Northward expansion of the invasive green algae Codium fragile spp . fragile ( Suringar ) Hariot , 1889 into coastal waters of Newfoundland , Canada

Invasive green alga Codium fragile spp. fragile (Suringar) Hariot, 1889 thalli were first detected in Newfoundland (Canada) within Mortier Bay (southwestern Placentia Bay) in August 2012. Subsequent SCUBA surveys in 2013 found Codium attached to subtidal substrates in Arnold’s Cove (northeastern Placentia Bay) and on the northern coast of central Newfoundland near Pilley’s Island (Notre Dame Bay), which is currently the most northern location of Codium in Atlantic Canada. Due to large distances between confirmed sightings, further surveys are recommended to determine the distribution range of Codium and its possible dispersal vectors in Newfoundland coastal waters. Implications of an invasion by Codium on coastal Newfoundland ecosystems are yet to be determined but potential concerns include the replacement of native kelp species, as previously documented elsewhere in Atlantic Canada.


Introduction
Non-indigenous species represent an increasing global threat to coastal marine biodiversity and community structure (Ruiz et al. 1997;Cohen and Carlton 1998).The green, branching macroalga, the oyster thief, Codium fragile (Suringar) Hariot, 1889 (hereafter called Codium) is a notorious, global, invasive seaweed (Trowbridge 1998).In coastal ecosystems, Codium can influence biodiversity and community structure by outcompeting native seaweeds and exploiting open space created by local disturbances within native kelp beds (Scheibling and Gagnon 2006).Although Codium has mostly been studied in association with rocky shores and attached to hard substrates (including shellfish), recent research has documented Codium thriving attached to eelgrass (Zostera marina Linneaus, 1753) in sandy habitats (Drouin et al. 2012).In the shellfish aquaculture industry, Codium can foul gear and product (e.g.oysters or mussel lines) generating increased labour and impacting the movement of seed stock or product (Bird et al. 1993).Such opportunistic fouling, in addition to various reproductive strategies (e.g.sexual, asexual, and vegetative) (Mathieson et al. 2003), and capacity to disperse long distances (Watanabe et al. 2009) contribute to the invasive success and ecological and economic concerns associated with Codium.
Codium is native to the northwest Pacific but invaded coastal European waters by 1900 (Van Goor 1923 cited by Silva 1955) and was first reported near Long Island, New York (United States) in 1957, where it was likely introduced through transfers of shellfish product (Bouck and Morgan 1957;Malinowski and Ramus 1973;Carlton and Scanlon 1985).Cold waters were once thought to limit the spread of Codium into Atlantic Canada (Bird et al. 1993).Nevertheless, it has since spread into the Gulf of Maine (Mathieson et al. 2003) and Nova Scotia (Canada) since 1989 (Bird et al. 1993;Scheibling and Gagnon 2006), and along the Gulf of St. Lawrence coast since 1996 (Garbary et al. 1997).In fact, at many sites along the Atlantic coast of Nova Scotia native kelps and other seaweeds have been replaced by Codium as the dominant canopy species (Scheibling and Gagnon 2006).Morphological and molecular evidence suggests that two subspecies of Codium fragile are present in Atlantic Canada, ssp.atlanticum (Cotton) Silva and ssp.fragile (formerly referred to as ssp.tomentosoides [Van Goor] Silva) (Hubbard and Garbary 2002;Kusakina et al. 2006).The distribution and morphology of Codium has been well described in southern Atlantic Canada (e.g.Hubbard and Garbary 2002;Kusakina et al. 2006;Lyons and Scheibling 2009), but this paper describes the first observations of Codium in Newfoundland (at its current northern limit in the northwest Atlantic).
Surveys to detect non-indigenous species in coastal Newfoundland waters have been conducted since 2006 by the Department of Fisheries and Oceans (Canada) Aquatic Invasive Species (DFO AIS) Program in collaboration with the Department of Ocean Sciences, Memorial University of Newfoundland (MUN) (Callahan et al. 2010).In total 231 locations, primarily harbours, throughout Newfoundland have been surveyed for nonnative, and in particular aquatic invasive species (C.McKenzie, unpublished data).Site selection was based on previous reports of aquatic invasive species, areas expected to be of high risk to introductions (e.g.high boat traffic and low wave energy), and consideration of local ecological knowledge.

Materials and methods
The Newfoundland DFO AIS program conducted surveys comprised of shoreline and underwater surveys (by SCUBA divers from DFO and MUN), including transects conducted perpendicular to shore to assess biological communities, along with visual inspections of wharf structures, boat hulls, other artificial materials, and natural subtidal and intertidal substrates.Following detection of Codium, divers conducted further visual surveys (20 min) to assess cover of Codium in the area.Analyses of underwater video determined percent cover of Codium within 10 m sections along each transect.Temperature data were collected from 2010 to 2013 from Arnold's Cove (Ma 2012), Little Bay, Come by Chance (Smart Bay 2013), and Notre Dame Bay to provide preliminary comparisons of environmental conditions between locations.
To determine the subspecies of C. fragile in Newfoundland we considered indicative features such as the size and shape of the utricles and mucron.Divers collected Codium specimens from Arnold's Cove and transported them in seawater to the Northwest Atlantic Fisheries Centre (St.John's, Newfoundland).Thalli were maintained at 5°C prior to analyses and 13 morphometric (4 macroscopic and 9 microscopic) traits were measured on ten thalli (based on Hubbard and Garbary 2002).Utricles were sampled approximately 2 cm from the tip of the plant (based on Trowbridge 1998; Hubbard and Garbary 2002) and five utricles were measured from each plant (one tip).Unfortunately, Codium specimens collected earlier from other locations were frozen upon collection, which affected cell structure and decreased accuracy of morphological measurements.Features were assessed qualitatively for these collections.

Distribution of Codium in Newfoundland
Thalli of Codium were detected washed onshore in Mortier Bay,Newfoundland,in Spanish Room (47.195ºN,55.075ºW)on 22 August 2012, and small fragments (< 10 cm) of Codium washed onshore Woody Island (47.784ºN, 54.180ºW) were collected on 27 September 2012 (Figure 1).No Codium was detected in underwater transects or during rapid dive surveys at 18 locations surveyed throughout Placentia Bay between August and October 2012.Follow-up targeted surveys discovered thalli attached to the subtidal seabed in Mortier Bay in Marystown (47.166 ºN, 55.148 ºW) on 27 November 2012 and Little Bay (47.164ºN, 55.115ºW) on 6 June 2013 (approximately 5 and 7 km from Spanish Room, respectively) (Figure 1).In Marystown, divers observed scattered plants at < 7 m depths.In 2013, seawater in Little Bay ranged from 1.2 C (March) up to 18.1 C (August), although March was the earliest temperature data recorded in 2013.
In 2013, DFO divers detected sparsely distributed (i.e.no meadow formation evident) Codium near the public wharf in Arnold's Cove (47.756ºN, 53.988ºW) on 30 August 2013 (Figure 1).Average (± SE) cover of Codium across all transects (8 in total) at two sites in Arnold's Cove conducted on 1 October 2013 was estimated at 1.30 ± 0.24 % and was at most 3.26 ± 0.85 %, which occurred between 60-70 m along the transect for both sites.Most (> 75%) of the Codium at Arnold's Cove  S1.
was unattached; however, thalli were observed attached to anthropogenic debris (e.g.bottles), horse mussels (Modiolus modiolus Linneaus, 1758), sea scallops (Placopecten magellanicus Gmelin, 1791), the shell of a hermit crab (Pagurus sp.), other shell remains, the holdfast of a native kelp (Saccharina longicruris Kuntz, 1891), and small rocks.When Codium was observed attached to horse mussels, it most often attached to the posterior end of the mussel, which protruded from the sediment while it was attached by byssal threads to small rocks below the sediment surface.
Seawater temperature was 14.4°C during August surveys, but ranged between 0.

Morphology of Codium
Thalli from Arnold's Cove were irregularly dichotomous and grew up to 69 cm high (Figure 2, Table 1).Microscopically, utricles were long with a slight constriction near the centre and mucrons on the top of utricles were long and pointed, characteristic of Codium fragile ssp.fragile (Figure 2).The microscopic characteristics examined showed similarities to specimens from more southern locations in Atlantic Canada (Hubbard and Garbary 2002)  So far, Codium has not formed dense stands in subtidal regions in Newfoundland comparable to those recorded along the east coast of Nova Scotia (Canada) (Mathieson et al. 2003).However, areas with high numbers of thalli (i.e.Arnold's Cove) suggests close proximity to a point of initial establishment and a source from which new populations may spread within Newfoundland.In Placentia Bay, a counter clockwise gyre can disperse Codium in a southwest direction (Ma et al. 2012) Buoyant fragments of Codium can be influenced by environmental variables (such as light intensity) and may play an integral role in long-range dispersal of this alga (Gagnon et al. 2011).
Although Codium can disperse up to 70 km yr -1 (Trowbridge 1998), the large distance between Arnold's Cove and Mortier Bay, combined with the high number of drift thalli washed onshore Spanish Room in 2012, suggests that undetected populations may exist nearby.Further surveys are required to determine the precise distribution of Codium in Placentia Bay, particularly between Arnold's Cove and Mortier Bay.The proximity of known Codium populations to areas of high boat traffic or aquaculture activities suggest that anthropogenic vectors may have contributed to Codium transfer within Placentia Bay and from Placentia Bay to the northern coast of central Newfoundland (i.e.Pilley's Island).
The global spread of non-indigenous species has been accelerated by human-mediated pathways including ballast water exchange, vessel hull fouling, recreational boating, aquarium trade, and movement of aquaculture gear and product (Cohen and Carlton 1998;Bax et al. 2003;Blakeslee et al. 2010;Clarke Murray et al. 2012).International translocation and domestic range discontinuities of Codium have most likely resulted from transport through fouling of ship hulls or shellfish and use as packaging for transport of product (Carlton and Scanlon 1985;Trowbridge 1998) Codium can tolerate a wide range of environmental conditions including temperatures from -2 to 33°C, salinities from 12 to 40, and relatively short periods (< 6 h) of desiccation and exposure to freshwater (Trowbridge 1998;Kim and Garbary 2007).The establishment of Codium in Arnold's Cove and near Pilley's Island suggests that Codium on both north and south coasts has survived at least one Newfoundland winter and, in the case of thalli near Pilley's Island, considerable ice cover.However, populations in the southern Gulf of St. Lawrence also survive under extended periods of ice cover every year thus presence of ice, in itself, does not appear to be a limiting factor.Therefore, further establishment of Codium in Newfoundland is unlikely to be precluded by seawater temperatures, although growth may be slower at lower water temperatures.Studies in the northwest Atlantic have observed optimal growth at 24°C (Trowbridge 1998), but minimal growth thresholds have been reported between 10 and 13°C (Malinowski and Ramus 1973) and limited growth at 6°C (Hanisak 1979).For example, a field study in Nova Scotia demonstrated that Codium growth peaked at 5.6 cm month -1 in August-September in water temperatures > 20 °C, but was only 2.6 cm month -1 in May-June in water temperatures averaging 11°C (Bégin and Scheibling 2003).Based on temperature data from Placentia Bay, it is likely that Codium may grow more slowly in Newfoundland than in some southern Atlantic Canadian locations.Therefore, the largest plants observed in Arnold's Cove (up to 69 cm in height) likely grew over two or three years.Although, fragmentation during colder months (Fralick and Mathieson 1972) may extend the time required for Codium buds to become large branched fronds.Seawater temperatures in the most northern regions of Newfoundland only reach 10 to 15°C and occur for short and variable intervals (Caines and Gagnon 2012).Therefore, we suggest such conditions may lead to truncated periods of growth in Codium.Similarly, Codium reproduction may be limited in cold waters.Observations in the northwest Atlantic indicate gametangia are produced and released at temperatures ≥ 10°C (Trowbridge 1998); however, vegetative propagation may occur through budding and fragmentation, which can further increase the dispersal of Codium (Bégin and Scheibling 2003).Additional studies are required to determine if environmental variability limits the formation of dense stands of Codium similar to populations elsewhere in Atlantic Canada.
Research in Nova Scotia has demonstrated that Codium can opportunistically exploit breaks in kelp canopy and prevent re-establishment of native kelps (Scheibling and Gagnon 2006).Codium has also been observed to invade soft-bottom estuaries dominated by eelgrass by attaching to rhizomes (Garbary et al. 2004;Drouin et al. 2012).Such physical changes in subtidal macrophyte canopies can exert significant changes to benthic communities, including limitation of movement and recruitment of fish and invertebrate species inhabiting kelp and eelgrass ecosystems (Levin et al. 2002).However, the invasion of Codium may also positively affect biodiversity as research by Drouin et al (2011) demonstrated increases in density and diversity of faunal assemblages within eelgrass beds invaded by Codium.
Invasions of Codium can impact the shellfish aquaculture industry (Bird et al. 1993).Settlement of Codium may reduce filtering in bivalves, affect growth and survival, foul equipment and raise cleaning costs, and introduce risks of further transport and spread of Codium, which can lead to novel protocols and permit restrictions (Fralick and Mathieson 1973;Carlton and Scanlon 1985;Bird et al. 1993).Although Codium has not been documented on aquaculture sites in Newfoundland, Codium detected near Pilley's Island is located adjacent to important mussel aquaculture sites (< 15 km).Overall, the effects of Codium in Newfoundland are currently unknown.Further sampling and research on this introduction is required to establish range boundaries and study large-scale and long-term effects of this introduction in the predominantly cold water ecosystems of Newfoundland.

Figure 1 .
Figure 1.Distribution of aquatic invasive species survey locations in Newfoundland indicating presence (open triangles) and absence (closed circles) of Codium fragile ssp.fragile.Survey locations with confirmed Codium are labelled and abbreviated (PI Pilley's Island; WI Woody Island; AC Arnold's Cove; SR Spanish Room; MT Marystown; LB Little Bay).For details see supplementary material TableS1.
8 and 16.7°C (2010 to 2011) in Arnold's Cove (Ma.2012) and attained temperatures up to 21.3 °C in August 2012 (between 2011 to 2013) in nearby Come by Chance (~ 7 km from Arnold's Cove, 47.789ºN, 54.049ºW) (Smart Bay).Seabed in Arnold's Cove was gently sloping and transects extended to depths up to 7 m.The substrate of one site was primarily mud and gravel with few scattered rocks, while the substrate at the second site was bedrock and boulder nearshore and transitioned to sand and small cobble (< 10 cm diameter) along each transect.Native kelp (S. longicruris) was the most common macroalgae, followed by Codium.Other benthic vegetation observed included brown algae (Laminaria digitata [Hudson] J.V. Lamouroux, 1813, Desmarestia sp., Ascophyllum nodosum (Linnaeus) Le Jolis, 1863, Fucus sp., Colpomenia peregrina Sauvageau, 1927), coralline algae, eelgrass (Z.marina), and various red and green (e.g.Ulva sp.) algae.

Figure 2 .
Figure 2. Photographs of Codium fragile ssp.fragile from Newfoundland showing (A) Codium attached to subtidal seabed in Arnold's Cove, (B) an example of dichotomy of drift thalli collected from Spanish Room, (C) utricle of Codium showing gametangium and pointed mucron, and (D) a close view of long and pointed mucron on top of utricle.Photographs by P. Sargent (A), T. Wells (B), and K. Matheson (C and D).

Table 1 .
Macroscopic and microscopic morphometric data of Codium (n=10) collected from Arnold's Cove (Newfoundland) with comparison to morphometric data of diagnostic features of C. fragile ssp.fragile collected from more southern locations in Atlantic Canada (from Hubbard and Garbary 2002).Divers from MUN discovered Codium along the northern coast of central Newfoundland near Pilley's Island (Notre Dame Bay; 49.477ºN, 55.725ºW) on 23 September 2013 (Figure1).Thalli were sparsely distributed and attached to an otherwise barren bedrock platform at depths up to 7 m.Although previously recorded temperature data near this location is limited, in 2004, sea water temperatures reached a high of 19.2°C (August) in Flat RockTickle (49.484ºN, 55.735ºW).
. Such a current suggests it is not likely that plants found onshore Mortier Bay drifted from nearby islands of St. Pierre and Miquelon (~ 90 km southwest of Mortier Bay), where Codium was first detected in 2009 (F.Urtizberea, pers.com.).Instead, if Codium in Arnold's Cove is the introduction source within Placentia Bay, a counter clockwise current may have contributed to dispersal of Codium to Woody Island (approximately 15 km west of Arnold's Cove) and Mortier Bay (i.e.Little Bay and Marystown, approximately 120 km southwest of Arnold's Cove).
. The presence of Codium in locations with relatively high vessel activity (i.e.Arnold's Cove and Mortier Bay) and the large geographic discontinuities between populations in Newfoundland (e.g.Placentia Bay and Pilley's Island) suggest that anthropogenic pathways may have contributed to introductions and subsequent distribution of Codium in Newfoundland.In fact, Arnold's Cove is a high risk location for non-indigenous species in Newfoundland exemplified by established populations of golden star tunicate (Botryllus schlosseri Pallas, 1766), European green crab (Carcinus maenas Linnaeus, 1758), coffin box bryozoan (Membranipora membranacea Linneaus, 1767), and sea potato (Colpomenia peregrina Sauvageau, 1927).Recent surveys have discovered these species in Mortier Bay (minus sea potato) (C.McKenzie, unpublished data), along with the detection of vase tunicate (Ciona intestinalis Linneaus, 1767 Type B) in 2012 (Sargent et al. 2013).Vessels from Mortier Bay often travel to other regions within Newfoundland, other Atlantic Canadian provinces, and to St. Pierre and Miquelon.Therefore, additional surveys are required to document secondary spread of Codium to nearby locations.