Settlement of native algal propagules
There was a significant inhibitory effect of 1,1,3,3-tetrabromo-2-heptanone on the amount of settled spores/germlings from the native macroalgal species C. virgatum, P. stricta and U. lactuca and settled cells from the native microalgal species Cylindrotheca fusiformis (Table 2 and Fig. 2). Post hoc tests showed that the lowest concentration for inhibitory effects were found at 0.06 μg cm−2 for C. virgatum and U. lactuca and 0.125 μg cm−2 for P. stricta and Cylindrotheca fusiformis (Dunnett's test at α = 0.05, Fig. 2). Further graphical examination revealed that the amount of successfully settled native spores were reduced to a minimum for all species tested already at concentrations well below B. hamifera's natural surface concentrations of 2–4 μg of 1,1,3,3-tetrabromo-2-heptanone per cm2 (Fig. 2). The compound also had a stronger effect on P. stricta at higher concentrations, compared to the other natives, reducing the amount of settled spores to a mere 1% at 0.5 μg cm−2, and no spores settled at the higher concentrations (Fig. 2a). Similarly, at 1 μg cm−2, the mean number of settled germlings of U. lactuca was below 1 and reduced to 0 at 2 and 4 μg cm−2, whereas C. virgatum and Cylindrotheca fusiformis continued to colonize the panels, although to low extents, also at the highest concentration (Fig. 2b–d).
Table 2. One-way anova on effects of 1,1,3,3-tetrabromo-2-heptanone on settlement of native algal propagules
|Error||40||83.15|| || |
| Cylindrotheca fusiformis |
|Error||56||91292.75|| || |
| Ulva lactuca |
|Error||32||197.53|| || |
| Polysiphonia stricta|
|Error||40||6.73|| || |
Figure 2. Effects of 1,1,3,3-tetrabromo-2-heptanone on settlement of propagules of native macroalgae (a) Polysiphonia stricta, (b) Ulva lactuca, (c) Ceramium virgatum and microalgae of (d) Cylindrotheca fusiformis. Asterisks indicate significant differences compared to controls at α = 0.05 using Dunnett's test. Data are presented as mean ± SE
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Transfer of 1,1,3,3-tetrabromo-2-heptanone from the exotic species to its native host algae
In the laboratory experiment, the measured mean surface concentrations of 1,1,3,3-tetrabromo-2-heptanone on the native host algae F. lumbricalis and C. officinalis placed adjacent to B. hamifera were 0.103 ± 0.013 and 0.194 ± 0.033 μg cm−2 (mean ± SE, n = 16), respectively (Fig. 3). In comparison, the mean surface concentration on C. officinalis in the field experiment was high (mean ± SE: 0.534 ± 0.191 μg cm−2; n = 8, Fig. 3). 1,1,3,3-Tetrabromo-2-heptanone was not present on C. officinalis collected in the field (i.e. controls, 0 μg cm−2; n = 8). When the mean surface concentrations from these experiments are compared to the levels of this compound affecting the settlement of native species (Fig. 2) described previously, it becomes clear that there would be a very large reduction in settlement for all four native species under these surface concentrations. The high concentration measured on C. officinalis in the field experiment corresponds to the 0.5 μg level, although slightly underestimated, where the settlement of Cylindrotheca fusiformis would be decreased by 68%, C. virgatum by 70%, U. lactuca by 87% and P. stricta by 97% compared to controls. These results show that the transfer of B. hamifera's secondary compounds to other surfaces has a high potential to reduce the settlement of native algal competitors.
Figure 3. Surface concentration of 1,1,3,3-tetrabromo-2-heptanone per cm2 on native algal host species F. lumbricalis and C. officinalis placed adjacent to Bonnemaisonia hamifera for 1 week, in field and laboratory experiments. Data are presented as mean ± SE
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In this study, we found that the settlement of native macroalgal propagules and microalgae is strongly inhibited on surfaces coated with the major defence compound from B. hamifera, 1,1,3,3-tetrabromo-2-heptanone, at concentrations much lower than the natural surface concentration of B. hamifera. We also show that this compound can be transferred from B. hamifera to the surface of its primary host algae, F. lumbricalis and C. officinalis, by both laboratory and field experiments. Comparisons between the results from these two experiments revealed that the amounts of 1,1,3,3-tetrabromo-2-heptanone transferred to the host algae is more than sufficient to prevent settlement by the native species tested. Together, these results provide support for the main prediction of the NWH, that is, that non-indigenous species become successful in the invaded range if they possess novel chemical weapons that can suppress native competitors.
In contrast to the results from our experiments with the recruiting stages, we found no effect of adult B. hamifera on growth rates of the adult native competitors C. virgatum, Cystoclonium purpureum, P. stricta, Pilayella littoralis, S. cirrosa and Cladophora rupestris. In general, there are remarkably few, if any, well-established examples of allelopathy among adult stages of marine macroalgae (Granéli & Pavia 2006), in strong contrast to the large number of reports on chemical defences against herbivory (Pavia et al. 2012) and biofouling (Steinberg, de Nys & Kjelleberg 2002;) in marine systems. There is, however, some circumstantial evidence for allelopathic effects among adult macroalgae. For instance, the green algae Chaetomorpha linum and Chaetomorpha aerea are never found at the same time in tide pools, most likely because they hinder the growth of one another, and both species have been reported to inhibit growth of the red algae C. virgatum (see Harlin 1987 and references therein). Furthermore, in a laboratory experiment, Cho et al. (2001) found that extracts from a few species, out of a wide range of green, brown and red algae tested, significantly inhibited the growth of the commonly epiphytic green algae Enteromorpha prolifera. It is also noteworthy that some of the most rigorous studies of allelopathy among benthic marine organisms have shown that macroalgae have the potential to suppress coral competitors through allelopathy (de Nys, Coll & Price 1991; Paul et al. 2011; Rasher et al. 2011), but similar well-documented examples for interactions among macroalgal adults are still lacking.
Allelopathic effects of adult macroalgae are commonly reported to have effects on early life stages of macroalgae, that is, propagules, spores and germlings (Råberg et al. 2005; Dworjanyn, de Nys & Steinberg 2006; Nylund et al. 2007), as well as on microalgae, bacteria and fungi (Harlin 1987; Ervin & Wetzel 2003; Gross 2003; Lane et al. 2009). Therefore, it is possible or even likely that the earliest life stages of native macroalgae are more heavily affected than adults by novel weapons in non-indigenous invasive macroalgae, in accordance with the results of the second experiment in this study. There was a very strong inhibitory effect of 1,1,3,3-tetrabromo-2-heptanone on the settlement of macroalgal spores of the native red algae C. virgatum and P. stricta and gametes from the green alga U. lactuca, as well as cells of the epiphytic microalga Cylindrotheca fusiformis. The surface concentration of 1,1,3,3-tetrabromo-2-heptanone in B. hamifera range from approximately 2 to 5 μg cm−2 in natural populations (Nylund et al. 2008). There were, however, significant negative effects on settlement of propagules from C. virgatum and U. lactuca already at 0.06 μg cm−2, which is about one-hundredth of the natural surface concentration. At concentrations of 0.5–1 μg cm−2, the settlement was reduced to a minimum, and at the natural surface concentration, no spores from C. virgatum and P. stricta settled on the coated surfaces.
This highly effective inhibition of settlement of natives parallels the reported negative effects on native plant propagules of compounds from the non-indigenous plants in North America, which gave rise to the NWH (Callaway & Ridenour 2004). Even though the specific mechanism causing the striking dominance of Centaurea stoebe (formerly maculosa) in the invaded range remains to be elucidated (Callaway et al. 2011), as the high levels of (−)-catechin initially reported for Centaurea stoebe are now revised (Bais et al. 2010), there are many other examples. For instance, Vivanco et al. (2004) showed that the active compound in root exudates of C. diffusa, 8-hydroxyquinoline, inhibits the root and shoot differentiation and germination of all the seven native species included in the study. Similarly, in the recent study by Inderjit et al. (2011a), volatile organic compounds (VOCs) from the leaf litter of the invader A. adenophora significantly reduced the seedling length and germination of the native plants Bidens biternata and Bambusa arundinacea. The invasive plant S. terebinthifolius has also been shown to decrease germination of seeds and seedling biomass for the native species Bidens alba and Rivina humilis through aqueous extracts from its leaves (Morgan & Overholt 2005), as well as shown to reduce the number of leaves on seedlings of the native mangroves Rhizophora mangle and Avicennia germinans grown in soil with natural levels of crushed S. terebinthifolius fruits (Donnelly, Green & Walters 2008). Thus, similar to previous studies on non-indigenous terrestrial plants, the chemical weapon of the macroalga in this study also shows very strong effects on early life stages of native species in the new range.
In our coating experiments, we could show both in the laboratory and in the field that 1,1,3,3-tetrabromo-2-heptanone can be transferred to a remarkably high extent to the surfaces of the native host algae F. lumbricalis and C. officinalis, on which B. hamifera commonly grows as an epiphyte. The concentration of the metabolite on C. officinalis measured in the field experiment was high enough to reduce virtually all settlement of all the native species that were tested. By spreading its highly inhibitory compound to surrounding surfaces, B. hamifera can thus prevent native species to settle in adjacent areas, which could be regarded as a form of effective pre-emptive competition. Pre-emptive competition is a fundamental process in ecological communities (Schoener 1983) and has been shown to facilitate the invasion by amphipod species as they rapidly occupy available habitats and make them inaccessible to natives (van Riel et al. 2007). To our knowledge, the only related aquatic example of transferred allelochemicals concerns fischerellin A from the benthic cyanobacteria Fischerella musicola, which inhibits photosynthesis of other cyanobacteria (Gross, Wolk & Jüttner 1991). This compound was argued to be directly transferred by cell–cell contact, because it could be extracted from lipophilic beads but not the surrounding culture medium (Gross 1999). Hence, although not discussed by the authors, if the compound adheres to lipophilic surfaces and remains active long enough to affect settled cells, this could be a form of pre-emptive competition among microorganisms.
The ability of B. hamifera to spread its secondary metabolites to the surrounding environment also shows interesting possible similarities to mechanisms of invasion by exotic plants in terrestrial systems. Recent studies show that invading plants can alter the composition of microbes in the soil through exudation of defence compounds and thereby affect soil–plant interactions (Reinhart & Callaway 2006). More specifically, Alliaria petiolata has been shown to reduce both the survival and settlement of native plant species by inhibiting the growth of soil microbes through its root exudates (Stinson et al. 2006; Callaway et al. 2008). In marine systems, biofilms of substrata could be regarded as the equivalence to soil microbe communities, and the composition of biofilms has been shown to affect settlement of marine invertebrate and algal propagules (Steinberg, de Nys & Kjelleberg 2002). In natural populations of B. hamifera, settlement inhibition of epiphytes could also be related to the fact that the 1,1,3,3-tetrabromo-2-heptanone has strong negative effects against marine bacteria (Nylund et al. 2007;; Nylund et al. 2008) and alters the bacterial composition of biofilms in field experiments (Persson et al. 2011). However, regardless if the settlement inhibition of natives is related to altered biofilms or more direct allelopathic effects, the ability to ‘reserve’ space is likely to be of equal, or higher, importance in benthic sessile communities, where free substratum is a major limiting resource and competition for space is severe (Paine 1966; Lubchenco & Menge 1978; Petraitis, Latham & Niesenbaum 1989).
In conclusion, we show that the non-indigenous invasive red alga B. hamifera has strong allelopathic effects on its common native competitors. The secondary compound 1,1,3,3-tetrabromo-2-heptanone of B. hamifera strongly inhibited the settlement of native propagules, and it has previously been shown to strongly deter native grazers and to have significant effects on native bacteria and biofilms. We also show, for the first time, that an ecologically active secondary metabolite can be transferred from a macroalga to adjacent surfaces at inhibitory concentrations. Due to the strong effects on different groups of native species of the multipurpose defence compound 1,1,3,3-tetrabromo-2-heptanone, we conclude that the NWH is a viable explanation for the invasive success of B. hamifera in the Northern Atlantic.