Effects of invasive colonial tunicates and a native sponge on the growth, survival, and light attenuation of eelgrass (Zostera marina)

We examined the effects of invasive colonial tunicates (golden star, Botryllus schlosseri; violet Botrylloides violaceus), and the native breadcrumb sponge (Halichondria panicea) on the growth, survival, and light attenuation of eelgrass (Zostera marina). Eelgrass shoot growth and survival were higher for unfouled shoots than for fouled shoots, and dependent on fouling species identity. Growth was lowest for shoots with violet tunicate fouling, and survival was lowest for shoots with sponge fouling. A large proportion (0.20-0.38) of fouled shoots marked for growth measurements were not found during retrieval compared to unfouled plants (0.08), suggesting that fouling led to premature breaking away of shoots and blades. Transmission of incident photosynthetically active radiation (PAR) through fouled blades decreased exponentially with increasing biomass of most fouling species. Relatively low biomass (1-1.5 dry mg cm of blade) of the fouling organisms reduced light transmission by up to 98 % compared to unfouled blades. The proportion of incident PAR attenuated by the sponge and orange and burgundy morphs of the violet tunicate increased hyperbolically across their biomass, and reached a plateau at ~0.5 dry mg cm-2 of blade. The golden star tunicate and cream morph of the violet tunicate attenuated incident PAR linearly across their biomass. For the range of biomass examined, all fouling species attenuated 65-95 % of incident PAR prior to it reaching the blade. The reduction in light transmission was likely the causal mechanism underlying reduced growth of fouled shoots. Fouling of eelgrass by the invasive colonial tunicates and the native sponge will have numerous ecological consequences, including reduced productivity and coverage of eelgrass beds.


Introduction
Seagrass beds are valuable coastal habitats that provide numerous important ecological functions and services, including organic carbon production, enhanced biodiversity, nutrient cycling, and nursery habitat (Heck et al. 1995;Duarte and Chiscano 1999;Heck et al. 2003;Orth et al. 2006;Schmidt et al. 2011).Over the past century, global seagrass coverage has experienced widespread declines (Short and Wyllie-Echeverria 1996;Orth et al. 2006, Waycott et al. 2009), with global loss of areal coverage outpacing gains by more than tenfold (Waycott et al. 2009).Multiple stressors on the global, regional, and local scales are responsible for loss of seagrass meadows (Short and Wyllie-Echeverria 1996;Duarte 2002;Orth et al. 2006;Waycott et al. 2009).Degradation of water quality is well documented to reduce seagrass coverage because enhanced biomass of phytoplankton, epiphytes, macroalgae, and suspended solids shade and smother seagrass plants (Hauxwell et al. 2003;Kemp et al. 2005;Walker et al. 2006).Disease, dredging, vessel grounding, and threats from global climate change (e.g., increased sea surface temperature and storm frequency, sea level rise) also have demonstrable negative impacts on seagrass coverage (Short and Wyllie-Echeverria 1996;Short and Neckles 1999;Orth et al. 2006).Additionally, new mechanisms of seagrass decline continue to emerge.In particular, recent reviews suggest that invasive species may be an important factor leading to seagrass loss (Orth et al. 2006;Williams 2007).
The rate of reported species invasions in marine ecosystems have increased exponentially over the last two centuries (Ruiz et al. 2000).In seagrass ecosystems, at least 56 non-native species have been introduced worldwide (Williams 2007).The majority of invasive species in seagrass beds are invertebrates (63 %), and include cnidarians, ascidians, molluscs, polychaetes, crustaceans, and ectoprocts (Williams 2007;Carmen and Grunden 2010).Despite the prevalence of invasive invertebrates, most studies of invasive species in seagrass beds have focused on effects of non-native macroalgae (Williams 2007;e.g., den Hartog 1997;Ceccherelli et al. 2000;Eklöf et al. 2005;Drouin et al. 2011).The few studies of invasive invertebrates in seagrass beds have found that they have predominantly negative effects on seagrass coverage either directly through plant fouling, or indirectly, through feeding or habitat formation.For example, invasive anemones (Bunodeopsis sp.Andres, 1881) and bryozoans [Zoobotryon verticillatum (Della Chiaje, 1822)] can foul eelgrass (Zostera marina Linnaeus, 1758), reducing light reaching the blades and weighing blades down causing canopy collapse (Sewell 1996).Invasive green crabs [Carcinus maenas (Linnaeus, 1758)] can reduce the survival of transplanted eelgrass shoots through feeding and bioturbation (Davis et al. 1998), while high abundances of invasive tube-building polychaetes [Neodexiospira brasiliensis (Grube, 1872)] may reduce seagrass growth (Critchley et al. 1997).
Seagrass beds in the Canadian Maritime provinces are primarily composed of eelgrass (Zostera marina), and are often characterized by well established populations of invasive invertebrates.The three most common invasive species are the European green crab (Carcinus maenas), the colonial golden star tunicate [Botryllus schlosseri (Pallas, 1766)], and the colonial violet tunicate [Botrylloides violaceus (Oka, 1927)] (Wong unpubl.data).Tunicate fouling of eelgrass blades likely has effects similar to those from epiphyte fouling: the amount of photosynthetically active radiation (PAR) reaching the blade will be reduced, and blades will be weighed down, leading to self shading and breaking away of shoots (Bulthuis and Woelkerling 1983;Brush and Nixon 2002).Tunicate fouling would thus result in reduced shoot growth, photosynthesis, and shoot survival (Short et al. 1995;Moore and Wetzel 2000), with the degree of impact dependent on tunicate biomass.Although eelgrass beds on the Atlantic coast of Nova Scotia are rarely heavily fouled by epiphytes, blades are sometimes fouled by the native breadcrumb sponge [Halichondria panicea (Pallas, 1766)] (Wong pers.obs.).Invasive colonial tunicates may thus have important ecological consequences that did not exist previously for eelgrass beds in some areas of the Maritime provinces.To better understand the effect of fouling on eelgrass by these species, we determined (i) the growth, survival, and chlorophyll a content of unfouled eelgrass blades and those fouled with the invasive colonial tunicates (golden star tunicate and violet tunicate) and the native breadcrumb sponge, (ii) the reduction in transmission of incident PAR through fouled blades across a gradient of fouling organism biomass, and (iii) the attenuation of incident PAR by the fouling organisms themselves across a gradient of their biomass.Using this knowledge, we discuss the potential ecological consequences of the invasive colonial tunicates and native sponge for eelgrass beds that usually experience minimal epiphyte fouling.

Study site and organisms
Field measurements were conducted in August and September 2010 at an eelgrass bed in Port l'Hebert, on the south shore of Nova Scotia, Canada (GPS coordinates: N43.86805, W64.96280).The eelgrass bed was located in the inner portion of the bay, which is characterized by low water currents and high sediment deposition (M.Wong, unpub. data).Percent silt content and organic content of sediments was 52.8 and 18.5%, respectively, and was determined from 6 replicate samples processed as in Bale and Kenny (2005) and Wong et al. (2011).The bed was a monospecific stand of eelgrass, with shoot densities of ~1000 shoots m -2 and percent cover of ~90 %, determined from 10 randomly placed quadrats (0.5m × 0.5m) during the experimental period.Water depth at mean low water ranged from 0 to 0.35 m, and at mean high water from 1.0 to 1.5 m.Water temperature during the field work ranged from 15 to 30°C.
Invasive colonial tunicates and a native sponge were commonly observed fouling eelgrass blades.Tunicates were the golden star tunicate and violet tunicate.The golden star colour morph was brown.The violet tunicate was present in 3 colour morphs: orange, burgundy, and cream.No actual violet colour morphs were observed.The native breadcrumb sponge was pale yellow, and formed smooth thin crusts on the eelgrass blades.Other fouling epifauna or epiphytes on eelgrass blades were usually not observed during the field work or at other times of year (M.Wong, pers. obs.).

Effects of tunicates and sponge on eelgrass growth and survival
Growth of fouled and unfouled eelgrass shoots was determined using the plastochrone method (Short and Duarte 2001).On three different dates between August and September 2010 (i.e., D1: August 20, D2: September 2, D3: September 9), unfouled shoots, and shoots with either sponge, golden star tunicate, or one of the three violet tunicate colour morphs on the leaf blades were found by snorkelling at low tide.On each date, a large sewing needle was used to mark the shoots by making a small hole through all the leaves in the middle of the sheath.Marked plants were located >1 m apart to avoid marking plants on the same rhizome.Marked plants were tagged using plastic tie-warps and flagged with surveyor tape to aid in recovery.Individually numbered flags were inserted into the sediment next to the marked plants to allow identification of the original fouling organism and the location of marked plants in case they broke free prior to harvesting.
Marked plants were harvested 19-22 days after marking.On retrieval, the number of marked plants alive and missing was recorded.Retrieved plants were transported to the laboratory and refrigerated in the dark until processing.Plants were rinsed in fresh water, each shoot taken apart at the meristem and the presence of fouling organisms and condition of the leaves (i.e., dead or alive) was recorded.To determine leaf growth per shoot, the number of new, unmarked leaves was counted, and the dry weight of the full length third leaf was determined by drying at 60°C for 24 hours.The leaf plastochrone interval (i.e., time required to produce a new leaf) was calculated by dividing the growth period by the number of new leaves produced.Leaf growth was then calculated for each marked shoot as: Leaf growth (mg shoot -1 d -1 ) = weight of 3rd leaf (mg shoot -1 ) / plastochrone interval (d) (Short and Duarte 2001).The number of shoots marked for each fouling species ranged from 10-26 on each sampling date, and depended on our ability to find appropriate shoots.Violet tunicates were not observed until the end of August, and thus were only marked on the last two marking dates.

Light attenuation of fouled and unfouled eelgrass blades
We measured the transmission of photosynthetically active radiation (PAR) through fouled and unfouled eelgrass blades using an underwater quantum sensor (LICOR,  and data logger (LICOR, LI-1400).Unfouled blades of eelgrass and blades with varying degrees of tunicate or sponge fouling were collected from the eelgrass bed by snorkeling.On shore and under natural light, the light sensor was suspended directly under the water surface in a large plastic tank.Black electrical tape was used to adjust the width of the light sensor to be similar to that of a typical eelgrass blade, allowing the sensor to be fully covered when determining light transmission through blades.For measurements of the transmission of incident PAR through each fouled blade, incident irradiance at the water surface (I 0 ) was first recorded (Figure 1).One side of the fouled blade was then gently scraped clean, the clean side submersed directly over the light sensor, and the irradiance transmitted through the blade + fouling organism (Trans b,f ) was recorded.Scraping one side clean prior to measurements was necessary to directly measure light transmitted through the blade.The fouling organism that covered the top side of the blade was then removed by gently scraping and irradiance transmitted through the blade alone (Trans b ) was recorded.The proportion of incident light transmitted through the fouled blade (i.e., blade + tunicate or sponge) was calculated as Trans b,f /I 0 .The proportion of incident light attenuated by the fouling organisms themselves (Atten f /I 0 ) was calculated as (I 0 -Trans b,f -Atten b )/I 0 , where Atten b is light attenuated by an unfouled blade (Figure 1b).The effect of scraping on light transmission was determined by comparing transmission through non-fouled blades before and after scraping.Fouled blades with patchy cover by the fouling organism were not used.
The tunicate or sponge removed for measurements of Trans b was placed in ethanol, and later dried at 60°C for 24 hours to determine dry mass per area of blade.The portion of the blade that covered the light sensor was clipped, and frozen until processed for chlorophyll a content.Chlorophyll a was extracted from the blade samples using 90% acetone, and chlorophyll a per area of blade determined by the Welschmeyer technique (Welschmeyer 1994).

Statistical analyses
To examine the effect of different fouling species on shoot growth, two-way analyses of variance (ANOVAs) with fouling species and marking date as fixed factors and leaf growth per shoot as the dependent variable were used.Because violet tunicates were not observed on the first marking date, two different ANOVAs were conducted.The first ANOVA used 3 levels of fouling species (none, golden star, sponge) and 3 levels of marking date (D1, D2, D3).The second ANOVA used 4 levels of fouling species (none, golden star, sponge, violet) and 2 levels of date (D2, D3).The three colour morphs of violet tunicate were pooled to provide enough data for the analyses.Type III sums of squares based on unweighted marginal means were used to account for the unequal number of replicates per fouling species (Quinn and Keough 2002).
We used linear least-squares regression to determine the relationship between the proportion of incident PAR transmitted through the fouled blades with biomass of the fouling organism.The proportion of incident PAR transmitted was log 10 transformed to linearize the data which showed exponential decay with tunicate or sponge biomass.Tunicate and sponge biomass were standardized by area of blade covering the light sensor.Regressions were fit to data for each fouling species separately.Separate regression lines were compared using ANCOVA, with fouling species as the categorical independent variable (5 levels: sponge, golden star tunicate, or the 3 violet tunicate colour morphs), fouling organism biomass as the continuous independent variable (i.e., covariate), and the proportion of incident PAR transmitted as the dependent variable.A significant interaction term between the categorical and continuous (covariate) predictor variables would indicate that the regression coefficients (i.e., slopes) differ among groups (Quinn and Keough 2002), meaning that transmission of incident PAR differs with fouling species (Quinn and Keough 2002).The effect of blade scraping on incident PAR transmission was determined using a one-way ANOVA, with blade scraping (3 levels: no scrape, 1 scrape, 2 scrapes) as the independent variable and proportion of incident PAR transmitted as the dependent variable.
To determine the relationship between light attenuation by the sponge or each tunicate species across their respective biomass gradient, we used non-linear least squares regression to fit the hyperbolic function: Linear least squares regression was used to determine the relationship between chlorophyll a concentration in fouled blades with tunicate or sponge biomass.Because no clear relationship existed, a one-way ANOVA with fouling species (6 levels) as the independent variable and chlorophyll a concentration as the dependent variable was conducted.For all regressions, ANOVAs, and the ANCOVA, residual plots were examined to determine if the underlying assumptions of homogeneity of variance and normality were violated (Draper and Smith 1998).If violations occurred, they were corrected by transforming the data using the square root or log 10 (Draper and Smith 1998).Significant main effects and interactions were examined using Tukey's test.All statistical analyses were done using R v.2.8.1 statistical software.

Field observations
The golden star tunicate and the sponge were visible on eelgrass blades beginning in mid-July.All colour morphs of violet tunicates were observed later, beginning in late August.The distribution of tunicate colonies and the sponge was very patchy, with larger patches becoming more prevalent at the end of September.Tunicate colonies and the sponge grew on single eelgrass blades or spread across numerous blades of neighbouring plants, and often formed mats ranging from 5 to 30 cm in diameter.Both alive and dead blades were present in the mats, and the mats weighed the eelgrass blades down towards the sea bottom.Blades were sometimes fouled with several species of organisms, but these blades were not used in our light or growth measurements.Floating eelgrass detritus on the water surface and shore often contained blades fouled with the tunicates or sponge.

Effects of tunicates and sponge on eelgrass survival and growth
The proportion of marked shoots that were retrieved and showed growth was highest for unfouled blades, followed in decreasing order by those fouled with golden star tunicate, violet tunicate, and sponge (Table 1).Large proportions (0.205-0.375) of fouled marked plants were not found during retrieval compared to unfouled marked plants (0.086).The highest proportion of marked plants retrieved that were dead were those fouled by sponge (0.333), while the proportion of dead plants retrieved that were originally fouled by golden star tunicate (0.180) did not differ from unfouled plants (0.171).Interestingly, marked plants that were originally fouled on one or more blade were not always retrieved still fouled, and no marked plants that were originally unfouled were retrieved fouled.
Eelgrass shoot growth was significantly affected by both fouling species and marking date (Figure 2).This effect was evident when the ANOVA included 3 fouling species (none, sponge, golden star tunicate) and 3 marking dates (square root transformed; fouling species: F 2,55 = 3.52, p = 0.037; date: F 2,55 = 5.49, p = 0.007), and when the ANOVA included the 4 fouling species and 2 marking dates (log 10 transformed; fouling species: F 3,50 = 8.28, p = 0.008; date: F 1,50 = 1.31, p = 0.021).For both analyses, shoot growth was significantly highest for unfouled blades, followed by blades fouled by the sponge and golden star tunicate (Tukey's test, p < 0.05).Shoot growth was lowest for blades fouled by the violet tunicate when this fouling species was included in the analysis (Tukey's test, p < 0.05).Growth of shoots fouled by golden star and sponge tended to be similar (Tukey's test, p > 0.05).Shoot growth for plants marked on D1 and D2 was significantly higher than for plants marked on D3 (Tukey's test, p < 0.05).
Chlorophyll a concentration of blades was significantly affected by fouling species (F 5,214 = 14.3, p < 0.0001; Figure 3), and was highest for blades fouled with the cream morph of violet tunicate and the unfouled eelgrass, intermediate in blades with golden star tunicate and the orange and burgundy morphs of violet tunicate, and lowest in blades with the sponge (Tukey's test, p < 0.05).Chlorophyll a concentration was not significantly affected by scraping of the blades (F 1,45 = 0.99, p = 0.324).

Light attenuation of fouled and unfouled eelgrass blades
The proportion of incident PAR transmitted through eelgrass blades fouled with invasive colonial tunicates or the native sponge decreased significantly as fouling biomass increased for all fouling species, except for the golden star tunicate where the trend was only marginally significant (Table 2a, Figure 4).With the exception of blades fouled by golden star tunicate, the proportion of incident PAR transmitted decreased exponentially across fouling organism biomass, and log 10 transformed data were used for the linear regressions.The proportion of incident PAR transmitted through eelgrass blades fouled with golden star tunicate tended to decrease linearly with increased tunicate biomass.For unfouled eelgrass blades, the proportion of incident PAR that was transmitted was 0.30 ± 0.009 (mean ± SE, n = 33).Even blades fouled with minimal biomass (<0.20 dry mg cm -2 blade) of the sponge or tunicates transmitted less incident PAR compared to unfouled blades (0.05 -0.25).When biomass of fouling organisms was >1.0 dry mg cm -2 blade, the proportion of incident PAR transmitted through the fouled blades ranged from 0.05 to 0.1.Comparison of regression lines indicated that the proportion of incident PAR transmitted through fouled eelgrass blades differed significantly among fouling species (interaction term: F 4,165 = 6.06, p = 0.00014), with regression intercepts and slopes for blades fouled with sponge and all violet tunicate morphs differing significantly (p < 0.05) from those for golden star tunicate.Scraping of eelgrass blades to remove tunicates did not affect light transmission (F 2,30 = 0.64, p = 0.534).
The proportion of incident PAR attenuated by the sponge and the orange and burgundy morphs of the violet tunicate increased hyperbolically with their dry biomass (Table 2b, Figure 5).For the golden star tunicate and the cream colour morph of the violet tunicate, this relationship tended to increase linearly.For all fouling species, the maximum proportion of incident PAR attenuated was 0.9 to 0.95 for the range of biomass examined.For the three species fit with the hyperbolic model, the proportion of incident PAR attenuated tended to plateau at approximately 0.5 dry mg cm -2 blade.

Discussion
Field observations made during our study indicated that invasive colonial tunicates and a native sponge were the main fouling organisms of eelgrass at our study site.These fouling organisms had negative effects on eelgrass growth, survival, and light transmission.Shoot growth of fouled plants was clearly reduced compared to unfouled plants.Interestingly, this effect was dependent on the identity of the fouling species, with growth lowest for shoots fouled by the violet tunicate.Also, the effects of the fouling organisms on shoot growth were dependent on the growing season of both the eelgrass and fouling species.In temperate regions, eelgrass growth is strongly influenced by seasonal fluctuations in ambient light and temperature (Robertson and Mann 1984;Hemminga and Duarte 2000).Eelgrass biomass, density, and growth on the Atlantic coast of Nova Scotia are usually highest from late May until the end of August, with declines beginning by September (Robertson and Mann 1984;Wong unpub. data).Although tunicate zooids and sponges are likely present on eelgrass blades when shoot growth is at a maximum (Carver et al. 2006), colonies are not large enough to detect until later in the season when eelgrass growth is  beginning to slow.Despite the incomplete overlap in the major eelgrass growth season with heavy fouling by the tunicates and sponge, effects of the fouling species on shoot growth were still detected.Tunicate and sponge fouling persisted well into the late fall at the field site (Wong unpub.data), and to influence eelgrass growth dynamics late in the growing season.
The most likely mechanism underlying reduced growth of fouled shoots was the reduction of incident PAR reaching fouled blades.For most fouling species, the proportion of incident PAR transmitted through the fouled eelgrass blades decreased exponentially with increasing biomass.All fouling species attenuated high proportions of incident PAR prior to the light reaching the eelgrass blades.The sponge and the orange and burgundy morphs of the violet tunicate attenuated incident PAR hyperbolically across increasing biomass.An increase in light attenuation that plateaus at high biomass of the fouling species has also been observed for epiphytes fouling blades of several seagrass species (as reviewed by Brush and Nixon 2002;Alcoverro et al. 2004;Frankovich and Zieman 2005).The exact form of the saturating function (i.e., plateau levels off or continues to increase slightly in height) can vary (Brush and Nixon 2002), and is influenced in part by how the data weight the regression (Frankovich and Zieman 2005).In our study, a thickening of the tunicate colony or sponge at high biomass caused the plateau in light attenuation, which begins at ~0.5 dry mg cm -2 of blade.At this biomass, 85 -95% of the incident light is attenuated, so further thickening contributes little to further PAR attenuation.This differs from studies of light attenuated by epiphytes, where the plateau in light attenuation at high biomass results because the excess plants tend to grow or float from the sides of the blade, and do not intercept incident PAR that can be attenuated by the leaf (Brush and Nixen 2002).In addition to a hyperbolic relationship, we also identified linear relationships between light attenuation and biomass of the golden star tunicate and the cream morph of the violet tunicate.Similar relationships have been observed for bryozoans on eelgrass blades (Glazer 1999).Linear relationships may indicate that a larger range in biomass should be sampled to better capture the plateau in light attenuation at high fouling organism biomass.Clearly, the relationship between light attenuation and biomass of fouling organisms is species specific, and depends on how the species grow on the leaves as well as their physical structure.
Our measurements of light attenuation by colonial tunicates and the sponge were highly variable compared to measurements of epiphyte light attenuation in other studies (Cebrián et al. 1999; as reviewed by Brush and Nixon 2002;Alcoverro et al. 2004;Frankovich and Zieman 2005).The irregular physical structure of the tunicates and sponge contributed to this variability.Colonial tunicates consist of a polysaccharide matrix in which individual zooids are embedded in distinct patterns (Hirose et al. 1991;Carver et al. 2006), while the sponge skeleton is interspersed with siliceous and calcareous spicules (Ruppert et al. 2003).The portion of the tunicate or sponge structure laid across the light sensor (i.e., the amount of zooids or calcareous structures vs. matrix covering the sensor) would have influenced measurements of light attenuation.It is unlikely that additional measurements would have reduced this inherent variability.Tunicate zooids were often overlapping and differed in size, and structures within the sponge integument were not visible to the naked eye.Thus, it was not possible to standardize measurements by the number of zooids or sponge structures covering the sensor.
When our measurements of light attenuation by the tunicates and sponge were compared to attenuation by erect epiphytes at similar biomass (as reviewed by Brush and Nixon 2002), the encrusting organisms in our study attenuated higher proportions of incident PAR.A similar result was found by Cebrián et al. (1999) when light attenuation of a red encrusting algae and an erect brown algae was directly compared.Generally, 1 dry mg cm -2 of blade of seagrass epiphytes attenuates 10-50% of incident PAR (Brush and Nixon 2002), which is much less than the 90-95% attenuated by the same biomass of tunicates and sponge in our study.Even a minimal amount of tunicate or sponge fouling on eelgrass will substantially affect light attenuation by the blades.The overall effect of reduced light will not only be influenced by biomass of the fouling organisms, but also by their optical properties.Examination of their optical properties using a spectrophotometer would elucidate how the tunicates and sponge change the light spectrum reaching the eelgrass blades, and in particular, would indicate if they act as neutral filter or if they are attenuating the red and violet wavelengths most harvested by eelgrass for photosynthesis (Cummings and Zimmerman 2003).
Fouling by the colonial tunicates and the sponge cause low light conditions for the fouled plants as well as for neighbouring plants shaded by the tunicate or sponge mats.Shading by fouled plants or environmental variables such as suspended sediment can reduce seagrass nutrients, growth, survival, and photosynthetic processes (Ralph et al. 2007;Biber et al. 2009;Collier et al. 2009;Ochieng et al. 2010).Eelgrass plants can acclimate to low light conditions by increasing chlorophyll content to enhance photon capture efficiency (Cummings and Zimmerman 2003;Ralph et al. 2007).However, this mechanism is not completely compensatory, because increased chlorophyll content does not scale proportionally with changes in light absorption (i.e., the "package" effect; Dubinsky et al. 1986).In our study, measurements of chlorophyll a content in eelgrass blades fouled by the cream and orange morph of the violet tunicate may have indicated an early response to light reduction, because chlorophyll a content was similar to unfouled blades yet higher than in blades with other fouling species.However, this may also simply reflect the later settlement and growth of violet tunicate colonies compared to the other fouling species.Chlorophyll a content in blades fouled with the golden star tunicate, sponge, and burgundy morph of the violet tunicate was significantly lower than in unfouled blades, suggesting blade senescence and reduced blade lifespan.
In addition to the reduction in light reaching fouled eelgrass blades, tunicate and sponge fouling also had a physical impact on eelgrass plants.The relatively low retrieval rate of marked fouled plants indicates that fouling likely caused shoots to break away.Also, many marked fouled plants were retrieved unfouled, indicating that fouled blades broke away and were replaced with new growth.Observations during plant marking indicated that shoot and blade loss were not restricted to the marked plant that was originally fouled, because the fouling organisms often spread to neighbouring shoots.The replacement of lost shoots and blades with new growth may have negative effects on bed productivity, because stored carbohydrate reserves are used that would otherwise be allocated for plant maintenance during the winter and at the beginning of the growing season (Zimmerman et al. 1995;Lee and Dunton 1997;Alcoverro et al. 2001).The fouling organisms themselves likely benefited from the breaking away of shoots and blades, because it provided a transport mechanism (i.e."rafting") allowing colonization of new areas in the eelgrass bed and of new eelgrass beds in neighbouring bays (Worcester 1994).
Numerous ecological consequences for eelgrass beds are likely when shoot growth, survival, and light attenuation are reduced by fouling from the invasive colonial tunicates and the native sponge.The reduction in incident PAR reaching fouled eelgrass blades documented in our study will lower photosynthesis within all parts of the plant (Williams and McRoy 1982;Beer et al. 1998).This will reduce the number of leaves per shoot, shoot growth rate, and shoot density in the bed (Short et al. 1995;Moore and Wetzel 2000;Ralph et al. 2007).A reduction in photosynthetic rate also impairs root functioning, because flow of oxygen to the belowground components is lowered (Hemminga and Duarte 2000).Light limitation may change the ratio of aboveground to belowground components to reduce the maintenance cost of nonphotosynthetic tissues (Olesen and Sand-Jensen 1993).The degree of impact of reduced photosynthesis on aboveground and belowground plant components will depend in part on the amount of carbohydrate reserves available for maintenance of plant metabolism (Hemminga and Duarte 2000), and how well the tunicates and sponge act as a physical barrier to gas exchange and nutrient uptake.The reduction of eelgrass primary production is not compensated by the tunicates or sponge as it is by fouling epiphytes, which often contribute significantly to the total primary productivity of seagrass beds (Moncreiff et al. 1992;Nelson and Waaland 1997;Wear et al. 1999).Clearly, fouling of eelgrass by invasive colonial tunicates and a native sponge may have numerous negative ecological consequences resulting from reduced shoot growth, survival, and light attenuation of fouled blades.The severity of these impacts depends on environmental factors controlling the population dynamics of the fouling organisms as well as the seagrass.These impacts may not have previously existed in seagrass beds that are usually free of heavy epiphyte fouling, such as those in the Canadian Maritime provinces.

Figure 1 .
Figure 1.Calculation of PAR attenuated by the fouling organisms, where (a) I 0 -Atten b -Trans b,f = Atten f , and (b) I 0 -Trans b = Atten b .I 0 = incident PAR, Atten f = PAR attenuated by the fouling organism, Atten b = PAR attenuated by the blade, Trans bf = PAR transmitted through the fouled blade, Trans b = PAR transmitted through the unfouled blade.Parameters with an asterisk indicate those directly measured with light sensor.

where
Atten f / I 0 = the proportion of incident PAR attenuated by the fouling species, M = dry mass of the fouling species, and a and b are parameters.

Figure 4 .
Figure 4. Proportion of incident PAR transmitted (log 10 transformed) through eelgrass blades fouled by invasive colonial tunicates or the native sponge across a gradient of their biomass.n = 23-44.

Figure 5 .
Figure 5. Proportion of incident PAR attenuated by the invasive colonial tunicates or the native sponge across a gradient of their biomass.n = 23-44.

Table 1 .
Survival of marked unfouled and fouled eelgrass shoots pooled across all marking dates.Data for all colour morphs of the violet tunicate were also pooled.All plants that showed growth were also still alive.