An invasive foundation species enhances multifunctionality in a coastal ecosystem.

While invasive species often threaten biodiversity and human well-being, their potential to enhance functioning by offsetting the loss of native habitat has rarely been considered. We manipulated the abundance of the nonnative, habitat-forming seaweed Gracilaria vermiculophylla in large plots (25 m2) on southeastern US intertidal landscapes to assess impacts on multiple ecosystem functions underlying coastal ecosystem services. We document that in the absence of native habitat formers, this invasion has an overall positive, density-dependent impact across a diverse set of ecosystem processes (e.g., abundance and richness of nursery taxa, flow attenuation). Manipulation of invader abundance revealed both thresholds and saturations in the provisioning of ecosystem functions. Taken together, these findings call into question the focus of traditional invasion research and management that assumes negative effects of nonnatives, and emphasize the need to consider context-dependence and integrative measurements when assessing the impact of an invader, including density dependence, multifunctionality, and the status of native habitat formers. This work supports discussion of the idea that where native foundation species have been lost, invasive habitat formers may be considered as sources of valuable ecosystem functions.

Differentiation of haploid and diploid fertilities in Gracilaria chilensis affect ploidy ratio.

BACKGROUND: Algal isomorphic biphasic life cycles alternate between free-living diploid (tetrasporophytes) and haploid (dioicious gametophytes) phases and the hypotheses explaining their maintenance are still debated. Classic models state that conditional differentiation between phases is required for the evolutionary stability of biphasic life cycles while other authors proposed that the uneven ploidy abundances observed in the field are explained by their cytological differences in spore production. RESULTS: We monitored the state and fate of individuals of the red seaweed Gracilaria chilensis periodically for 3 years in five intertidal pools from two sites with distinct conditions. We tested for differentiation in fecundity and spore survival among the gametophyte males and females (haploids) and the tetrasporophytes (diploids). We tested for the influence of fecundity and spore survival on the observed uneven ploidy abundances in recruits. The probability of a frond becoming fecund was size-dependent, highest for the haploid males and lowest for the haploid females, with the diploids displaying intermediate probabilities. Fecund diploids released more tetraspores than carpospores released by the haploid females. Spore survival depended on ploidy and on the local density of co-habiting adult fronds. An advantage of diploid over haploid germlings was observed at very low and very high adult fronds densities. CONCLUSIONS: Neither spore production nor spore survival determined the highly variable ploidy ratio within G. chilensis recruits. This result invalidates the hypothesis of natural cytological differences in spore production as the only driver of uneven field ploidy abundances in this species. Diploid spores (carpospores) survived better than haploid spores (tetraspores), especially in locations and time periods that were associated with the occurrence of strong biotic and abiotic stressors. We hypothesise that carpospore survival is higher due to support by their haploid female progenitors passing-on nutrients and chemical compounds improving survival under stressful conditions.

Nonnative Gracilaria vermiculophylla tetrasporophytes are more difficult to debranch and are less nutritious than gametophytes.

Theory predicts that the maintenance of haplodiplontic life cycles requires ecological differences between the haploid gametophytes and diploid sporophytes, yet evidence of such differences remain scarce. The haplodiplontic red seaweed Gracilaria vermiculophylla has invaded the temperate estuaries of the Northern Hemisphere, where it commonly modifies detrital and trophic pathways. In native populations, abundant hard substratum enables spore settlement, and gametophyte:tetrasporophyte ratios are ~40:60. In contrast, many non-native populations persist in soft-sediment habitats without abundant hard substratum, and can be 90%-100% tetrasporophytic. To test for ecologically relevant phenotypic differences, we measured thallus morphology, protein content, organic content, "debranching resistance" (i.e., tensile force required to remove a branch from its main axis node), and material properties between male gametophytes, female gametophytes, and tetrasporophytes from a single, nonnative site in Charleston Harbor, South Carolina, USA in 2015 and 2016. Thallus length and surface area to volume ratio differed between years, but were not significantly different between ploidies. Tetrasporophytes had lower protein content than gametophytes, suggesting the latter may be more attractive to consumers. More force was required to pull a branch from the main axis of tetrasporophytes relative to gametophytes. A difference in debranching resistance may help to maintain tetrasporophyte thallus durability relative to gametophytes, providing a potential advantage in free-floating populations. These data may shed light on the invasion ecology of an important ecosystem engineer, and may advance our understanding of life cycle evolution and the maintenance of life cycle diversity.

Nutrients, but not genetic diversity, affect Gracilaria chilensis (Rhodophyta) farming productivity and physiological responses.

In terrestrial plants, it is well known that genetic diversity can affect responses to abiotic and biotic stress and have important consequences on farming. However, very little is known about the interactive effects of genetic and environmental factors on seaweed crops. We conducted a field experiment on Gracilaria chilensis to determine the effect of heterozygosity and nutrient addition on two southern Chilean farms: Ancud and Chaica. In addition to growth rate and productivity, we measured photosynthetic responses, photosynthetic pigment concentration (chlorophyll a and phycobiliproteins), C:N ratio (C:N), and epiphytic load. Nutrient addition affected the growth rate, productivity, phycobilin, and C:N content, but not the epiphytic load. These results were independent of the heterozygosity of the strains used in the experiments. Interestingly, depending on the sampled sites, distinct photosynthetic responses (i.e., maximal quantum yield, Fv /Fm , and maximal electron transport rate, ETRmax ) to nutrient addition were observed. We propose that thallus selection over the past few decades may have led to ecological differentiation between G. chilensis from Chaica and Ancud. The lack of effect of heterozygosity on growth and physiological responses could be related to the species domestication history in which there is a limited range of genetic variation in farms. We suggest that the existing levels of heterozygosity among our thalli is not sufficient to detect any significant effect of genetic diversity on growth or productivity in Metri bay, our experimental site located close to the city of Puerto Montt, during summer under nitrogen limiting conditions.

The effect of polar auxin transport on adventitious branches formation in Gracilaria lichenoides in vitro.

Seaweed tissue culture (STC) is an important micropropagation tool that has been applied for strain improvement, micropropagation and genetic engineering. Because the mechanisms associated with STC are poorly understood, its application to these organisms lags far behind that of tissue culture propagation of higher plants. Auxin, calcium (Ca2+ ) and hydrogen peroxide (H2 O2 ) fluxes all play key roles during plant growth and development. In this study, we therefore measured indole-3-acetic acid, Ca2+ and H2 O2 fluxes of Gracilaria lichenoides explants during adventitious branches (ABs) formation for the first time using noninvasive micro-test technology. We confirmed that polar auxin transport (PAT) also occurs in the marine red alga G. lichenoides. We additionally found that N-1-naphthylphthalamic acid may suppress auxin efflux via ABCB1 transporters and then inhibit ABs formation from the apical region of G. lichenoides segments. The involvement of Ca2+ and H2 O2 fluxes in PAT-mediated AB formation in G. lichenoides was also investigated. We propose that complex feedback among Ca2+ , H2 O2 and auxin signaling and response systems may occur during ABs polar formation in G. lichenoides explants, similar to that in higher plants. Our results provide innovative insights that should aid future elucidation of mechanisms operative during STC.

Marine heatwaves alter competition between the cultured macroalga Gracilariopsis lemaneiformis and the harmful bloom alga Skeletonema costatum.

Seaweed cultivation can inhibit the occurrence of red tides. However, how seaweed aquaculture interactions with harmful algal blooms will be affected by the increasing occurrence and intensity of marine heatwaves (MHWs) is unknown. In this study, we run both monoculture and coculture systems to investigate the effects of a simulated heatwave on the competition of the economically important macroalga Gracilariopsis lemaneiformis against the harmful bloom diatom Skeletonema costatum. Coculture with G. lemaneiformis led to a growth decrease in S. costatum. Growth and photosynthetic activity (Fv/Fm) of G. lemaneiformis was greatly reduced by the heatwave treatment, and did not recover even after one week. Growth and photosynthetic activity of S. costatum was also reduced by the heatwave in coculture, but returned to normal during the recovery period. S. costatum also responded to the stressful environment by forming aggregates. Metabolomic analysis suggests that the negative effects on S. costatum were related to an allelochemical release from G. lemaneiformis. These findings show that MHWs may enhance the competitive advantages of S. costatum against G. lemaneiformis, leading to more severe harmful algal blooms in future extreme weather scenarios.

Flow enhances photosynthesis in marine benthic autotrophs by increasing the efflux of oxygen from the organism to the water.

Worldwide, many marine coastal habitats are facing rapid deterioration due in part to human-driven changes in habitat characteristics, including changes in flow patterns, a factor known to greatly affect primary production in corals, algae, and seagrasses. The effect of flow traditionally is attributed to enhanced influx of nutrients and dissolved inorganic carbon (DIC) across the benthic boundary layer from the water to the organism however, here we report that the organism's photosynthetic response to changes in the flow is nearly instantaneous, and that neither nutrients nor DIC limits this rapid response. Using microelectrodes, dual-pulse amplitude-modulated fluorometry, particle image velocimetry, and real time mass-spectrometry with the common scleractinian coral Favia veroni, the alga Gracilaria cornea, and the seagrass Halophila stipulacea, we show that this augmented photosynthesis is due to flow-driven enhancement of oxygen efflux from the organism to the water, which increases the affinity of the RuBisCO to CO(2). No augmentation of photosynthesis was found in the absence of flow or when flow occurred, but the ambient concentration of oxygen was artificially elevated. We suggest that water motion should be considered a fundamental factor, equivalent to light and nutrients, in determining photosynthesis rates in marine benthic autotrophs.

Pollinators of the sea: A discovery of animal-mediated fertilization in seaweed.

The long-held belief that animal-mediated pollination is absent in the sea has recently been contradicted in seagrasses, motivating investigations of other marine phyla. This is particularly relevant in red algae, in which female gametes are not liberated and male gametes are not flagellated. Using experiments with the isopod Idotea balthica and the red alga Gracilaria gracilis, we demonstrate that biotic interactions dramatically increase the fertilization success of the alga through animal transport of spermatia on their body. This discovery suggests that animal-mediated fertilization could have evolved independently in terrestrial and marine environments and raises the possibility of its emergence in the sea before plants moved ashore.

Did pollination exist before plants?

Research shows that seaweeds depend on crustaceans for fertilization.

Impact of exotic macroalga on shorebirds varies with foraging specialization and spatial scale.

Exotic species may increase or decrease native biodiversity. However, effects of exotic species are often mixed; and indirect pathways and compensatory changes can mask effects. Context-specific assessments of the indirect impacts of exotic species are also needed across multiple spatial scales. Agarophyton vermiculophyllum (previously Gracilaria vermiculophylla), an exotic, invasive macroalga, has established throughout the western hemisphere with reported positive or neutral impacts on biodiversity. Shorebirds are an important group for conservation in areas invaded by A. vermiculophyllum. We assess the impacts of this invader on shorebirds by measuring behavior and habitat selection at spatial scales ranging from algal patches to the entire study region. Birds were considered either flexible-foragers that used diverse foraging techniques, or specialized-foragers that employed fewer, more specialized foraging techniques. Responses were scale dependent, with patterns varying between spatial scales, and between behavior and habitat selection. However, a general pattern of habitat selection emerged wherein flexible-foraging shorebirds preferred A. vermiculophyllum habitat, and for specialized-foragers, habitat selection of A. vermiculophyllum was mixed. Meanwhile, flexible-foraging birds tended to neutrally use or avoid uninvaded habitat, and specialized-foraging birds mostly preferred uninvaded habitat. Shorebird behavioral response was less clear; with flexible-foragers spending less time on bare sediment than expected, the only significant response. Shorebird response to A. vermiculophyllum differed by foraging mode; likely because flexible, opportunistic species more readily use invaded habitat. Increases in A. vermiculophyllum could result in functional homogenization if the bare habitat preferred by specialized-foragers is reduced too greatly. We hypothesize the effect of scale is driven by differences among tidal flats. Thus, tidal flat properties such as sediment grain size and microtopography would determine whether foraging from A. vermiculophyllum was optimal for a shorebird. Specialization and spatial scale are important when assessing the biodiversity conservation impacts of invasive A. vermiculophyllum.

Multiple effects of a Gracilaria vermiculophylla invasion on estuarine mudflat functioning and diversity.

The invasive Japanese seaweed Gracilaria vermiculophylla has become established over the past several years in numerous European estuaries, from Portugal to Norway. In the Faou estuary (48.295°N-4.179°W, Brittany, France), it forms a dense population at the mud's surface. The effects of G. vermiculophylla on metabolism, diversity, and the food web were studied. Community gross primary production (GPP) and respiration (CR) during emersion, chlorophyll-a content, macrofaunal and meiofaunal diversity and abundance, and stable isotopes (δ13C and δ15N) of representative macrofaunal species and main food sources were measured at low tide in winter, spring, summer 2014, and winter 2015. Results show significant seasonal variation in GPP and CR. Moreover, GPP was significantly higher in areas where G. vermiculophylla was present than in the control area (bare mud). However, this high GPP appeared to be linked to the increase in biomass in primary producers, with their efficiency (primary productivity, i.e. assimilation number) remaining relatively stable compared with the control area. Significant variation in abundance of meiofauna and macrofauna was also detected and new epifaunal species were collected, mainly in Gracilaria-colonized areas. Isotopic food-web Bayesian mixing models strongly suggested that G. vermiculophylla plays a major role in the diet of some dominant species. Mechanisms interacting with the functioning and diversity of the mudflat are discussed. Finally, the invasive seaweed G. vermiculophylla affected the mudflat ecosystem in three ways: as a new primary producer (increase in metabolism), as a habitat-forming species (changes in diversity and abundance of macrofauna and meiofauna), and as a new abundant food source, likely through the detrital pathway.

The photosynthetic responses to stocking depth and algal mat density in the farmed seaweed Gracilaria lemaneiformis (Gracilariales, Rhodophyta).

The branches and mass of Gracilaria lemaneiformis increase with growth season, and the thalli sink to deeper depths with increasing biomass density during maricultivation. The changing depth and algal mat density may affect the physiology of the algae. In the present study, the photosynthetic behaviors regarding different biomass densities in G. lemaneiformis thalli collected from different stocking depths were determined, to examine how photosynthesis of this farmed alga was affected by the growth depths and algal mat densities. Our results showed that the chlorophyll a (Chl a), carotenoids (Car), phycoerythrin (PE) contents, and irradiance-saturated maximum photosynthetic rates (P max) of the deeper layer-grown algae were significantly increased relative to the surface layer-grown algae. The P max, apparent photosynthetic efficiency (α) and dark respiration rate (R d) of G. lemaneiformis thalli, were reduced, whereas the irradiance saturation points (I k) were increased, with the increasing algal mat density. We proposed that appropriate measures are needed to trade off the stocking depth and biomass density, in an effort to maintain a relative high photosynthetic productivity during G. lemaneiformis maricultivation.

Effects of season on the yield and quality of agar from Gracilaria species (Gracilariaceae, Rhodophyta).

The effect of season on yield and physical properties of agars extracted from Gracia gracilis and G. bursa-pastoris were determined. The agar yield from G. gracilis was maximum during spring (30%) and minimum during autumn (19%). In G. bursa-pastoris, the agar yield was greatest in summer (36%) and lowest in winter (23%). Agar yield from G. bursa-pastoris was positively correlated with temperature (r=0.94; P<0.01) and salinity (r=0.97; P<0.01) and negatively with nitrogen content (r=-0.93; P<0.01). Agar gel strengths fluctuated from 229 to 828 gcm(-2) and 23 to 168 gcm(-2) for G. gracilis and G. bursa-pastoris, respectively. The gelling temperature showed significant seasonal variation for both species. Chemical analysis of agar from the two seaweeds indicated variation in 3,6-anhydrogalactose and sulfate content (P<0.01). Furthermore, there was an inverse correlation between the two chemical variables. In general, agar extracted from G. gracilis possessed better qualities than agar extracted from G. bursa-pastoris and can be considered a candidate for industrial use.

Growth and nitrogen uptake characteristics reveal outbreak mechanism of the opportunistic macroalga Gracilaria tenuistipitata.

Macroalgae has bloomed in the brackish lake of Shenzhen Bay, China continuously from 2010 to 2014. Gracilaria tenuistipitata was identified as the causative macroalgal species. The aim of this study was to explore the outbreak mechanism of G. tenuistipitata, by studying the effects of salinity and nitrogen sources on growth, and the different nitrogen sources uptake characteristic. Our experimental design was based on environmental conditions observed in the bloom areas, and these main factors were simulated in the laboratory. Results showed that salinity 12 to 20 ‰ was suitable for G. tenuistipitata growth. When the nitrogen sources' (NH4+, NO3-) concentrations reached 40 µM or above, the growth rate of G. tenuistipitata was significantly higher. Algal biomass was higher (approximately 1.4 times) when cultured with NH4+ than that with NO3- addition. Coincidentally, macroalgal bloom formed during times of moderate salinity (∼12 ‰) and high nitrogen conditions. The NH4+ and NO3- uptake characteristic was studied to understand the potential mechanism of G. tenuistipitata bloom. NH4+ uptake was best described by a linear, rate-unsaturated response, with the slope decreasing with time intervals. In contrast, NO3- uptake followed a rate-saturating mechanism best described by the Michaelis-Menten model, with kinetic parameters Vmax = 37.2 µM g-1 DM h-1 and Ks = 61.5 µM. Further, based on the isotope 15N tracer method, we found that 15N from NH4+ accumulated faster and reached an atom% twice than that of 15N from NO3-, suggesting when both NH4+ and NO3- were available, NH4+ was assimilated more rapidly. The results of the present study indicate that in the estuarine environment, the combination of moderate salinity with high ammonium may stimulate bloom formation.

MIPS: a calmodulin-binding protein of Gracilaria lemaneiformis under heat shock.

To study the Ca(2+)/Calmodulin (CaM) signal transduction pathway of Gracilaria lemaneiformis under heat stress, myo-inositol-1-phosphate synthase (MIPS), a calmodulin-binding protein, was isolated using the yeast two-hybrid system. cDNA and DNA sequences of mips were cloned from G. lemaneiformis by using 5'RACE and genome walking procedures. The MIPS DNA sequence was 2,067 nucleotides long, containing an open reading frame (ORF) of 1,623 nucleotides with no intron. The mips ORF was predicted to encode 540 amino acids, which included the conserved MIPS domain and was 61-67 % similar to that of other species. After analyzing the amino acid sequence of MIPS, the CaM-Binding Domain (CaMBD) was inferred to be at a site spanning from amino acid 212 to amino acid 236. The yeast two-hybrid results proved that MIPS can interact with CaM and that MIPS is a type of calmodulin-binding protein. Next, the expression of CaM and MIPS in wild-type G. lemaneiformis and a heat-tolerant G. lemaneiformis cultivar, "981," were analyzed using real-time PCR under a heat shock of 32 °C. The expression level displayed a cyclical upward trend. Compared with wild type, the CaM expression levels of cultivar 981 were higher, which might directly relate to its resistance to high temperatures. This paper indicates that MIPS and CaM may play important roles in the high-temperature resistance of G. lemaneiformis.

NH4+ enrichment and UV radiation interact to affect the photosynthesis and nitrogen uptake of Gracilaria lemaneiformis (Rhodophyta).

Solar ultraviolet radiation (UVR, 280-400 nm) is known to inhibit the photosynthesis of macroalgae, whereas nitrogen availability may alter the sensitivity of the algae to UVR. Here, we show that UV-B (280-315 nm) significantly reduced the net photosynthetic rate of Gracilaria lemaneiformis. This inhibition was alleviated by enrichment with ammonia, which also caused a decrease in dark respiration. The presence of both UV-A (315-400 nm) and UV-B stimulated the accumulation of UV-absorbing compounds. However, this stimulation was not affected by enrichment with ammonia. The content of phycoerythrin (PE) was increased by the enrichment of ammonia only in the absence of UVR. Ammonia uptake and the activity of nitrate reductase were repressed by UVR. However, exposure to UVR had an insignificant effect on the rate of nitrate uptake. In conclusion, increased PE content associated with ammonia enrichment played a protective role against UVR in this alga, and UVR differentially affected the uptake of nitrate and ammonia.

Potential effects of the invasive species Gracilaria vermiculophylla on Zostera marina metabolism and survival.

The potential threat to seagrasses of the invasive algae, Gracilaria vermiculophylla was assessed through metabolic indicators under experimental conditions. Net leaf photosynthesis (LNP) and dark respiration (LDR) were measured from leaf segments of Zostera marina shoots under different loads of G. vermiculophylla (control, low 2.2kg FW m(-2) and high 4kg FW m(-2)) in mesocosm experiments separated in tanks at four temperatures (19, 23.5, 26 and 30 degrees C). LNP decreased in the presence of the high density G. vermiculophylla mat (25% on average), being the most severe reductions at 30 degrees C (35% less in high). LDR did not respond significantly to differences in algal biomass, whereas a progressive increase was found with increasing temperatures (3.4 times higher at 30 degrees C than at 19 degrees C). Sulphide in porewater was measured weekly in order clarify the role of sediment conditions on seagrass metabolism, and increased both with algal biomass (29% in high) and temperature (from 0.5mM at 26 degrees C to 2.6mM at 30 degrees C), but changes in LNP and LDR were not correlated with sulphide concentrations. Seagrass survival rates showed decreasing trend with algal biomass at all the temperatures (from 74% to 21% survival). G. vermiculophylla showed harmful effects on Z. marina metabolism and survival with synergistic effects of temperature suggesting greater impact of invasive species under future higher water temperatures.

Use of UV-A energy for photosynthesis in the red macroalga Gracilaria lemaneiformis.

UV radiation is known to inhibit photosynthetically active radiation (PAR)-driven photosynthesis; however, moderate levels of UV-A have been shown to enhance photosynthesis and growth rates of some algae. Here, we have shown that UV-A alone could drive photosynthetic utilization of bicarbonate in the red alga Gracilaria lemaneiformis as evidenced in either O(2) evolution or carbon fixation as well as pH drift. Addition of UV-B inhibited the apparent photosynthetic efficiency, raised the photosynthetic compensation point and photosynthesis-saturating irradiance level, but did not significantly affect the maximal rate of photosynthetic O(2) evolution. The electron transport inhibitor, DCMU, inhibited the photosynthesis completely, reflecting that energy of UV-A was transferred in the same way as that of PAR. Inorganic carbon acquisition for photosynthesis under UV alone was inhibited by the inhibitors of carbonic anhydrase. The results provided the evidence that G. lemaneiformis can use UV-A efficiently to drive photosynthesis based on the utilization of bicarbonate, which could contribute significantly to the enhanced photosynthesis in the presence of UV-A observed under reduced levels of solar radiation.

Effect of nutrient supply on photosynthesis and pigmentation to short-term stress (UV radiation) in Gracilaria conferta (Rhodophyta).

The effects of increased photosynthetic active radiation (PAR), UV radiation (UVR), and nutrient supply on photosynthetic activity, pigment content, C:N ratio and biomass yield were studied in tank cultivated Gracilaria conferta (Rhodophyta). Electron transport rate (ETR) and biliprotein content were higher under high nutrient supply (HNS), obtained from fishpond effluents, compared to low nutrient supply (LNS), in contrast to mycosporine-like amino acids (MAAs) dynamic. The high MAA content in LNS-algae could be explained by higher UVR penetration in the thallus and by the competition for the use of nutrients with other processes. Effective quantum yield decreased after short-term exposure to high irradiance whereas full recovery in shade was produced only under slightly heat shock. UVA radiation provoked an additional decrease in photosynthesis under high water temperature. UVB radiation reversed UVA's negative effect mainly with HNS. Results support that nutrient-sufficiency help G. conferta to resist environmental changes as short-term temperature increase.

[Physiological responses of Gracilaria lemaneiformis to copper stress].

Gracilaria lemaneiformis was exposed to 0, 25, 50, 100, 250 and 500 microg x L(-1) of Cu2+ to study its physiological responses to Cu2+ stress. When the Cu2+ concentration was > or = 50 microg x L(-1), the relative growth rate (RGR) of G. lemaneiformis decreased significantly, and the optimal quantum yield (Fv/Fm), the maximum relative electron transfer rate (rETRmax), and the relative electron transfer efficiency (alpha) exhibited the same variation trend, compared with the control. With the increase of Cu2+ concentration, the maximum net photosynthetic rate (Pmax) and light saturation point (LSP) decreased significantly, light compensation point (LCP) had a significant increase, while chlorophyll a, carotenoid, and phycobiliprotein contents decreased after an initial increase. When the Cu2+ concentration reached 500 microg x L(-1), the chlorophyll a, carotenoid, and phycobiliprotein contents decreased significantly. It was suggested that G. lemaneiformis could tolerate low concentration Cu2+ stress, but its physiological activities were inhibited markedly when exposed to > or =50 microg x L(-1) of Cu2+.