Caulerpa taxifolia in seagrass meadows: killer or opportunistic weed?

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1 Biol Invasions (213) 15: DOI 1.17/s ORIGINAL PAPER Caulerpa taxifolia in seagrass meadows: killer or opportunistic weed? Tim M. Glasby Received: 28 May 212 / Accepted: 17 October 212 / Published online: 26 October 212 Ó Springer Science+Business Media Dordrecht 212 Abstract Seagrass habitats are being lost throughout the world and the invasive alga C. taxifolia has often been implicated in seagrass declines. Although C. taxifolia can impact a variety of species, evidence for its effects on seagrasses is largely correlative. This study combined observational studies and manipulative experiments done over many years to test hypotheses about effects of C. taxifolia on two Australian seagrasses, namely Posidonia australis and Zostera capricorni. Results indicated that C. taxifolia is not having adverse impacts on the coverage of these seagrasses in the sites studied. Rather, C. taxifolia appears to be an opportunist, persisting longer and its coverage being greater in previously non-vegetated sediments than amongst seagrasses. C. taxifolia co-existed with P. australis and did not cause reductions in the cover of the seagrass. Outcomes of experimental manipulations of C. taxifolia amongst Z. capriconi were less clear due to losses of Z. capriconi in all plots, regardless of the presence of C. taxifolia. It was possible that C. taxifolia may have enhanced the decline in canopy cover of Z. capricorni, but the presence of alga did not alter the final fate of Z. capricorni. There was also no evidence that longterm areal coverage of P. australis or Z. capriconi has T. M. Glasby (&) New South Wales Department of Primary Industries, Port Stephens Fisheries Institute, Locked Bag 1, Nelson Bay, NSW 2315, Australia tim.glasby@dpi.nsw.gov.au been affected by the introduction of C. taxifolia in the embayments studied. A review of literature on effects of species of Caulerpa on seagrasses provided limited experimental evidence for negative impacts of this genus on seagrass abundance. Keywords Invasive species Caulerpa taxifolia Marine algae Seagrass Experimental Review Introduction There are numerous examples of negative correlations between abundances of introduced and native species (Parker et al. 1999; Ruiz et al. 1999; Bruno et al. 25; Simberloff 29), but there are fewer instances for which there is experimental evidence that an introduced species has caused the decline of a native (i.e. been the driver of change; MacDougall and Turkington 25). Removal experiments can be useful for testing whether an introduced species is driving ecological change (removal would result in the recovery of the native assemblage, e.g. Bulleri et al. 21), although results could be confused if impacts are long-term (e.g. recovery is slow, Glasby and Underwood 1996), or if there have been historical or multiple interacting drivers of change (Didham et al. 25). Moreover, due to varying degrees of hysteresis, recovery after the removal of an invader might elicit community responses that are quite different from

2 118 T. M. Glasby those associated with the initial invasion (Díaz et al. 23). A more direct test of impacts of invasion, therefore, is to use experimental additions of an introduced species, noting that this can potentially raise ethical concerns and impacts might still take a long time to manifest. Marine macroalgae account for some 2 % of the known introductions of marine species globally (Schaffelke et al. 26). The ecological effects of introduced marine macroalgae have been reviewed comprehensively over the last few years (e.g. Schaffelke et al. 26; Williams and Smith 27; Thomsen et al. 29) and the consensus is that there is a dearth of experimental data and hence limited understanding of their impacts. The few high profile invasive macroalgae that have been studied experimentally can have negative effects on native biota, but impacts are not always detected (Williams and Smith 27; Thomsen et al. 29). In comparison to other marine habitats, the effects of introduced species in seagrass has received relatively little attention (Williams 27). When seagrass beds are invaded, it is often by macroalgae and in situations where the seagrass beds have already been disturbed (Williams 27). Species of the genus Caulerpa are the best known macroalgal invaders of seagrass beds and one of the better studied species is Caulerpa taxifolia. Much has been written about C. taxifolia since its rise to prominence in the mid 198s. C. taxifolia is a highly invasive green alga, capable of spreading rapidly over large areas and creating dense mats (Meinesz et al. 21; Wright and Davis 26). C. taxifolia can invade sheltered or exposed marine habitats (Meinesz et al. 1993), including seagrass beds (Ceccherelli and Cinelli 1998), non-vegetated soft sediments (Jaubert et al. 1999) and rocky reefs (Bellan-Santini et al. 1996; Ceccherelli et al. 22). Beds of C. taxifolia can affect the feeding behaviour and distribution of benthic fishes (Levi and Francour 24; Longepierre et al. 25) and support assemblages of fish (York et al. 26) and invertebrates (McKinnon et al. 29; Gribben et al. 29a) that differ from those in adjacent native habitats. The recruitment of clams can be enhanced in beds of C. taxifolia relative to non-vegetated habitats, but the growth, survivorship and reproductive capacity of those clams can be reduced by the alga (Gribben and Wright 26; Wright et al. 27; Gribben et al. 29b; Byers et al. 21). Various authors have asserted that C. taxifolia can cause the regression of seagrasses (e.g. Boudouresque et al. 1995; Glardon et al. 28; Francour et al. 29; Lapointe and Bedford 21; Peirano et al. 211), but most evidence for this comes from correlative studies. Observations of Posidonia oceanica beds in the Mediterranean Sea soon after they were invaded by C. taxifolia indicated that the seagrass showed signs of etiolation, chlorosis and had reduced numbers of leaves compared to an uninvaded site (Meinesz et al. 1993;de Villèle and Verlaque 1995). After C. taxifolia invaded sites in southern California, Williams and Grosholz 22) documented considerably reduced biomass of the seagrass Ruppia maritima in patches where C. taxifolia was abundant. Manipulative experiments in Italy demonstrated that the shoot density of the seagrass Cymodocea nodosa was decreased by C. taxifolia over a period of 4 months (Ceccherelli and Cinelli 1997), but a subsequent 13 month study indicated no significant effects (Ceccherelli and Sechi 22). There is evidence that abundances of seagrasses at some sites in the Mediterranean declined prior to the introduction of C. taxifolia (Chisholm et al. 1997) and that seagrass coverage has not in fact declined since the introduction of the alga (Jaubert et al. 1999; Ceccherelli and Sechi 22). Given the limited experimental evidence for impacts of C. taxifolia on seagrasses (see Table 1), it is quite possible that the alga may, at least in some cases, be responding opportunistically to reductions of seagrass that are caused by some other disturbance. What seems clear is that it is inappropriate to generalise about any impacts of C. taxifolia on seagrasses. Caulerpa taxifolia was first discovered in the temperate waters of New South Wales (NSW), Australia, in 2 and is currently present in 13 estuaries or coastal lakes. It appears that the strain of C. taxifolia in NSW is different from that in the Mediterranean (Meusnier et al. 24), although genetic data suggest there have been several introductions of C. taxifolia into NSW (Schaffelke et al. 22). C. taxifolia was declared noxious in NSW after its appearance in isolated patches some 75 km south of native subtropical populations in Queensland, and its subsequent rapid spread (Glasby and Creese 27). Observations in NSW indicated that, as in the Mediterranean (Ceccherelli and Cinelli 1999), there were negative associations between abundances of seagrasses

3 Caulerpa taxifolia in seagrass meadows 119 Table 1 Putative effects of Caulerpa spp. on seagrasses based on mensurative studies Seagrass species Putative effect Evidence Apparent response of seagrass Reference Posidonia oceanica Posidonia australis Cymodocea nodosa Zostera noltii Zostera capricorni Ruppia maritima Halodule wrightii -ve C Shorter, narrower leaves at one site with C. taxifolia cf another without. Necrosis of leave base where C. taxifolia smothering seagrass -ve C P. oceanica leaves produced more phenolic compounds (presumed to be a stress response) at one site with C. taxifolia cf another without -ve C Longevity of P. oceanica leaves reduced where C. taxifolia was dense. At some times of sampling, shorter leaves at one site with C. taxifolia cf another without. Leaf productivity greater (thought to be a stress response) in presence of C. taxifolia n C Areal coverage of seagrass unchanged 8 years after invasion of C. taxifolia de Villèle and Verlaque (1995) Cuny et al. (1995) Dumay et al. (22) Jaubert et al. (23) n C Number of leaves similar in areas with or without C. taxifolia Dumay et al. (22) n C No indication that rhizome growth or shoot density where C. taxifolia present were worse than uninvaded sites -ve C Tendency for reduced meristematic activity in P. oceanica when mixed with C. taxifolia or C. prolifera -ve C Leaves of P. oceanica smaller (and leaf turnover enhanced) when in the presence of C. taxifolia -ve C More plagiotropic shoots (thought to be a stress response) at one seagrass site with C. taxifolia cf another without -ve C Negative associations between P. oceanica shoot density and biomass of three species of Caulerpa (C. prolifera, C. racemosa, C. taxifolia) Peirano et al. (25) Garcias-Bonet et al. (28) Pergent et al. (28) Molenaar et al. (29) Holmer et al. (29) n E Cover of seagrass not affected by C. taxifolia Current study -ve E Reduced seagrass shoot density in presence of C. taxifolia (over 4 months) Ceccherelli and Cinelli (1997) -ve E Reduced seagrass shoot density in presence of C. racemosa (over 14 months) Ceccherelli and Campo (22) n E No effect of C. taxifolia on seagrass shoot density after 13 months Ceccherelli and Sechi (22)?ve E Increased seagrass shoot density in presence of C. racemosa (over 14 months) n(?) E Some indication that decline of seagrass canopy was exacerbated by C. taxifolia, but Z. capricorni died in the absence of C. taxifolia Ceccherelli and Campo (22) Current study -ve C Biomass of R. maritima less in presence of C. taxifolia Williams and Grosholz (22) -ve E Shoot density and biomass of H. wrightii were reduced in the presence of C. prolifera (native) over 6 months -ve E Where C. prolifera (native) was particularly abundant, the alga colonised gaps before H. wrightii and potentially excluded the seagrass over 15 month Taplin et al. (25) Stafford and Bell (26) Negative effect (-ve), positive (?ve) or neutral (n) C correlative, E experimental (P. australis and Z. capricorni) and C. taxifolia. A possible explanation for such a pattern is that sparse (\5 % coverage) seagrass is susceptible to impacts from C. taxifolia, whereas dense ([5 %) seagrass is not because it negatively affects C. taxifolia (e.g. through competition for resources such as light).

4 12 T. M. Glasby Hypotheses derived from this model were tested using small-scale manipulative experiments (with limited spatial replication given the official declaration of the species as noxious), in conjunction with descriptions of changes in abundance of C. taxifolia and seagrasses over larger spatial and temporal scales. Materials and methods Study sites and invasion history All experiments were done in two estuaries near Sydney in SE Australia, namely the commercial port of Botany Bay (34 S, E) and Port Hacking (34 4 S, E), the latter being bounded by residential properties to the north and national park to the south. Two sites were used in both Port Hacking (Gunnamatta Bay and Fishermans Bay, separated by 2.5 km) and Botany Bay (both in Quibray Bay, separated by 8 m), each site being sheltered from oceanic swell. The predominant native seagrasses were the perennials Posidonia australis and Z. capricorni, and the annual Halophila ovalis, all of which grew to a maximum depth of *5 m. The invasive alga C. taxifolia was first verified in Port Hacking in 2 and in Botany Bay in 21 (Glasby et al. 25), although there is anecdotal evidence that it was present in parts of Port Hacking in 1998 (Grey 21). Estuarywide surveys indicated that C. taxifolia reached its peak abundance in Botany Bay in 23 and has declined steadily ever since (unpublished data). In Port Hacking, the area of C. taxifolia has fluctuated greatly among years, but its abundance has been trending downwards since 24. In each site, C. taxifolia grew amongst seagrass and was particularly dense immediately adjacent to seagrass beds (i.e. on soft sediment that was previously non-vegetated). C. taxifolia stolons intertwined to form a matrix *4 mm thick, with fronds extending up a further 3 65 mm. In the field, invasive strains of C. taxifolia are known only to reproduce via asexual fragmentation (Wright 25). Temporal patterns of seagrasses and C. taxifolia at the bay scale To test the hypothesis that seagrass coverage has declined since C. taxifolia was introduced, boundaries of seagrass beds were identified from orthorectified aerial photographs (at 1:1,5) for multiples times before and after C. taxifolia was discovered in Quibray Bay and Gunnamatta Bay (see Fig. 1). Field-based ground truthing was used to validate and augment maps for all years except 197 and 198 (hence species could not be distinguished for these years). Ground truthing was done using a real-time computer mapping system, a GPS, an underwater video system and an acoustic single beam depth sounder. Data on seagrass areas for Gunnamatta Bay from 1977 to 1999 were obtained from Meehan et al. (25) who used the same mapping methods. While the majority of seagrass beds were monospecific, area estimates for P. australis presented herein include mixtures of P. australis and H. ovalis or Z. capricorni, whilst areas for Z. capricorni include mixtures of Z. capricorni and H. ovalis. C. taxifolia areal coverage was estimated in summer and winter in by towing divers behind a boat on a manta board in a zigzag pattern around the perimeter of each bay and recording bed boundaries. This method overestimated the amount of C. taxifolia as the alga rarely covered 1 % of the substratum and was typically sparsely distributed within each bay. Associations between C. taxifolia and seagrasses To investigate spatial relationships between C. taxifolia and seagrasses, percentage covers of each were estimated in cm quadrats using a grid of 1 regularly-spaced points. Covers of primary foliage (i.e. shoots) and canopy foliage (i.e. leaves) were estimated. Observations suggested that the length of C. taxifolia was greater amongst dense seagrass than where there was little or no seagrass. This was tested by measuring in situ the lengths of 15 haphazardlychosen C. taxifolia fronds (stolon to tip of frond) per quadrat to the nearest mm using a ruler. Average length of frond per quadrat was calculated for comparisons with percentage cover of seagrass canopy. Quadrats were positioned haphazardly in areas where C. taxifolia was present, at depths of.5 2 m below mean low water. Sampling was done between March and May 23 in a total of 136 quadrats across the four sites (*34 per site). Pearson correlations were used to test for associations between C. taxifolia cover and seagrass cover. One factor permutational ANOVA (PERMANOVA) was used to compare C. taxifolia cover and frond length among plots with no seagrass, sparse (1 49 %

5 Caulerpa taxifolia in seagrass meadows 121 canopy cover) seagrass or dense (5 1 % canopy cover) seagrass. Data were transformed to square roots before calculating a Euclidian distance resemblance matrix and using 9999 unrestricted permutations of the raw data with Type III partial sums of squares. Differences among treatments were investigated using pair-wise t-tests. Monitoring of fixed plots To monitor vegetative expansion of C. taxifolia beds and test for corresponding declines in seagrass, permanent plots (5 9 5 cm) were established in two sites in Quibray Bay in May 23 and sampled 15 times over 8 years (until March 211). Plots were marked with stakes in two diagonally opposite corners, which enabled a quadrat to be re-located in the same place over each plot so percentage covers of vegetation and bare space could be estimated using a grid of 1 points. Plots were on the boundaries of beds of C. taxifolia and beds of seagrass. At each site, n = 3 replicate plots were positioned in each of the following habitats: primarily P. australis, P. australis mixed with C. taxifolia, and primarily C. taxifolia. Habitats were contiguous, with plots in each habitat not more than 2 m apart. At one of the sites and additional habitat, Z. capricorni mixed with C. taxifolia, was also sampled. At the time, no areas could be found in Quibray Bay that had Z. capricorni but no C. taxifolia. Plots were sampled approximately every 3 6 months in the first 3 years, then less regularly (8 18 mo intervals) thereafter (see Fig. 3). At each time of sampling, percentage covers of seagrass or C. taxifolia canopies were estimated and lengths of up to 15 haphazardly-chosen C. taxifolia fronds (or all fronds if there were \15 in total) were measured per quadrat. Experimental transplantation of C. taxifolia Two separate transplant experiments were done, one in Quibray Bay, the other in Gunnamatta Bay, to test the hypothesis that C. taxifolia impacts beds of sparse seagrass (P. australis or Z. capricorni) by reducing their canopy covers (i.e. numbers and/or lengths of leaves), but not dense seagrass beds. C. taxifolia was sourced from a m area (*5 8 % cover of C. taxifolia),.8 m deep some 15 m from the experimental plots. Intact C. taxifolia (with attached rhizomes, stolons and fronds) was removed by hand and kept in seawater on the boat for a maximum of 3 min. Stolons were cut into 15 cm fragments (with 8 14 fronds) and four were planted in each experimental plot (described below) by gently pushing rhizoids into the sediment and using two U-shaped plastic coated wire stakes to pin the stolon to the sediment (as per Ceccherelli and Cinelli 1999). These fundamentals of each experiment were the same, but as described below there were slight differences in treatments. Transplant experiment 1 was set up in Quibray Bay in water.5 1 m below mean low tide and ran from December 23 to January 28. Eleven treatments were established, with n = 3 replicate plots (5 9 5 cm) per treatment, each separated by 1 5 m. Four treatments involved transplanting four fragments of C. taxifolia (to create a C. taxifolia coverage of *1 % in plots) into sparse seagrass (P. australis and Z. capricorni; canopy cover 2 3 %) and dense seagrass (P. australis and Z. capricorni; canopy cover 8 9 %). To test for the effects of C. taxifolia in relation to the seagrass canopy per se, C. taxifolia was also transplanted into plots of P. australis or Z. capricorni that had had their canopies cut to change them from dense to sparse. This involved removing leaves (cutting with scissors) at the tops of some of the sheaths such that canopy cover in the plot was reduced from 8 to 9 % down to 2 3 %. These canopy cutting treatments helped distinguish effects of canopy cover per se from other effects (e.g. sediment properties) that might be associated with dense seagrass. There were also two seagrass control treatments (one P. australis, one Z. capricorni each 5 % canopy cover) that had no C. taxifolia added. The final three treatments were controls to test for artefacts associated with handling the C. taxifolia; namely undisturbed C. taxifolia plots (*1 % cover), C. taxifolia that had been cut into 15 cm sections in situ (without removing algae from the sediment), and C. taxifolia fragments that had been moved to non-vegetated plots (to test for the effects of moving C. taxifolia to a new place, as was being done when it was transplanted to seagrass). Plots without C. taxifolia were weeded whenever they were sampled and a 3 cm C. taxifolia exclusion buffer was maintained around those plots. C. taxifolia was nevertheless able to invade (e.g. via drifting fragments) most plots at some stage of the experiment.

6 122 T. M. Glasby Transplant experiment 2 was done to test whether results were consistent at a second site and included more controls to test for natural changes in sparse versus dense seagrass. This second experiment was set up in Gunnamatta Bay (November 24 September 29) in water.3.8 m below mean low tide and did not involve P. australis treatments. Seven treatments were established, with n = 3 replicate plots (5 9 5 cm) per treatment, each separated by 1 5 m. C. taxifolia was transplanted into naturally sparse Z. capricorni (2 3 % canopy cover) and dense Z. capricorni (8 9 % canopy cover). C. taxifolia was also transplanted into plots of Z. capricorni that had had their canopies cut to change them from dense to sparse. Z. capricorni control treatments were: naturally sparse Z. capricorni with no C. taxifolia added, dense Z. capricorni with no C. taxifolia added, and cut sparse Z. capricorni with no C. taxifolia added. The latter treatment enabled a test of the effects of cutting the Z. capricorni canopy on the growth of the seagrass. The final treatment consisted of undisturbed C. taxifolia plots (with no seagrass). Procedural controls to test for effects of manipulating the C. taxifolia were not included as results from experiment 1 indicated no such experimental artefacts (see results). All other procedures were the same as the first experiment. Repeated measures analyses were used to compare percentage covers of seagrasses and C. taxifolia among treatments over time. Analyses were done using PERMANOVA with Euclidean distance resemblance matrices, as described above. Results Temporal patterns of seagrasses and C. taxifolia at the bay scale Fluctuations in the total area of seagrass in Quibray Bay since 197 were driven by changes in Z. capricorni (Fig. 1a). Such infrequent mapping of Z. capricorni may not accurately depict changes in its abundance, but nevertheless, the emergence and spread of C. taxifolia occurred sometime during the period that Z. capricorni appears to have declined. Notably, the area of Z. capricorni in 28 was essentially the same as that estimated some 2 years before the introduction of C. taxifolia. When the coverage of C. taxifolia was maximal (25.9 ha) in 25, it covered 1 % of Quibray Bay compared to seagrass which covered *3 % of the bay over the four mapping times. In Gunnamatta Bay the total area of seagrass was almost totally driven by P. australis (hence it was not distinguished from total seagrass in Fig. 1b), with the area of Z. capricorni being trivial in comparison (Fig. 1b). The areas of P. australis and Z. capricorni remained relatively stable during the period that C. taxifolia appeared and became abundant. At its peak, C. taxifolia was found in 2.5 % (25 ha) of Gunnamatta Bay which was substantially more than the average seagrass coverage (12 ha, 1 % of bay) or the maximal seagrass coverage (18 ha, 15 % of bay). This latter result was due to C. taxifolia colonising large areas of previously non-vegetated soft sediments. It is noteworthy that the Z. capricorni beds at both the Gunnamatta and Quibray Bay sites used in this study appear to have disappeared in the past. The small patch of Z. capricorni sampled in the current study in Gunnamatta Bay was not mapped by West et al. (1985), while maps for Quibray Bay indicate that the beds of Z. capricorni used in Quibray Bay were present in 1942 and 1985 (Larkum and West 199), but not in the early 197s (Larkum 1976). Associations between C. taxifolia and seagrasses In areas where C. taxifolia and seagrass co-occurred, there was a negative correlation between the percentage cover of C. taxifolia and canopy covers of P. australis (r =-.42, 47 df, P \.1) and Z. capricorni (r =-.41, 48 df, P \.1). Similar negative correlations existed between C. taxifolia and primary (shoot) covers of P. australis (r =-.4, 47 df, P \.1) and Z. capricorni (r =-.56, 48 df, P \.1). Percentage cover of C. taxifolia differed significantly among habitat categories (pseudo F 4,131 = 15.98, P =.1), being greatest where there was no seagrass or a sparse canopy of seagrass (Fig. 2a). C. taxifolia coverage was restricted to \4 % where seagrass canopy was dense ([5 %; Fig. 2a). The average maximum length of C. taxifolia fronds differed significantly among habitats (pseudo F 4,16 = 13.29, P =.1), with longer fronds occurring in plots with a dense P. australis canopy

7 Caulerpa taxifolia in seagrass meadows Fig. 1 Areas (in hectares) of seagrass (all species and separated into P. australis and Z. capricorni) and C. taxifolia (columns) in a Quibray Bay and b Gunnamatta Bay. Arrow on x-axis indicates when C. taxifolia was first discovered. C. taxifolia areas for each year are an average of summer and winter estimates (no data for 28). P. australis is not plotted in b as it could not be distinguished from total seagrass. Note different scale for Z. capricorni on right y-axis in b Area (ha) (a) Quibray Bay Total seagrass Posidonia Zostera No data 3 (b) Gunnamatta Bay Area (ha) Total seagrass Zostera area (ha) 5 Zostera No data (Fig. 2b). The same pattern was apparent for the average length of C. taxifolia fronds (65 mm for dense P. australis, 3 4 mm for all other habitats), although estimates were not as precise as those for average maximum frond length. Monitoring of fixed plots Percentage cover of P. australis canopy (leaves) fluctuated considerably throughout the study, whereas the primary cover (shoots) was relatively stable. These temporal patterns were similar whether C. taxifolia was essentially absent (never[4 % cover, Fig. 3a) or abundant (up to 5 % cover, Fig. 3b). P. australis canopy cover declined between Dec 23 and Mar 24 with a concomitant increase in C. taxifolia in those plots where C. taxifolia was present (Fig. 3b). But a similar magnitude of decline in canopy cover occurred where C. taxifolia was absent (Fig. 3a), indicating that C. taxifolia was not the cause of the decline in P. australis canopy. A small amount of C. taxifolia managed to invade when the P. australis canopy cover was reduced to *6 % (Fig. 3a), while C. taxifolia cover increased markedly (up to 5 %)

8 124 T. M. Glasby (a) a a % cover Caulerpa a* b 2 c No seagrass Posidonia Zostera Posidonia Zostera (b) 1-49% canopy 5-1% canopy b Caulerpa frond length (mm) a a a a 1 No seagrass Posidonia Zostera Posidonia Zostera 1-49% canopy 5-1% canopy Fig. 2 a Mean percentage cover (?S.E.) and b average maximum frond length of Caulerpa taxifolia in plots where there was no seagrass, a sparse canopy (1 49 %) of P. australis or Z. capricorni, or a dense canopy (5 1 %) of P. australis or Z. capricorni. Letters (a c) indicate results of pairwise tests bars with the same letter are not significantly different. a* indicates this treatment could not be logically separated from the when P. australis canopy declined to 5 % (Fig. 3b). Around mid 25, there was a sharp decline in cover of C. taxifolia in all plots and declines in P. australis canopy were again evident in some plots where C. taxifolia was absent (Mar 211, Fig. 3b). P. australis primary cover was relatively stable throughout the study and showed no correlation with changes in C. taxifolia cover. other two marked a (i.e. significantly different from areas with no seagrass, but not different from sparse P. australis). n = 2 36 quadrats per habitat for a and n = quadrats per habitat (with each replicate being the mean of up to 15 C. taxifolia fronds per quadrat) for b. Replicates pooled across four sites in Quibray Bay and Gunnamatta Bay The cover of Z. capricorni declined dramatically over the first 6 months after which it remained uncommon in all plots for the next 7 years (Fig. 3c). As with P. australis, the cover of C. taxifolia was [3 % only when the Z. capricorni canopy was \5 %. C. taxifolia coverage was often greater than 5 % when Z. capricorni canopy was non-existent (Fig. 3c, d). Note that Z. capricorni was often still

9 Caulerpa taxifolia in seagrass meadows 125 Fig. 3 Mean percentage covers (?S.E.) of seagrass and Caulerpa taxifolia in fixed plots (5 9 5 cm) averaged across two sites in Quibray Bay. Plots were situated in areas that were initially a dominated by P. australis, b a mixture of P. australis and C. taxifolia, c a mixture of Z. capricorni and C. taxifolia or, d primarily C. taxifolia. Dotted lines indicate seagrass canopy covers (leaves), solid lines indicate primary cover (shoots) of P. australis (closed triangles), Z. capricorni (closed squares) or C. taxifolia (open circles) % cover (a) Posidonia habitat May Sep Dec Mar Jul Jan Apr Aug Dec Apr Dec Jan Oct Aug Mar (c) Zostera habitat (b) Posidonia / Caulerpa boundary May Sep Dec Mar Jul Jan Apr Aug Dec Apr Dec Jan Oct Aug Mar (d) Caulerpa habitat 8 8 % cover May Sep Dec Mar Jul Jan Apr Aug Dec Apr Dec Jan Oct Aug Mar May Sep Dec Mar Jul Jan Apr Aug Dec Apr Dec Jan Oct Aug Mar present in plots (with leaves\5 mm tall) even when no canopy cover was recorded. Because it was not possible to find plots with Z. capricorni but no C. taxifolia, this study on its own does not indicate whether the increase in C. taxifolia might have caused the decline in Z. capricorni, or been a response to this decline (as indicated for P. australis). However, it was clear that (i) there was typically a negative association between Z. capricorni and C. taxifolia, (ii) Z. capricorni and C. taxifolia coverage showed large temporal variability and (iii) Z. capricorni was capable of sprouting in plots where C. taxifolia was present (Fig. 3d). Effects of C. taxifolia on Z. capricorni were tested directly in the manipulative experiment described below. Another species of seagrass, H. ovalis, appeared at certain times in all the plots that had no P. australis. Moreover, the peaks in abundance of H. ovalis (2 6 % cover) occurred at times when there was \2 % cover of any other vegetation. Experimental transplantation of C. taxifolia Transplant experiment 1: Quibray Bay Caulerpa taxifolia was apparently not affected by any of the experimental procedures relating to cutting the stolons into fragments or moving the fragments to new areas. The patterns of C. taxifolia percentage cover over the first five times of sampling (i.e. 13 months) did not differ significantly among the procedural control, translocation control or the undisturbed control (pseudo F 8,3 =.6, P =.99). Thus, results of the experimental transplantation can be assumed to be indicative of what would occur following natural colonisation by C. taxifolia (i.e. no experimental

10 126 T. M. Glasby Fig. 4 Mean percentage covers (?S.E.) of P. australis australis and C. taxifolia over time in plots with varying covers of seagrass canopy. a 5 % P. australis canopy with no C. taxifolia added, b Sparse P. australis canopy with C. taxifolia added, c dense P. australis with C. taxifolia added and d P. australis that had canopy cut to make it sparse. Dashed lines represent canopy cover of P. australis, solid lines represent primary cover of P. australis (triangles) or C. taxifolia (circles) % cover (a) 5% Posidonia control Dec Mar May Sep Jan Apr Aug Dec Apr Dec Jan (c) Dense Posidonia + Caulerpa (b) Sparse Posidonia + Caulerpa Dec Mar May Sep Jan Apr Aug Dec Apr Dec Jan (d) Cut sparse Posidonia + Caulerpa % cover Dec Mar May Sep Jan Apr Aug Dec Apr Dec Jan Dec Mar May Sep Jan Apr Aug Dec Apr Dec Jan artefacts were evident). After the first 13 months, C. taxifolia disappeared from most treatments (i.e. the initial experimental conditions were essentially ended), before recolonising and covering similar areas in the different C. taxifolia treatments. It is noteworthy that by Jan 25, P. australis had started to grow into each of the C. taxifolia control treatments, reaching by Jan 28 a mean canopy cover of 4, 27 and 32 % in the undisturbed, procedural and translocation C. taxifolia controls, respectively. Z. capricorni shoots also appeared (max 4 % primary cover) in some of the C. taxifolia control plots towards the end of the experiment but remained short and so did not create a canopy. The average cover of C. taxifolia in the P. australis control plots across all times (mean 4.3 % ± s.e. 2.2) was significantly less than in sparse (18.9 % ± 3.) or dense (8.6 % ± 2.9) P. australis plots to which C. taxifolia had been added (pesudo F 3,3 = 9.17, P =.1). Primary cover of P. australis showed little change throughout the study for any treatment, whereas P. australis canopy cover and primary cover of C. taxifolia were extremely variable (Fig. 4). There was no evidence that C. taxifolia had an adverse effect on the coverage of sparse P. australis. P. australis canopy increased from 5 to 8 % in control plots to which no C. taxifolia was added (despite C. taxifolia managing to invade at various times; Fig. 4a) and a similar increase in canopy occurred in the sparse P. australis plots (from 2 to 8 %) despite C. taxifolia coverage reaching up to 6 % (Fig. 4b). C. taxifolia cover was typically less that 3 % in plots that had an average P. australis canopy of[5 % (Fig. 4). For the first two sampling times after the experiment was set up (March May 24), the cover of C. taxifolia in plots that had sparse P. australis canopy (either naturally sparse or cut to be sparse) was significantly less than in plots with a dense P. australis canopy (pseudo F 9,32 = 2.6, P =.3). This is consistent with the notion that C. taxifolia growth is limited by the P. australis canopy per se, not the

11 Caulerpa taxifolia in seagrass meadows 127 Fig. 5 Mean percentage covers (?S.E.) of Z. capricorni capricorni and C. taxifolia over time in plots with varying covers of seagrass canopy. a 5 % Z. capricorni canopy with no C. taxifolia added, b Sparse Z. capricorni canopy with C. taxifolia added, c dense Z. capricorni with C. taxifolia added and d Z. capricorni that had canopy cut to make it sparse. Dashed lines represent canopy cover of Z. capricorni, solid lines represent primary cover of Z. capricorni (squares) or C. taxifolia (circles) % cover (a) 5% Zostera control Dec Mar May Sep Jan Apr Aug Dec (c) Dense Zostera + Caulerpa 1 Apr Dec Jan (b) Sparse Zostera + Caulerpa Dec Mar May Sep Jan Apr Aug Dec Apr Dec Jan (d) Cut sparse Zostera + Caulerpa % cover Dec Mar May Sep Jan Apr Aug Dec Apr Dec Jan Dec Mar May Sep Jan Apr Aug Dec Apr Dec Jan primary (shoot) cover or other features of areas where dense P. australis happens to grow. After this time, covers of C. taxifolia declined in most treatments and typically remained small (but variable) in plots that had a dense canopy cover ([5 %) of P. australis (Fig. 4). Despite efforts to exclude C. taxifolia, the alga invaded all the Z. capricorni control plots and reached *6 % cover at various times (Fig. 5a). The average cover of C. taxifolia across all times did not differ significantly among controls (29.2 % ± 5.) or sparse (29.4 % ± 5.1) or dense (26.5 % ± 4.4) Z. capricorni treatments, meaning there was no good test of potential effects of C. taxifolia on Z. capricorni. Z. capricorni canopy covers declined substantially in all treatments over the course of the experiment and each such decline was either coincident with or immediately prior to an increase in C. taxifolia (Fig. 5). This decline in Z. capricorni canopy (which resulted in all Z. capricorni plots having \25 % canopy cover after May 24) possibly caused patterns of C. taxifolia coverage over time to be similar in all the Z. capricorni treatments (Fig. 5). As such, the comparison of naturally sparse Z. capricorni and Z. capricorni that was cut to make artificially sparse was not particularly relevant, but nevertheless these treatments were remarkably consistent throughout the experiment (Fig. 5b, d). Maximum lengths of C. taxifolia fronds were measured for the first six times of sampling and differed significantly with time and treatment (pseudo F 4,18 = 1.56, P =.41). Pairwise comparisons did not identify any consistent patterns among treatments for any of the times and changes in canopy cover of seagrass prevented any proper test of hypotheses regarding length of C. taxifolia fronds. It was, however, evident that the average maximum frond length did not differ significantly among treatments at the start of the experiment, yet for the next five times of sampling, the longest C. taxifolia fronds were

12 128 T. M. Glasby (a) Sparse Zostera control 1 (b) Sparse Zostera + Caulerpa % cover (c) Dense Zostera control 1 (d) Dense Zostera + Caulerpa % cover Nov Feb Apr Aug Dec May Dec Sep Nov Feb Apr Aug Dec May Dec Sep Fig. 6 Mean percentage covers (?S.E.) of Z. capricorni capricorni and C. taxifolia over time in plots with varying covers of seagrass canopy. a Sparse Z. capricorni canopy with no C. taxifolia added, b Sparse Z. capricorni canopy with C. taxifolia added, c dense Z. capricorni with C. taxifolia added and d Dense Z. capricorni canopy with C. taxifolia added. Dashed lines represent canopy cover of Z. capricorni, solid lines represent primary cover of Z. capricorni (squares) or C. taxifolia (circles) always in the P. australis treatments (both sparse and dense). Relatively few C. taxifolia fronds were available to be measured in this experiment compared to the initial descriptive study meaning precision was greatly compromised. Transplant experiment 2: Gunnamatta Bay Dense and sparse Z. capricorni control plots remained relatively free of C. taxifolia throughout the experiment, although some plots were invaded in late 25 (Fig. 6a, c). Comparisons of sparse and dense Z. capricorni canopy treatments were compromised by the large decline in dense Z. capricorni treatments in the early stages of the experiment. Nevertheless, the average cover of C. taxifolia in the Z. capricorni control plots (1.4 % mean ±.1 s.e.) was significantly less than in dense (3.2 % ± 1.4) or sparse (7.6 % ± 1.9) Z. capricorni plots to which C. taxifolia was added, or C. taxifolia control plots (44.5 % ± 8.2). The decline in Z. capricorni cover over time was similar among Z. capricorni controls and Z. capricorni plots to which C. taxifolia had been added, despite the large difference in mean covers and prevalence of C. taxifolia in these treatments (Fig. 6). This indicates that C. taxifolia was not responsible for the decline in Z. capricorni and that some other process caused a large-scale decline at the site. There was no indication that cutting the Z. capricorni canopy had adverse effects on the seagrass, but rather stimulated the growth of the canopy compared to other Z. capricorni

13 Caulerpa taxifolia in seagrass meadows 129 (a) Cut sparse Zostera control 1 (b) Cut sparse Zostera + Caulerpa 1 (c) Caulerpa control % cover Nov Feb Apr Aug Dec May Dec Sep Nov Feb Apr Aug Dec May Dec Sep Nov Feb Apr Aug Dec May Dec Sep Fig. 7 Mean percentage covers (?S.E.) of Z. capricorni capricorni and C. taxifolia over time in plots with varying covers of seagrass canopy. a Z. capricorni that had canopy cut to make it sparse, no C. taxifolia added, b Z. capricorni that had canopy cut to make it sparse, with C. taxifolia added, c C. taxifolia plots with no seagrass. Dashed lines represent canopy cover of Z. capricorni, solid lines represent primary cover of Z. capricorni (squares) orc. taxifolia (circles) treatments where canopy was not cut (compare November 24 February 25 in Figs. 6a, b to 7a, b). Again, changes in the cover of cut Z. capricorni over time were generally comparable in plots with C. taxifolia and those without (Fig. 7a, b), with the exception of the patterns described below for Z. capricorni canopy cover. Although Z. capricorni clearly declined in the absence of C. taxifolia, there were some suggestions that C. taxifolia may have sped up the decline of sparse Z. capricorni, particularly its canopy. For example, Z. capricorni canopy cover decreased slightly (28 18 %) from February to April 25 in sparse Z. capricorni plots that had\1 % cover of C. taxifolia (Fig. 6a), yet decreased markedly (22 4 %) over the same period in sparse Z. capricorni plots to which C. taxifolia had been added (Fig. 6b). Likewise, plots that had artificially sparse (cut) Z. capricorni but no C. taxifolia, increased their canopy covers from August to December 25 (24 38 %) (Fig. 7a), yet Z. capricorni canopy covers declined (19 8 %) in plots where C. taxifolia had been added to artificially sparse Z. capricorni (Fig. 7b). This latter pattern occurred despite Z. capricorni primary cover increasing over this same period in plots with (Fig. 7b) or without (Fig. 7a) C. taxifolia. Zostera capricorni was able sprout and grow in plots that had large covers of C. taxifolia (Fig. 7c), but the leaves did not grow long enough during the experiment to create a canopy. The site was visited in September 21 and there was still no Z. capricorni in any plot, but some C. taxifolia was present in a subset of plots (mean 1.3 % ±.6 across all plots). Covers of C. taxifolia in the artificially sparse (cut) Z. capricorni plots were similar to those in the naturally sparse Z. capricorni plots, and less than in the dense Z. capricorni plots, suggesting that the canopy of Z. capricorni per se can limit the cover of C. taxifolia. At some times there were increases in C. taxifolia after declines in Z. capricorni, but by December 26, C. taxifolia and Z. capricorni had disappeared from all plots. H. ovalis was uncommon in all plots at all times, never reaching more than 1 % cover and typically\5 % cover. Discussion There were clear negative associations between C. taxifolia and the seagrasses Posidonia australis and Zostera capriconi in the NSW estuaries examined here, which matches patterns documented in numerous other countries. Experimental transplantations of C. taxifolia, however, provided no evidence that the alga is adversely affecting the growth of P. australis, nor did monitoring over 7 years indicate a decline in seagrass associated with the presence of C. taxifolia. The cover of Z. capricorni declined dramatically over the course of this study in all experimental plots, regardless of the presence of C. taxifolia. There was some indication that C. taxifolia may have enhanced the decline of the Z. capricorni canopy, but this did not

14 13 T. M. Glasby alter the final fate of Z. capricorni, which disappeared from all plots. Importantly, both Z. capricorni and P. australis were found to sprout in plots that had large covers of C. taxifolia for over a year (although notably the newly-sprouted Z. capriconi did not grow large enough to form a canopy). At the bay-wide scale (*1 km 2 ), the areal coverages of Z. capricorni and P. australis were not different from what they were many years prior to the introduction of C. taxifolia. C. taxifolia in NSW is most likely behaving as an opportunistic weed which rapidly colonises previously non-vegetated areas (where it reaches large densities and affects infauna; Wright and Gribben 28; Gribben et al. 29b; McKinnon et al. 29; Byers et al. 21) and periodically colonises gaps in seagrass beds with no apparent affects on seagrass coverage. This study appears to be the first experimental investigation (albeit with limited replication) of effects of C. taxifolia on the growth of a species of Posidonia. Moreover, by using experimental additions of C. taxifolia, this study provides a direct test of the effects of invasion by the alga on seagrasses. A review of the published literature (Table 1) indicates that all the evidence for negative impacts of C. taxifolia on Posidonia spp. is correlative. In fact, the only published experimental evidence for negative impacts of C. taxifolia on seagrass density seems to be a 4 month study by Ceccherelli and Cinelli (1997), and importantly the same authors reported no such effect over the longer-term (Table 1). This is not to say that C. taxifolia might not be having sub-lethal effects on seagrasses, for example by altering sediment characteristics which can affect seagrass growth in some way (Calleja et al. 27). But as yet, evidence for such sediment effects by C. taxifolia comes only from correlative studies (e.g. Holmer et al. 29; Eyre et al. 211). Experimental studies with other species of Caulerpa have also provided limited evidence for negative effects on seagrasses (Table 1). C. racemosa caused decreased shoot density of Cymodocea nodosa over 14 months, yet increased shoot density of the smaller seagrass Zostera noltii (Ceccherelli and Campo 22). In its native habitat, C. prolifera was implicated in declines in shoot densities of the sympatric Halodule wrightii (Taplin et al. 25) and outcompeted the seagrass for space over 15 months (Stafford and Bell 26). Thus, the published literature provides little evidence that species of Caulerpa are capable of driving changes in seagrass abundance. There are indications that species of Caulerpa compete with seagrasses, either via interference competition, (i.e. pre-empting space), or exploitative competition (i.e. utilising limited resources), as has been demonstrated for C. prolifera in its native environment. More experimental investigations are needed to determine whether competition between invasive Caulerpa and seagrasses could be any more severe than competition among sympatric native species (e.g. Williams 1987). The thickness of the C. taxifolia beds in the present study was around 4 mm of stolons, plus fronds. It is possible that far thicker stolon mats, as have occurred at some Mediterranean sites (Thibaut et al. 24), could have negative effects on seagrasses by creating anoxic conditions (Holmer et al. 29; Eyre et al. 211). Much thicker mats of C. taxifolia (e.g. 2 3 mm high mats of stolons) have been observed in some NSW estuaries, but never amongst seagrasses. In some Mediterranean sites where C. taxifolia was so abundant that it smothered seagrass, P. oceanica showed signs of necrosis at the base of the shoots (de Villèle and Verlaque 1995). There is certainly evidence that smothering is an important means by which Caulerpa spp. impact macroalgae and invertebrates (Piazzi et al. 25; Gribben et al. 29b; Cebrian et al. in press). Additionally, ambient nutrient levels might influence the outcome of competition between Caulerpa spp. and seagrasses, given that the former can potentially thrive in eutrophic conditions (Lapointe and Bedford 21; Burke and Grime 1996; Gennaro and Piazzi 211; but see Ceccherelli and Sechi 22), while many seagrasses can be adversely affected (Chisholm et al. 1997; Cardoso et al. 24). Propagule density could also influence the outcome between C. taxifolia and seagrasses. It is possible that if more C. taxifolia had been added to seagrass plots in the present study there could have been detectable negative effects on the seagrasses. But this seems unlikely given that C. taxifolia increased from 1 % of the substratum (initial experimental coverage) to 5 % in sparse P. australis plots in Quibray Bay, yet coverage of the seagrass was not affected. There was evidence from this study of a threshold of seagrass canopy cover (*5 %), which tended to restrict C. taxifolia cover to less than 4 % of the substratum. This was likely a shading effect of the native canopy; scouring of the sediment by the canopy

15 Caulerpa taxifolia in seagrass meadows 131 was unlikely as the denser the seagrass canopy, the more intertwined the leaves resulting in reduced physical contact with the substratum. Experimental reductions in the canopy of P. australis and Z. capricorni resulted in increased covers of C. taxifolia relative to plots with naturally dense seagrass canopies. Thus, the native canopy reduces survivorship of the invader, as demonstrated for the invasive alga Sargassum muticum (Britton-Simmons 26) and some terrestrial plants (Corbin and D Antonio 24). The native P. australis canopy also appeared to cause the length of C. taxifolia fronds to increase, as described for the Mediterranean (de Villèle and Verlaque 1995; Ceccherelli and Cinelli 1998); probably an effect of reduced light levels (i.e. etiolation). Thus, it could be argued that dense seagrass beds could provide optimal conditions and hence a refuge for C. taxifolia (Stafford and Bell 26), but for the reasons discussed below, the results of this study do not support such a conclusion. Various authors have reported that C. taxifolia and C. racemosa can invade only sparse seagrass (Meinesz et al. 1993, de Villèle and Verlaque 1995, Ceccherelli and Cinelli 1999; Ceccherelli et al. 2) or degraded seagrass beds (Chisholm et al. 1997; Occhipinti- Ambrogi and Savini 23; Ruitton et al. 25; Bulleri et al. 211; Kiparissis et al. 211). Indeed plant invasions are often correlated with the physical structure of the recipient community, for example forests with an intact canopy can have relatively few invaders (Mack et al. 2), while non-vegetated ground can be more susceptible to invasion than well vegetated areas (Harrison and Bigley 1982; Crawley 1986; Burke and Grime 1996; but see Thomsen et al. 26). Dense seagrass beds seem better able to resist colonisation by C. taxifolia, suggesting that maintaining healthy populations of seagrasses will help minimise colonisation of these beds by C. taxifolia and other invaders (Williams 27). But importantly, C. taxifolia can rapidly colonise non-vegetated sediments, so invasion of estuaries by this alga need not be related at all to the health of seagrass beds. Indeed the present study found that previously non-vegetated sediments seem a better habitat for C. taxifolia than are seagrass beds the alga typically persisted longer and reached larger densities in the former habitat (see also Holmer et al. 29). In this regard, patterns of C. taxifolia abundance were similar to those of the small native seagrass Halophila ovalis. The major difference was that C. taxifolia bloomed more frequently than did H. ovalis and the invader reached greater percentage covers. The patterns of colonisation and abundance of C. taxifolia in NSW estuaries seem similar to those of species of Caulerpa in their native habitats (Williams et al. 1985; KirkmanandKuo199; Short et al. 26; Burfeind and Udy 29), i.e. colonising non-vegetated soft sediments, co-occurring at low densities with seagrasses and having stochastic population dynamics. In some cases, the previously non-vegetated areas that C. taxifolia invades may have been non-vegetated historically (i.e. the empty niche hypothesis, e.g. Elton 1958; MacArthur 197), while in others they may be periodically non-vegetated (i.e. the fluctuating resource hypothesis, e.g. Johnstone 1986; Davis et al. 2) due to natural fluctuations in seagrasses (Duarte et al. 26) or anthropogenic disturbances (Walker et al. 26). But importantly, both of the aforementioned invasion hypotheses describe situations where the invader persists and dominates, whereas C. taxifolia in NSW often disappears from sites, or in some cases, entire estuaries (Glasby unpublished data). Experimental work has demonstrated that other invasive macroalgae such as Undaria pinnatifida and Caulerpa racemosa are opportunistic invaders of algal beds on rocky reefs (Valentine and Johnson 23, 24; Bulleri et al. 21). Both of these introduced algae colonised gaps that were created amongst native canopy-forming algae. But U. pinnatifida subsequently died off after the native algal canopy re-grew (Valentine and Johnson 23), whilst C. racemosa was capable of persisting and preventing the recovery of the native assemblage (Bulleri et al. 21). In the present study, C. taxifolia amongst P. australis behaved similarly to U. pinnatifida in that it could occupy space when the native canopy cover was low, but was then outcompeted as the native canopy increased (see also Burfeind and Udy 29). The most typical outcome involving the smaller seagrass species (Z. capricorni), however, was that both it and C. taxifolia eventually disappeared from experimental plots. As yet, neither the native seagrass nor the invader has recovered and come to dominate these plots. There are many documented cases of competitively inferior invasive habitat-formers responding favourably to disturbances (such as removal of vegetation) and preventing the recovery of a native (e.g. Hobbs and Huenneke 1992; Corbin and D Antonio 24; Bando 26; Williams 27), but it would seem that other factors are at play in these NSW sites.