USA.

The clubbed tunicate Styela clava (Herdman, 1881) is an invasive, solitary, club-shaped organism native to the western Pacific. S. clava has spread far beyond its native range into temperate waters worldwide, including those of Long Island Sound (LIS) on the northeastern coast of the United States. A visual inspection of public and private commercial and recreational marinas and docks located along the shores of LIS was conducted between June 2011 and September 2013. The wrack lines of public beaches and other public coastal areas were also surveyed. S. clava was found at 77.8 % of the sites surveyed indicating it has now fully colonized the Sound. It is strongly suggested that managers of neighboring waters be vigilante, and be prepared to take steps to mitigate negative impacts, should the species spread further south along the Atlantic Coast of the United States.


Introduction
The clubbed tunicate Styela clava (Herdman 1881) is an invasive, solitary, club-shaped tunicate native to the marine and estuarine waters of the western Pacific from the Sea of Okhotsk southward along the coasts of Japan, Korea, and China to Shanghai (Cohen 2005;Darbyson et al. 2009).Today, S. clava can be found in temperate marine waters worldwide, including those of Asia, Europe, North America, Australia and New Zealand (Goldstien et al. 2011).The adult tunicate is easily identifiable by its brown, leathery, outer covering and the two siphons that protrude from the top of the tunic (Figure 1).Although considered a solitary tunicate, it is often found in extremely dense clusters consisting of thousands of individual organisms (Osman and Whitlatch 2000;Kluza et al. 2006).These dense clusters may foul lines, buoys, pipes, oyster cages, docks and other submerged natural or artificial objects and can become serious concerns in regions where Styela has invaded (Arsenault et al. 2009).Given that Styela has few known natural predators and is an effective filter feeder, the introduction and spread of this species into an area may result in deleterious effects to the native species and cause serious economic consequences for boaters, aquaculture businesses, and marinas.
The spread of S. clava outside of its native range is thought to have begun in the 1920s in Newport Bay, California, although it was not formally identified there until 1932 (Abbott and Johnson 1972;Cohen 2005).From Newport Bay, it spread along the eastern Pacific coast of North America and is now found from Mexico north to Canada (Clarke and Therriault 2007;BNZ 2008;Cohen 2005).S. clava made its first appearance in Great Britain in the 1950s (Carlisle 1954) where it is assumed to have been transported on ships returning from Korea.From there it spread into the waters of Denmark and France in the 1960s, Ireland and the Netherlands in the 1970s, and Belgium, Germany, and Spain during the last decades of the 20 th Century (Clarke and Therriault

2007
).In the early 1970's, S. clava was reported in New York waters (Berman et al. 1992) although this appears to have been an isolated occurrence.It was next observed in Beverly, Massachusetts, in the 1970s (Berman et al. 1992), and in Rhode Island, New Hampshire, and Maine in the 1980s (Karney and Rhee 2009).By 1998, S. clava had entered the waters around Prince Edward Island, Canada (Clarke and Therriault 2007).
There are some disagreements as to when S. clava actually reached Long Island Sound (LIS), a body of water located between the states of Connecticut (CT) and New York (NY).According to Whitlatch et al. (1995) and Berman, et al. (1992), S. clava reached the eastern end of LIS in 1973 while Clarke and Therriault (2007) suggest that it did not reach LIS until the 1990s.Nevertheless, by the year 2000, large populations of S. clava could be found fouling docks and lines in Groton, CT (Figure 1; Osman andWhitlatch 2000, Fofonoff, et al. 2003).Pederson et al. (2005) identified S. clava at three additional sites within LIS (Mystic, CT, Milford, CT, and Stamford, CT) and in the Peconic Bay estuary (Jamesport, NY and Greenport, NY), which is located at the eastern end of Long Island, NY.These sightings have resulted in LIS being classified as a site where S. clava is considered introduced but not fully colonized (Fofonoff et al. 2003).Anecdotal evidence suggests that S. clava has spread to other sites in the Sound over the past decade (Brunetti and Cuomo, personal observations).This study was undertaken in an effort to document the distribution of S. clava within LIS in order to better understand the present status of its invasion and to identify the most likely vectors by which it is spreading in the Sound.

Methods
A visual inspection of public and private commercial and recreational marinas and docks located along the shores of LIS (Figure 3) was conducted between June 2011 and September 2013 beginning in easternmost Connecticut, extending west to New York City, and east again along the southern coast of LIS to Mount Sinai, New York.Marinas were chosen based on accessibility and willingness to allow data collection and represent a range of sizes and types of users.The wrack lines of public beaches and other public coastal areas were also surveyed for the presence of S. clava.A total of 45 sites were surveyed within 25 towns: 29 sites in Connecticut and 16 sites in New York (Table 1; Figure 3).
Data recorded at the time of the survey included: location, presence/absence of Styela, average size of Styela when present, and average density of Styela when present.A visual inspection was made of submerged lines, the underside of docks, submerged marine equipment, and wrack lines for the presence of S. clava.When found, individuals were measured and, where possible, density was determined within a 60 cm diameter circular area using a single target organism as a centroid.Abundances were classified as High when > 50 organisms were found, Low when < 10 organisms were found, and Medium when 10-50 organisms were found.Multiple measurements were taken at each site and the average abundance per site was recorded.In addition, marina owners, workers, and patrons were shown photographs of S. clava and interviewed as to whether or not they had ever seen them on lines or docks in the vicinity

Results
The Connecticut sites covered approximately 110 miles of coastal area while the New York state sites covered approximately 260 miles of coastal area.S. clava was found at 77.8% (35 out of 45) of the sites surveyed (Table 1) and in 97.8% of the towns surveyed (Table 1; Figure 4).It was common for S. clava to be found at one marina in a town but not at an adjacent marina in the same town (Table 1).Three of the recorded Styela observations were made with organisms that had been washed ashore onto beaches in Connecticut (Table 1).Therefore, abundance data was not recorded for these three sites as the actual attachment sites for Styela in these areas was not observed.The greatest abundance of S. clava was recorded at three sites located in Stonington and Groton in eastern Connecticut and at one site, Port Jefferson, in New York State (Figure 4).Abundance of S. clava was classified as low at 20 sites while 9 sites contained medium abundances.
The average size (Table 1) of individual Styela at 29 sites was > 5 cm and these should be considered as populations with reproducing adults.

Discussion
This study demonstrates that S. clava has fully invaded the LIS estuary.These tunicates can be found on both coasts of LIS, and they appear to have spread to all of these areas within the past  transport of Styela larvae into LIS is less likely owing to their short (~ 48 hour) larval phase (Clarke and Therriault 2007); however, it cannot be completely discounted.
Transport of adult Styela on lines, ship hulls, cages, and other moveable marine equipment is thought to be the primary long-distance mode by which S. clava spreads from one region to another (Darbyson et al. 2009).If adults are transported to an area favoring their survival and reproduction, they are able to establish new populations there, which, in turn, grow and expand within the local area via larval settlement.The spread of Styela in this manner should result in spatially isolated  populations throughout a region.Such populations should be associated with recreational and commercial vessels, marinas, docks, and pilings.The distribution of Styela populations within LIS appears to reflect this pattern of spatially isolated populations associated with marinas and docks throughout LIS.A close examination of the data reveal that even within marinas located in the same town, Styela occurs at one and not at the other supporting the idea that Styela colonization of areas within LIS has mainly been due to adult transport.
The general flow of surface currents in LIS is from East to West along the North shore of the Sound and West to East along the South shore of the Sound (Codiga and Aurin 2007).If Styela was simply spreading westward via larval dispersal from the Groton, CT area, one would expect it to be spreading relatively linearly, in a westward direction, along the north shore of the Sound.As well, one might expect denser populations to be found in the northeastern and north-central portions of the Sound corresponding to the length of time a colony was established.The less dense populations and possibly isolated individuals would be more typical of the northwestern and southern regions of the Sound.The present distribution and abundance of S. clava in LIS supports the concept that the spread of Styela along the north shore of LIS was the result of simple larval dispersion from the initial adult population located in Groton, CT.Similarly surface current transport of larvae from West to East might explain the distribution of S. clava along the south shore of LIS.Surface current transport of larvae, however, does not totally explain the high abundance of Styela found in Port Jefferson, NY, at a ferry terminal that is situated at the mid-point, of the sites located along the south shore of LIS.The number of occurrences of Styela at marinas observed in this study and the relative paucity of Styela observed on natural rock outcrops, along with the fact that Styela often was found in one marina but not in an adjacent marina, points to the potential importance of human-mediated transportation of Styela spread in the Sound.At this point, it appears highly likely that both vectors -larval transport and adult transport -are responsible for the spread of S. clava throughout the Sound.
Regardless of the vector, S. clava now appears to be firmly established throughout LIS although most populations have not yet reached the fouling densities (> 1000/m 2 ) found elsewhere (Minchin and Duggan 1988).High fouling densities of Styela are associated, worldwide, with negative impacts on native populations of filter-feeding organisms and fouling of docks, lines, pipes and marine-related equipment (Clarke and Therriault 2007).Dense populations of Styela rapidly deplete local waters of plankton, leaving little behind for native filter-feeders in the region, including many commercially important bivalves (LeBlanc et al. 2007).Additionally, massive Styela populations also out-compete native species for space (Stoecker 1978).At present, few dense Styela populations have been identified in LIS (this study).Whether or not the existing LIS populations of Styela will ever develop into massive populations remains an open question.At present, the colony density does not appear to pose a threat to native filter-feeding organisms nor to have significant economic impacts in the region.
The invasive tunicate S. clava has fully invaded the LIS estuary of the United States.Given that the known temperature tolerances for breeding populations of S. clava range from 15 to 23°C (Buizer 1980;Cohen 2005;Minchin et al. 2006;Global Invasive Species Database 2009;NIMPIS 2013), LIS might be expected to represent the southernmost area likely to be fully colonized along the east coast of North America.However, there have already been sightings of S. clava in the coastal waters of New Jersey, suggesting that S. clava may be able to invade even further south along the east coast of the United States.Preliminary work suggests that adult Styela may, in fact, be able to tolerate temperatures as high as 30°C (Cuomo, unpublished data) although whether or not adults can reproduce at these temperatures is unknown.It is strongly suggested that managers responsible for LIS waters, as well as those of neighboring waters, closely monitor the status of the populations within the Sound and be prepared to take steps to mitigate negative impacts should the populations become dense or spread further south along the Atlantic Coast of the United States.

Figure 2 .
Figure 2. Historical records of Styela clava in Long Island Sound (data obtained from MIT Sea Grant College Program (MITSG), Massachusetts Bays National Estuary Program, the U.S. Environmental Protection Agency (EPA) and the U.S. Geological Survey).
20 years.The spread of S. clava within the Sound raises several questions: a) what are the main vectors responsible for its spread; b) how much of a threat does the presence of S. clava pose to other organisms living within LIS; c) what are its potential economic impacts on the LIS region; and d) what is the likelihood that this tunicate might spread further south along the eastern Atlantic coast of the United States?Once S. clava is established, its expansion and development within that region is accomplished via both larval dispersion and adult transport.Larval dispersion requires an adult breeding population, temperatures within the range of 15 to 23°C (Buizer 1980; Cohen 2005; Minchin et al. 2006; Global Invasive Species Database 2009; NIMPIS 2013), salinities of 25 to 35 psu (Cohen 2005; Davis and Davis 2007; Global Invasive Species Database 2009; NIMPIS 2013), and suitable habitats for settlement.All of these conditions are met within the LIS estuary and have no doubt contributed to the spread of Styela.Ballast water

Figure 3 .
Figure 3.Long Island Sound study area with sampling sites marked by site identification number listed in Table1.

Figure 4 .
Figure 4. Relative abundance of Styela clava in Long Island Sound (by site).

Table 1 .
Location, site description, coordinates, Styela clava presence/absence, abundance, and average size of individuals for survey sites in Long Island Sound.