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Comparative time-courses of photoacclimation by Hawaiian native and invasive species of Gracilaria (Rhodophyta)
Aquatic Botany 163 (2020) 103210
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Comparative time-courses of photoacclimation by Hawaiian native and invasive species of Gracilaria (Rhodophyta)
T
Ken Hamel*, Celia M. Smith Department of Botany, University of Hawai‘i at Mānoa, 3190 Maile Way, Honolulu, HI 96822, USA
ARTICLE INFO
ABSTRACT
Keywords: Gracilaria salicornia Gracilaria coronopifolia Invasive species trait Photoacclimation Coral reef macroalgae
Invasive seaweeds have rapidly increased in global distribution and abundance in recent years, presenting significant threats to nearshore ecosystems. Developing characteristics diagnostic of potential macroalgal invasiveness is an important step in controlling their spread. Studies have identified photoacclimation, the ability of the photosynthetic apparatus to adjust to changes in light, as one important attribute in the success of several invasive terrestrial and marine plants; several studies indicate that the rate of photoacclimation plays a role in the differential success of invasive plants at the expense of natives. In this experiment, we compare two closely related species that occur in Hawaiian coastal waters with similar distribution, habitat, and morphology, the invasive Gracilaria salicornia, and the native G. coronopifolia. Samples were grown in outdoor culture, acclimated to one of two irradiance regimes, and reciprocally transplanted. Photoacclimation rates were assessed using in vivo spectrophotometry and PAM fluorometry. Results indicate that G. salicornia has remarkable tolerance to irradiance extremes and experiences rapid growth when transplanted from low to high photosynthetic photon flux density environments, and that differential rates of overall photoacclimation are insufficient to explain the relative success of these two plants. Photoacclimation is achieved via several complex processes that do not necessarily occur contemporaneously. Future researchers should be wary of assuming its completion based on single indicators.
1. Introduction Invasive and bloom forming marine macroalgae are major threats to reef ecosystems in many regions (Smith et al., 2002; Williams and Smith, 2007; van Tussenbroek et al., 2017; Veazey, 2018; Anton et al., 2019). Proliferations of macroalgae can negatively impact nearshore marine environments in many ways, including reducing benthic species diversity by outcompeting corals, native macroalgae, and seagrasses, by altering environmental parameters such as water quality and substrate conditions to states unfavorable to pre-existing organisms, and by reducing demersal species diversity by interfering with established food webs (Olenin et al., 2007; Smith et al., 2001; Schaffelke and Hewitt, 2007). Identifying characteristics of algae that may play a role in their invasiveness may contribute to reducing future introductions by aiding appropriate characterization of potential invaders, and by aiding management efforts via deepening our understanding of what environmental conditions promote invasive behavior. Increasing the efficacy of prevention and management is particularly important in shallow reef environments at high risk of biological invasions as with Hawaiian coastal waters.
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One characteristic that may be instrumental in the invasiveness of plants is photoacclimation, the adjustment of the photosynthetic apparatus in response to changes in irradiance. There has been growing interest in examining photoacclimatory capacity to diagnose invasive potential in both terrestrial (Yamashita et al., 2002; Feng et al., 2007a; Liu et al., 2016) and marine (Raniello et al.;, 2006; Dailer, 2006; Mehnert et al., 2012) plants. Evidence suggests that plants that can acclimate to a broader range of light conditions exercise competitive advantage over other plants (Raniello et al., 2004; Feng et al., 2007a). High shade tolerance and a large proportional increase in photosynthetic rate after high-light exposure are also photoacclimatory attributes that appear to favor invasive plants (Yamashita et al., 2000; Feng et al., 2007b). Photoacclimation may refer to one or more reversible changes to the size and/or number of photosynthetic units (PSU) in a cell, changes in the relative pigment content within a cell, or changes in photochemistry as a result of the plant’s exposure to higher (“sun” state) or lower (“shade” state) photon flux densities (Falkowski and LaRoche, 1991; Cunningham et al., 1992). These changes allow a plant to both avoid the potentially damaging effects of high irradiances as well as allow for
Corresponding author at: Department of Marine Affairs, University of Rhode Island, Coastal Institute Room 117, 1 Greenhouse Rd, Kingston, RI 02881 USA. E-mail addresses: [email protected] (K. Hamel), [email protected] (C.M. Smith).
https://doi.org/10.1016/j.aquabot.2020.103210 Received 19 July 2019; Received in revised form 3 January 2020; Accepted 18 January 2020 Available online 22 January 2020 0304-3770/ © 2020 Elsevier B.V. All rights reserved.
Aquatic Botany 163 (2020) 103210
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increased light harvesting at lower irradiances. For rhodophytes, acclimation to changing irradiance includes reciprocal changes in the concentration of chlorophyll a (Chl a) and phycobilins, and corresponding changes in carotenoid concentration and photosynthetic output (Waaland et al., 1974; Levy and Gantt, 1988; Beach et al., 2000; Payri et al., 2001). Photoacclimation is often described as requiring several days to weeks to occur (Gantt, 1990), but acclimation time has in fact rarely been measured and the point at which acclimation is attained remains poorly defined. Some phytoplankters fully acclimate in a matter of hours (Prézelin and Matlick, 1980) to days (Fisher et al., 1996), while corals may take between a few days to several weeks (Titlyanov et al., 2001). The few measurements made of macroalgae photoacclimation time range from eight days for Gracilaria chilensis (Glogau Stevens, 1992) to about one month for the Hawaiian native Ahnfeltiopsis concinna (Beach et al., 1997). Among the various estimates there has been little congruence as to the methodology used to determine the photoacclimation time course, making comparisons among taxa challenging. There is some evidence that invasive algae acclimate to changing irradiance faster than non-invasive Hawaiian algae (Beach, 1996; Dailer, 2006), giving invasives a potential advantage in rebounding from disturbances that scour marine substrates or displace existing animal and plant species; physical disturbances that dislodge benthos can generate intense competition for newly exposed substrate, while outcompeting established natives for other resources such as limiting nutrients may allow introduction of a potential invader to novel environments (Williams and Smith, 2007). Rapid photoacclimation may also allow an invasive with the ability to adjust to a wide range of light regimes that might otherwise be occupied by less phenotypically flexible algae. Photoacclimation response time may be a factor in determining future threats in the form of both native and alien algae and if so, its study can help us make decisions regarding what to look for in terms of invasive characters. Gracilaria salicornia (C. Agardh) E.Y. Dawson is a non-indigenous intertidal alga invasive to Hawaii that is currently found along much of Oʻahu’s south and windward shores; it most often is found as a freestanding plant, reaching lengths of up to 18 cm, with axis 1.5–4 mm thick (Abbott, 1999). Along several south shore beaches, G. salicornia forms extensive mats that can be over 10 cm thick and 2 or more meters across (Beach et al., 1997; Larned, 1998). Within a mat, tissues range in color from yellow in the canopy regions where they may be exposed at low tide to a dark purple understory, a result of self-shading (Beach et al., 1997). A native congener of this species, Gracilaria coronopifolia (J. Agardh), is a comparatively small (usually <8 cm long and 1−2 mm in diameter; Abbott, 1999) shallow-subtidal alga that is considered endemic to the Hawaiian Islands (Abbott, 1999 but see e.g. Lobban and Tsuda, 2003). G. coronopifolia exhibits pale yellow to white tissue in areas with high exposure to sunlight, and pink to red when shaded. In contrast to G. salicornia, G. coronopifolia is increasingly uncommon, at least in part from overharvesting due to its popularity as a local food. Both species are restricted to waters above a 4 m depth, and though only G. salicornia is truly intertidal, both are euryhaline, tolerating wide ranges of nutrients and salinity (Amato et al., 2018), occur often in the same locations, sometimes < 1 m apart, and exhibit analogous ranges of pigmentation resulting from differential exposure to incident solar irradiance. Though both exhibit a tendency to local dominance (Smith et al. 2004, pers. obs.), there are no data documenting interspecific competition. Three past studies have tracked both photosynthetic and pigment acclimation in response to changes in irradiance over time in tropical rhodophytes (Glogau Stevens, 1992; Beach et al., 2000; Dailer, 2006), with each examining a single species. Researchers have generally evaluated the presence, importance or magnitude of photoacclimation but have rarely considered the speed at which it occurs, especially when compared to other species; this is the first comparison of photoacclimation time-course of two species of algae grown under the same
conditions. This study tests the hypothesis that an invasive alga acclimates faster to changes to exposure in solar irradiance than a native congener. To this end, two congeners with similar local distributions, habitat, and morphology were reciprocally transplanted in an experimental mesocosm, and several physiological parameters measured in vivo. We predicted that the invasive species, G. salicornia, would react more rapidly than the native species, G. coronopifolia, to both increases and decreases in light quantity. 2. Methods 2.1. Collection of experimental materials Non-reproductive, representative G. salicornia and G. coronopifolia plants were collected at the seaward end of Paiko Lagoon, on O‘ahu’s south shore and at Kualoa Beach on O‘ahu’s windward shore over the span of eight months, from May to December of 2011 (Table S1). Additional samples of G. salicornia were collected from Kaimana Beach on the south shore, and of G. coronopifolia from windward Malaekahana Beach. Samples were selected for sun- or shade-acclimated pigmentation, i.e. paler or darker, respectively. Species determinations for each population were made in the field using morphological characters, and in the laboratory using primers and methods after Sherwood and Presting (2007) for the universal plastid amplicon (UPA) marker, part of the plastid 23S rDNA. 2.2. Reciprocal transplants in outdoor culture Between May and November of 2011, reciprocal transplants were performed in outdoor culture in five trials. Each trial consisted of two replicates for each of four treatments (sun to shade with its corresponding shade control and shade to sun with its corresponding sun control) of each of two species. Trials were conducted with ten replicate plants per treatment for a total of 80 individuals for the entire experiment. Photoacclimation was tracked and its effects measured every two days via PAM fluorometry, in vivo spectrophotometry and wet weight, in that order. All measurements were made on plants in the same order and at the same time, on each day. Each sample was maintained in a separate aerated, filtered seawater-filled 5 l aquaria set in a mesocosm – outdoor water baths on the balcony of the St. John Plant Science Building, University of Hawai‘i at Mānoa. Seawater was collected adjacent to the Makai Pier, near the easternmost tip of O‘ahu, and enriched with 10 μM NaNO3 and 2 μM PO4, to avoid nutrient limitation (Beach, 1996). This enriched seawater was replaced every other day. Water temperature of the cultures was maintained between 25 and 30 °C, measured with HOBO pendant loggers (Onset Computer Corporation, Pocasset, MA). Salinity was monitored periodically with a Reichert temperature compensated refractometer and adjusted to 34‰ with additions of distilled water. Salinity varied between 34 and 38‰ during the duration of the experiment. Water baths were shielded from rain with transparent polymethylmethacrylate sheet roofs. Samples of both species were initially acclimated to either full sunlight, with daily maxima ranging from ca. 1500–3300 μmol m−2 s−1 PAR (mean = 2450 μmol m−2 s−1 PAR), or shade (covered in three layers of medium grade shade-cloth), resulting in daily maxima ranging from ca. 70–180 μmol m−2 s−1 PAR (mean = 115 μmol m−2 s−1 PAR). These irradiances closely approximated those reported for G. salicornia field conditions in Beach et al. (1997). This acclimation period lasted for 11–17 days or until relative uniformity of plant color was achieved among samples. PAR was continuously monitored with LI−COR LI-193 underwater spherical quantum sensors placed in aquaria amongst, but separate from, the samples. Mean irradiance was sampled at 15-min intervals and stored in LI−COR LI-1000 data loggers. After the initial acclimation period, samples from each irradiance treatment were 2
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1) rETRmax as the product of PAR, ETR-Factor, PPS2/PPPS and Y(II), where ETR-Factor is the ratio of photons absorbed by photosynthetic pigments to incident photons, PPS2/PPPS is the ratio of photons absorbed by PSII to photons absorbed by photosynthetic pigments, and Y(II), the effective photochemical quantum yield of PSII and 2) Ek as ETRmax/α, where α is the initial slope of the RLC. 2.4. In vivo absorbance analyses An in vivo absorbance spectrum (400–750 nm) was acquired from each sample immediately after each fluorometric measurement. Spectra were obtained using a Shimadzu UV Vis-2101 spectrophotometer with a 150 mm integrating sphere following Beach et al. (1997). Specimens were analyzed by placing a non-overlapping layer of tissue between two white cards with a mask window cut of 1.0 cm by 0.7 cm. Spectra were normalized to Chl a maxima at ∼678 nm after spectra acquisition after Smith and Alberte (1994); these pigment-to-chlorophyll ratios act as proxies for pigment concentrations in living tissue that could otherwise only be obtained via destructive solvent extraction. The specific absorbance maxima of accessory pigment-to-chlorophyll ratios that were selected for analysis were allophycocyanin (APC) at 649:678 nm, phycocyanin (PC) at 625:678 nm, phycoerythrin (PE) at 568:678, a combination of carotenoid and phycoerythrin (Car/PE) at 495:678 nm, and a combination of carotenoid and Chl a (Car/Chl) at 440:678 nm (Fig. 2). These pigment identities are based on published absorbance maxima (Haxo and Blinks, 1950; Smith and Alberte, 1994; Beach et al., 1997, 2000).
Fig. 1. Change in the ratio of 568:678 nm (PE) absorbance thru time for replicates of Gracilaria coronopifolia samples transferred from sun to shade environment (solid line). Samples were considered acclimated when parameter values ceased to be significantly different than control values (dashed line). *P < 0·05; **P < 0·01; ***P < 0·001. Mean ± SE, n = 10.
placed in their correspondingly alternate irradiance regimes, i.e. full sunlight (high light or HL) acclimated samples were placed in shaded (low light or LL) water baths and vice versa. 2.3. PAM fluorometry
2.5. Growth
PAM fluorometry parameters were measured using a Walz JUNIORPAM fluorometer every two days starting from the day that the samples were transplanted. Photosynthetic parameters were assessed by exposing the second or third node from the tip (ca. 3–6 cm from the tip for G. salicornia samples and 2–4 cm for G. coronopifolia) on each specimen to the fluorometer’s fiber optic cable, which was secured with a Walzsupplied magnetic leaf clip. Rapid light curves (RLC) were initiated immediately, without “dark-acclimation”, and generated by exposing tissue to a series of saturation pulses applied after 10 s exposures to actinic irradiance of increasing exposure ranging from 25 to 420 μmol m−2 s−1 PAR in LL acclimated samples and from 66 to 820 μmol m−2 s−1 PAR in HL acclimated samples (following Beer and Axelsson, 2004). Different PAR ranges were required for HL and LL acclimated samples as lower level PAR exposure in HL samples resulted in an incomplete RLC, while PAR pulses higher than 420 resulted in a depressed effective quantum ((Fm′-F)/Fm′) yield below a reliable threshold (as per Beer and Axelsson, 2004) in LL samples. The photosynthetic parameters rETRmax (relative maximum electron transport rate), and Ek (light saturation index) were obtained from RLC data with Wincontrol-3 software (Heinz Walz GmbH, Effeltrich, Germany). Wincontrol-3 calculates
Growth was measured by obtaining wet weight immediately after each spectrum was obtained. Tissue were blotted to remove excess water and specimens were weighed on a Sartorius analytical balance Model TE214S + 0.0001 gm. Mean relative growth rates (RGR) were measured using the method detailed by Hunt (1982): RGR = (lnWt2 -lnWt1)/(t2 -t1), where W is the total plant wet mass in grams and t is time in days. 2.6. Data analyses Two-way mixed ANOVA, with time as the within-subjects factor and light environment as the between-subjects factor, was used to determine whether treatment and control groups differed over time for photosynthetic parameters, pigment to chlorophyll ratios and growth rates using SPSS version 25 (IBM). When significant interactive effects occurred, a priori multiple comparisons with the False Discovery Rate correction (Benjamini and Hochberg, 1995) were conducted to determine how many days were needed for photosynthetic and pigment
Fig. 2. in vivo absorbance spectra of sun-acclimated (grey lines) and shade acclimated (black lines) G. salicornia (triangles) and G. coronopifolia (circles). All spectra normalized to chlorophyll a absorbance peak at 678 nm. n = 10. 3
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parameters of treatments to reach corresponding control values, and to determine differences between growth rates between treatments and controls.
Table 1 Summary of independent-samples t-tests of sun to shade treatments. n = 10. Values represent number of days samples of each species took to go from a HL state to a LL state for each parameter. For full graphical presentation of all data here summarized, see Figure S1.
3. Results Native G. coronopifolia did not perform as well as invasive G. salicornia in mesocosm culture; measuring days did not extend beyond 14 days for G. coronopifolia, in contrast to G. salicornia which was viable and measured for 16–20 days. One control and two treatment G. coronopifolia plants lost substantial mass in shaded culture by the end of their respective trials, though not before photoacclimation had been achieved, while the other samples maintained their vigor. G. salicornia plants responded more favorably to culture, with only one LL control sample losing substantial mass, although fragmentation often occurred as a result of dissolution between segments. The texture of plant bodies for both species became more flexible as they acclimated to high light and more turgid as the plants acclimated to low light. Treatments in this study took on the general visual appearance of their corresponding control, with regard to apparent pigmentation, with the exception of LL-HL samples of G. salicornia, which took on a light brown pigmentation with yellow branch tips rather than the yellow to orange exhibited by HL control samples and by sun-exposed plants in the field. Mixed ANOVA results revealed statistically significant interactions at the 0.05 level between light environment and time between each treatment and control pairing for both species across all PAM and pigment parameters except RGR, where only LL-HL G. salicornia demonstrated a difference (Table S2). Likewise, main effects for light environment were statistically significant between each treatment and control pairing for both species across all PAM and pigment parameters, reflecting the initial planned differences between control and treatment for each test. Main effects for time were statistically significant between each treatment and control pairing for both species across all PAM and pigment parameters, with three exceptions: G. salicornia shade to sun for PE and for Car/Chl, and for G. coronopifolia sun to shade for Car/ PE. Full acclimation for each parameter was considered to have been achieved at the first time when treatment values of each parameter ceased to be statistically different from corresponding control values (e.g. Fig. 1). The number of days required for this condition to be met varied by parameter, by treatment, and by species. Many treatment values that achieved acclimation did not remain static, but deviated from control values after first acclimating to the new light environment (Table S3). Similarly, parameter values for most control groups did not remain static, particularly for those samples exposed to prolonged periods of intense sunlight. Sun-acclimated samples exhibited high absorbance values at both carotenoid-associated absorbance maxima as well as reduced absorbance values at all phycobilin-specific maxima relative to shade-acclimated plants of both species (Fig. 2). Conversely, phycobilin absorbance values increased and the carotenoid absorbance decreased for shade acclimated plants.
G. coronopifolia G. salicornia
rETRmax
Ek
APC
PC
PE
Car/PE
Car/Chl
4 2
2 4
8 10
8 12
6 4
2 12
4 4
reached control levels faster in G. salicornia, while PC and PE response times were the same for both species, and Car/PE absorbance maxima adjusted faster in G. salicornia. Comparison of time course rates between species were indeterminable at the Car/Chl a absorbance maximum for G. coronopifolia as treatment values never reached those of controls; instead, both treatment and control increased as this maximum at about the same pace over the course of the experiment (Table 2). G. coronopifolia did not fully acclimate as judged by changes in the Car/Chl a maximum because treatment and control values reacted to their light environment at a similar rate, e.g. carotenoid values in the HL control never stabilized, but instead increased about as fast as carotenoid values in the LL-HL treatment. A similar phenomenon of increasing Car/Chl a absorbance in both treatment and control affected G. salicornia LL-HL results, but due to increased variance in the response of that species for that parameter, control and treatment ceased being statistically significant at day 6. RGRs were not significantly different between treatment and controls for G. coronopifolia. G. salicornia were significantly different between treatment and controls (Table S2), but differences did not emerge until after four days, with the treatment maintaining an increased mean RGR 51.72 % (±1.51 % SE) higher than that of the control for the duration of the experiment (Fig. 3). 4. Discussion This study compared rates of photoacclimation between two closely related, often co-occuring rhodophytes, one an invasive species, the other an increasingly uncommon native. Our goal was to better understand whether faster physiological responses to changes in ambient light can explain the relative success of the invader. This turned out not to be the case for these two Gracilaria species, though the results suggest that G. salicornia possesses other advantageous photoacclimatory characteristics not shared by its native congener. Feng et al. (2007a) compared several species of invasive and non-invasive embryophytes, determining that invasive plants could acclimate to a broader range of irradiances. Yamashita et al. (2000) found that an invasive tree recovered from a sudden increase in light faster than non-invasives. This study, the first to compare acclimation rates between native and invasive species of algae, found that while the invasive acclimated readily to a broader range of irradiance than did the native, it did not adjust faster to changing irradiance than the native plant; if judged by the overall rate of acclimation, i.e. the time at which all processes have finished acclimating, the invasive was slightly faster in adjusting to increased light, while the native was somewhat faster in adjusting to shade. However, the overall time is a simplistic approach to evaluating acclimation time-course. Photoacclimation is comprised of a series of complex processes, which past studies have shown often happen at different rates. Lightharvesting, photoprotection, and photosynthesis in red algae are processes requiring different suites of pigments that in turn require different resources to operate (Beach and Smith, 1996; Schubert et al., 2006; Zubia et al., 2014). These processes are physically separated in the rhodophyte chloroplast with carotenoids associated exclusively with Photosystem I, phycobilins exclusively with Photosystem II, and chlorophyll present within both (Gantt and Cunningham, 2001).
3.1. Sun to shade acclimation Pairwise comparisons indicated G. coronopifolia acclimated more rapidly than did G. salicornia, with the following exceptions: for both rETRmax and PE measurements, G. salicornia adjusted faster than G. coronopifolia, while Car/Chl a acclimated at the same time (Table 1). RGRs were not significantly different between HL-LL treatment and controls for either species. (Fig. 3) 3.2. Shade to sun acclimation Results of this treatment were more mixed: pairwise comparisons indicated rETRmax adjusted faster to increased light in G. coronopifolia, Ek adjusted at the same time in both species, APC treatment values 4
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Fig. 3. Relative growth rates (wet weight) in treatments (solid lines) and controls (dashed lines). a: G. salicornia LL → HL; b: G. salicornia HL → LL; c: G. coronopifolia LL → HL; G. coronopifolia HL → LL. Mean ± SE, n = 10.
pigments have adjusted (Anabaena variabilis in Collins and Boylen, 1982). In this study, no process could be said to be faster than another in a general sense, as response time varied across treatments and by species. Added to the question of which specific processes are relatively faster is whether shade acclimation is faster or slower than sun acclimation. Of the few studies that have measured both sun and shade acclimation, most concluded that these two processes were completed in the same amount of time (Collins and Boylen, 1982; Henley and Ramus, 1989; Sims and Pearcy, 1990; Glogau Stevens, 1992). Dailer (2006) reported contemporaneous acclimation for both directions, with the above notable exception. Beach et al. (2000) reported faster phycobilin acclimation for LL-HL than for HL-LL and Prézelin and Matlick (1980) found that the dinoflagellate Glenodinium sp. acclimated to low light within 12 h but took “several days” to acclimate to high light. In the present study, overall shade acclimation for G. coronopifolia took 10 days for sun acclimation and 8 days for shade acclimation, while the invasive G. salicornia took 8 days for sun acclimation and as long as 12 days for shade acclimation. Challenges in determining relative rates of photoacclimation are compounded by a general lack of agreement on how acclimation is should be assessed. A variety of acclimation rates have been reported for various species; however, comparing these values may not be appropriate, due to the variety in study design and methodology, the inconsistency of parameters assessed, and the lack of an agreed-upon delineation for the end-point of photoacclimation. The time course cited in a study is usually the overall rate, but in other instances a range of times is offered; as a result, the time course can be dependent on the number or types of processes measured. In early studies, and in some more recent ones only tangentially concerned with acclimation,
Table 2 Summary of independent-samples t-tests of shade to sun treatments. n = 10. Values represent number of days samples of each species took to go from a LL state to a HL state for each parameter. * indicates a failure to detect acclimation. For full graphical presentation of all data here summarized, see Figure S2.
G. coronopifolia G. salicornia
rETRmax
Ek
APC
PC
PE
Car/PE
Car/Chl
6 8
4 4
10 8
8 8
8 8
6 8
* 6
Although adjustment of pigment content can change in response to changing ambient irradiance, differential changes between pigment classes are not necessarily cotemporal. Changes in Chl a have been reported to stabilize before phycobilins (G. tikvahiae in Lapointe, 1981; Anacystis nidulans in Lönneborg et al., 1985) or after, as in G. chilensis (Glogau Stevens, 1992). Carotenoid levels have been reported to reach a state of acclimation before Ch a (G. tenuistipitata in Carnicas et al., 1999) or at the same time as Chl a (Collins and Boylen, 1982) in Anabaena variabilis, and faster than (G. tenuistipitata in Carnicas et al., 1999) or slower than (Ahnfeltiopsis concinna in Beach et al., 2000) phycobilins. Dailer (2006) found that rates of phycobilin acclimation to low light were twice that of carotenoids and Chl a in the summer compared to the winter in Eucheuma denticulatum, while rates for acclimation to high light for photosynthesis, phycobilins, carotenoids, and Chl a were all cotemporal regardless of season. Additionally, rates of changes in pigmentation, including chlorophyll, often appear to have little relationship to acclimation rates of photosynthesis, which often occurs well before that of pigmentation (Gonyaulax polyedra in Prézelin and Matlick, 1983; Anacystis nidulans in Lönneborg et al., 1985; Ahnfeltiopsis concinna Beach et al., 2000), but can also occur well after 5
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investigators often decided that samples were acclimated based on convenience, for example, after a preset amount of time had passed. Determining the importance of photoacclimation time course may require more consistency between studies. Although G. salicornia’s rates of change in photosynthetic responses do not appear to confer any advantage relative to G. coronopifolia, G. salicornia does respond positively to increases in light in a way that the native species does not. Transfers from shade to sun were not significantly different than controls for G. coronopifolia, but growth of G. salicornia treatments were over 50 % higher under the same conditions; this response may have implications for cases where disturbance forces changes in light regime. For example, G. salicornia mats experience wave action and other mechanical disturbance that often leads to breakage and the formation of canopy gaps and tumbleweed-like fragments. Attachment points left behind after breakage have the potential to regrow, while fragments that break off may settle and fuse in rocky crevices or other hard substrates (Smith et al., 2004), often in orientations inverted from their previous state (pers. obs.). Both exposed attachment points and reoriented fragments may present shadeacclimated tissue to a higher light environment. The results of this study suggest that this sudden increase in light may be followed by substantial increase in growth, similar to a study by Laing et al. (1989) that found the same phenomenon in G. chilensis. Opportunistic responses to gap formation have enabled other invasive plants to succeed at the expense of less photosynthetically plastic species (Yamashita et al., 2000; Liu et al., 2016); the tendency for shade-acclimated G. salicornia to experience rapid growth when introduced to high light levels may at least partially explain its success. The ability of G. salicornia to withstand prolonged shading may be related to its success at vegetative reproduction. In culture, shade-acclimated tissue is especially turgid and breakable relative to sun-acclimated tissue, and though this difference has not been evaluated in the literature in situ, this breakability may contribute to dispersal via fragmentation for which this species is known (Smith et al., 2004). As a result of prolonged light limitation, G. salicornia in this study often dissolved at the nodes, spontaneously fragmenting, where G. coronopifolia dissolved in indeterminate places. Although these fragments were not reintroduced to increased light, it is conceivable that they would regenerate; in this way, light limitation might be used to advantage for this species.
K. Hamel performed the experiments, analyzed the data, and wrote the manuscript. C.M. Smith provided supplies, equipment and editorial advice. Declarations of interest None. Acknowledgements The authors are indebted to Dr Amy Carlile and the Alison Sherwood Lab, University of Hawaiʻi for molecular identification of G. coronopifolia, to Ms. Nikolara Jansons, University of Hawaiʻi for field assistance, and to Dr. Julie Rose, NOAA Fisheries for consultation on statistical approach. Finally, the authors would like to acknowledge the thoughtful effort and constructive suggestions of the reviewers. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.aquabot.2020.103210. References Abbott, I.A., 1999. Marine Red Algae of the Hawaiian Islands. Bishop Museum Press, Honolulu. Amato, D., Smith, C., Duarte, T., 2018. Submarine groundwater discharge differentially modifies photosynthesis, growth, and morphology for two contrasting species of Gracilaria (Rhodophyta). Hydrology 5 (4), 65. https://doi.org/10.3390/ hydrology5040065. Anton, A., Geraldi, N.R., Lovelock, C.E., Apostolaki, E.T., Bennett, S., Cebrian, J., KrauseJensen, D., Marbà, N., Martinetto, P., Pandolfi, J.M., Santana-Garcon, J., 2019. Global ecological impacts of marine exotic species. Nat. Ecol. Evol. 3 (5), 787–800. https://doi.org/10.1038/s41559-019-0851-0. Beach, K.S., 1996. Photoadaptive Strategies of Hawaiian Macroalgae. PhD Dissertation. Botany Department, University of Hawai‘i at Mānoa, Honolulu. Beach, K.S., Smith, C.M., 1996. Ecophysiology of tropical rhodophytes I: microscale acclimation in pigmentation. J. Phycol. 32, 701–710. https://doi.org/10.1111/j.00223646.1996.00701.x. Beach, K.S., Borgeas, H.B., Nishimura, N.J., Smith, C.M., 1997. In vivo absorbance spectra and UV-absorbing compounds in tropical reef macroalgae. Coral Reefs 16, 21–28. https://doi.org/10.1007/s003380050055. Beach, K.S., Smith, C.M., Okano, R., 2000. Experimental analysis of rhodophyte photoacclimation to PAR and UV-Radiation using in vivo absorbance spectroscopy. Bot. Mar. 43, 525–536. https://doi.org/10.1515/BOT.2000.052. Beer, B., Axelsson, L., 2004. Limitations in the use of PAM fluorometry for measuring photosynthetic rates of macroalgae at high irradiances. Eur. J. Phycol. 39, 1–7. https://doi.org/10.1080/0967026032000157138. Benjamini, Y., Hochberg, Y., 1995. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc. Series B Stat. Methodol. 57 (1), 289–300. https://doi.org/10.1111/j.2517-6161.1995.tb02031.x. Carnicas, E., Jiménez, C., Niell, F.X., 1999. Effects of changes of irradiance on the pigment composition of Gracilaria tenuistipitata var. Liui Zhang et Xia. J. Photochem. Photobiol. B 50 (2–3), 149–158. https://doi.org/10.1016/S1011-1344(99)00086-X. Collins, C.D., Boylen, C.W., 1982. Ecological consequences of long-term exposure of Anabaena variabilis (Cyanophyceae) to shifts in environmental factors. Appl. Environ. Microbiol. 44, 141–148. Cunningham, F.X., Vonshak, A., Gantt, E., 1992. Photoacclimation in the red alga Porphyridium cruentum. Plant Physiol. 100, 1142–1149. https://doi.org/10.1104/pp. 100.3.1142. Dailer, M., 2006. Photoecological Strategies Influencing the Invasive Success of Marine Macrophytes Euchuema Denticulatum on Hawaiian Coral Reefs. Master’s Thesis. Botany Department, University of Hawai‘i, Honolulu. Falkowski, P.G., LaRoche, J., 1991. Acclimation to spectral irradiance in algae. J. Phycol. 27, 8–14. Feng, Y.L., Wang, J.F., Sang, W.G., 2007a. Irradiance acclimation, capture ability, and efficiency in invasive and non-invasive alien plant species. Photosynthetica 45, 245–253. https://doi.org/10.1007/s11099-007-0040-2. Feng, Y.L., Wang, J.F., Sang, W.G., 2007b. Biomass allocation, morphology and photosynthesis of invasive and noninvasive exotic species grown at four irradiance levels. Oecologia 31, 40–47. https://doi.org/10.1016/j.actao.2006.03.009. Fisher, T., Minnaard, J., Dubinsky, Z., 1996. Photoacclimation in the marine alga Nannochloropsis sp. (Eustigmatophyte): a kinetic study. J. Plankton Res. 18, 1797–1818. https://doi.org/10.1093/plankt/18.10.1797. Gantt, E., 1990. Pigmentation and photoacclimation. In: Cole, K.M., Sheath, R.G. (Eds.), Biology of the Red Algae. Cambridge University Press, Cambridge, U.K, pp. 203–220. Gantt, E., Cunningham, F.X., 2001. Algal pigments. Encyclopedia of Life Sciences. John Wiley, Oxford, UK. https://doi.org/10.1038/npg.els.0000323.
5. Conclusion The invasive potential of G. salicornia has been attributed to its ability to sequester nutrients (Larned, 1998), benefit from low herbivory, asexual reproduction via fragmentation, and broad tolerance to environmental conditions (Smith et al., 2004; Amato et al., 2018), including to a wide range of irradiance regimes (Beach et al., 1997). This study reinforces the notion that G. salicornia possesses broad tolerance to solar irradiance, thriving both in direct tropical sunlight and to long periods of deep shade. Moreover, G. salicornia’s tendency for rapid growth when transitioning from shade to sun may be of advantage following disturbances that create gaps in this species’ unique matforming morphology. In light of these findings, it seems unlikely that relative rates of photoacclimation alone can account for differential success of native and invasive species, though additional research along these lines might demonstrate otherwise. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Contributors K. Hamel and C.M. Smith conceived and designed the experiments. 6
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alga Caulerpa racemosa var. Cylindracea to depth and daylight patterns and a putative new role for siphonaxanthin. Mar. Ecol. 27, 20–30. https://doi.org/10.1111/j.14390485.2006.00080.x. Schaffelke, B., Hewitt, C.L., 2007. Impacts of introduced seaweeds. Botanica Marina 50, 397–417. https://doi.org/10.1515/BOT.2007.044. Schubert, N., García‐Mendoza, E., Pacheco‐Ruiz, I., 2006. Carotenoid composition of marine red algae 1. J. Phycol. 42 (6), 1208–1216. https://doi.org/10.1590/S0102695X2012005000075. Sherwood, A.R., Presting, G.G., 2007. Universal primers amplify a 23S rDNA plastid marker in eukaryotic algae and cyanobacteria. J. Phycol. 43, 605–608. https://doi. org/10.1111/j.1529-8817.2007.00341.x. Sims, D.A., Pearcy, R.W., 1990. Photosynthesis and respiration in Alocasia macrorrhiza following transfers to high and low light. Oecologia 86, 447–453. https://doi.org/10. 1007/BF00317615. Smith, C.M., Alberte, R.S., 1994. Characterization of in vivo absorption features of chlorophyte, phaeophyte and rhodophyte algal species. Mar. Biol. 118, 511–521. https://doi.org/10.1007/BF00350308. Smith, J.E., Smith, C.M., Hunter, C.L., 2001. An experimental analysis of the effects of herbivory and nutrient enrichment on benthic community dynamics on a Hawaiian reef. Coral Reefs 19, 332–342. https://doi.org/10.1007/s003380000124. Smith, J.E., Hunter, C.L., Smith, C.M., 2002. Distribution and reproductive characteristics of nonindigenous and invasive marine algae in the Hawaiian Islands. Pac. Sci. 56, 299–315. https://doi.org/10.1353/psc.2002.0030. Smith, J.E., Hunter, C.L., Conklin, E.J., Most, R., Sauvage, T., Squair, C., Smith, C.M., 2004. Ecology of the invasive red alga Gracilaria salicornia (Rhodophyta) on O‘ahu, Hawai‘i. Pac. Sci. 58, 325–343. https://doi.org/10.1353/psc.2004.0023. Titlyanov, E.A., Titlyanova, T.V., Yamazato, K., van Woesik, R., 2001. Photo-acclimation dynamics of the coral Stylophora pistillata to low and extremely low light. J Exp Mar Bio Ecol 263, 211–225. https://doi.org/10.1016/S0022-0981(01)00309-4. van Tussenbroek, B.I., Hernández Arana, H.A., Rodríguez-Martínez, R.E., EspinozaAvalos, J., Canizales-Flores, H.M., González-Godoy, C.E., Guadalupe Barba-Santos, M., Vega-Zepeda, A., Collado-Vides, L., 2017. Severe impacts of brown tides caused by Sargassum spp. On near-shore Caribbean seagrass communities. Mar Poll Bull 122, 272–281. https://doi.org/10.1016/j.marpolbul.2017.06.057. Veazey, L.M., 2018. Distribution Modeling of Mesophotic Corals and Algae to Further Ecological Understanding and Resource Management of Hawaiian Coral Reefs. PhD Dissertation. Zoology Department, University of Hawai‘i at Mānoa, Honolulu. Waaland, J.R., Waaland, S.D., Bates, G., 1974. Chloroplast structure and pigment composition in the red alga Griffithsia pacifica: regulation by light intensity. J. Phycol. 10, 193–199. https://doi.org/10.1111/j.1529-8817.1974.tb02697.x. Williams, S.L., Smith, J.E., 2007. A global review of the distribution, taxonomy, and impacts of introduced seaweeds. Annu. Rev. Ecol. Evol. Syst. 38, 327–359. https:// doi.org/10.1146/annurev.ecolsys.38.091206.095543. Yamashita, N., Ishida, A., Kushima, H., Tanaka, N., 2000. Acclimation to sudden increase in light favoring an invasive over native trees in subtropical islands, Japan. Oecologia 125, 412–419. https://doi.org/10.1007/s004420000475. Yamashita, N., Koike, N., Ishida, A., 2002. Leaf ontogenetic dependence of light acclimation in invasive and native subtropical trees of different successional status. Plant Cell Environ. 25 (10), 1341–1356. https://doi.org/10.1046/j.1365-3040.2002. 00907.x. Zubia, M., Freile-Pelegrín, Y., Robledo, D., 2014. Photosynthesis, pigment composition and antioxidant defences in the red alga Gracilariopsis tenuifrons (Gracilariales, Rhodophyta) under environmental stress. J. Appl. Phycol. 26 (5), 2001–2010. https://doi.org/10.1007/s10811-014-0325-3.
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Comparative time-courses of photoacclimation by Hawaiian native and invasive species of Gracilaria (Rhodophyta)
T
Ken Hamel*, Celia M. Smith Department of Botany, University of Hawai‘i at Mānoa, 3190 Maile Way, Honolulu, HI 96822, USA
ARTICLE INFO
ABSTRACT
Keywords: Gracilaria salicornia Gracilaria coronopifolia Invasive species trait Photoacclimation Coral reef macroalgae
Invasive seaweeds have rapidly increased in global distribution and abundance in recent years, presenting significant threats to nearshore ecosystems. Developing characteristics diagnostic of potential macroalgal invasiveness is an important step in controlling their spread. Studies have identified photoacclimation, the ability of the photosynthetic apparatus to adjust to changes in light, as one important attribute in the success of several invasive terrestrial and marine plants; several studies indicate that the rate of photoacclimation plays a role in the differential success of invasive plants at the expense of natives. In this experiment, we compare two closely related species that occur in Hawaiian coastal waters with similar distribution, habitat, and morphology, the invasive Gracilaria salicornia, and the native G. coronopifolia. Samples were grown in outdoor culture, acclimated to one of two irradiance regimes, and reciprocally transplanted. Photoacclimation rates were assessed using in vivo spectrophotometry and PAM fluorometry. Results indicate that G. salicornia has remarkable tolerance to irradiance extremes and experiences rapid growth when transplanted from low to high photosynthetic photon flux density environments, and that differential rates of overall photoacclimation are insufficient to explain the relative success of these two plants. Photoacclimation is achieved via several complex processes that do not necessarily occur contemporaneously. Future researchers should be wary of assuming its completion based on single indicators.
1. Introduction Invasive and bloom forming marine macroalgae are major threats to reef ecosystems in many regions (Smith et al., 2002; Williams and Smith, 2007; van Tussenbroek et al., 2017; Veazey, 2018; Anton et al., 2019). Proliferations of macroalgae can negatively impact nearshore marine environments in many ways, including reducing benthic species diversity by outcompeting corals, native macroalgae, and seagrasses, by altering environmental parameters such as water quality and substrate conditions to states unfavorable to pre-existing organisms, and by reducing demersal species diversity by interfering with established food webs (Olenin et al., 2007; Smith et al., 2001; Schaffelke and Hewitt, 2007). Identifying characteristics of algae that may play a role in their invasiveness may contribute to reducing future introductions by aiding appropriate characterization of potential invaders, and by aiding management efforts via deepening our understanding of what environmental conditions promote invasive behavior. Increasing the efficacy of prevention and management is particularly important in shallow reef environments at high risk of biological invasions as with Hawaiian coastal waters.
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One characteristic that may be instrumental in the invasiveness of plants is photoacclimation, the adjustment of the photosynthetic apparatus in response to changes in irradiance. There has been growing interest in examining photoacclimatory capacity to diagnose invasive potential in both terrestrial (Yamashita et al., 2002; Feng et al., 2007a; Liu et al., 2016) and marine (Raniello et al.;, 2006; Dailer, 2006; Mehnert et al., 2012) plants. Evidence suggests that plants that can acclimate to a broader range of light conditions exercise competitive advantage over other plants (Raniello et al., 2004; Feng et al., 2007a). High shade tolerance and a large proportional increase in photosynthetic rate after high-light exposure are also photoacclimatory attributes that appear to favor invasive plants (Yamashita et al., 2000; Feng et al., 2007b). Photoacclimation may refer to one or more reversible changes to the size and/or number of photosynthetic units (PSU) in a cell, changes in the relative pigment content within a cell, or changes in photochemistry as a result of the plant’s exposure to higher (“sun” state) or lower (“shade” state) photon flux densities (Falkowski and LaRoche, 1991; Cunningham et al., 1992). These changes allow a plant to both avoid the potentially damaging effects of high irradiances as well as allow for
Corresponding author at: Department of Marine Affairs, University of Rhode Island, Coastal Institute Room 117, 1 Greenhouse Rd, Kingston, RI 02881 USA. E-mail addresses: [email protected] (K. Hamel), [email protected] (C.M. Smith).
https://doi.org/10.1016/j.aquabot.2020.103210 Received 19 July 2019; Received in revised form 3 January 2020; Accepted 18 January 2020 Available online 22 January 2020 0304-3770/ © 2020 Elsevier B.V. All rights reserved.
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increased light harvesting at lower irradiances. For rhodophytes, acclimation to changing irradiance includes reciprocal changes in the concentration of chlorophyll a (Chl a) and phycobilins, and corresponding changes in carotenoid concentration and photosynthetic output (Waaland et al., 1974; Levy and Gantt, 1988; Beach et al., 2000; Payri et al., 2001). Photoacclimation is often described as requiring several days to weeks to occur (Gantt, 1990), but acclimation time has in fact rarely been measured and the point at which acclimation is attained remains poorly defined. Some phytoplankters fully acclimate in a matter of hours (Prézelin and Matlick, 1980) to days (Fisher et al., 1996), while corals may take between a few days to several weeks (Titlyanov et al., 2001). The few measurements made of macroalgae photoacclimation time range from eight days for Gracilaria chilensis (Glogau Stevens, 1992) to about one month for the Hawaiian native Ahnfeltiopsis concinna (Beach et al., 1997). Among the various estimates there has been little congruence as to the methodology used to determine the photoacclimation time course, making comparisons among taxa challenging. There is some evidence that invasive algae acclimate to changing irradiance faster than non-invasive Hawaiian algae (Beach, 1996; Dailer, 2006), giving invasives a potential advantage in rebounding from disturbances that scour marine substrates or displace existing animal and plant species; physical disturbances that dislodge benthos can generate intense competition for newly exposed substrate, while outcompeting established natives for other resources such as limiting nutrients may allow introduction of a potential invader to novel environments (Williams and Smith, 2007). Rapid photoacclimation may also allow an invasive with the ability to adjust to a wide range of light regimes that might otherwise be occupied by less phenotypically flexible algae. Photoacclimation response time may be a factor in determining future threats in the form of both native and alien algae and if so, its study can help us make decisions regarding what to look for in terms of invasive characters. Gracilaria salicornia (C. Agardh) E.Y. Dawson is a non-indigenous intertidal alga invasive to Hawaii that is currently found along much of Oʻahu’s south and windward shores; it most often is found as a freestanding plant, reaching lengths of up to 18 cm, with axis 1.5–4 mm thick (Abbott, 1999). Along several south shore beaches, G. salicornia forms extensive mats that can be over 10 cm thick and 2 or more meters across (Beach et al., 1997; Larned, 1998). Within a mat, tissues range in color from yellow in the canopy regions where they may be exposed at low tide to a dark purple understory, a result of self-shading (Beach et al., 1997). A native congener of this species, Gracilaria coronopifolia (J. Agardh), is a comparatively small (usually <8 cm long and 1−2 mm in diameter; Abbott, 1999) shallow-subtidal alga that is considered endemic to the Hawaiian Islands (Abbott, 1999 but see e.g. Lobban and Tsuda, 2003). G. coronopifolia exhibits pale yellow to white tissue in areas with high exposure to sunlight, and pink to red when shaded. In contrast to G. salicornia, G. coronopifolia is increasingly uncommon, at least in part from overharvesting due to its popularity as a local food. Both species are restricted to waters above a 4 m depth, and though only G. salicornia is truly intertidal, both are euryhaline, tolerating wide ranges of nutrients and salinity (Amato et al., 2018), occur often in the same locations, sometimes < 1 m apart, and exhibit analogous ranges of pigmentation resulting from differential exposure to incident solar irradiance. Though both exhibit a tendency to local dominance (Smith et al. 2004, pers. obs.), there are no data documenting interspecific competition. Three past studies have tracked both photosynthetic and pigment acclimation in response to changes in irradiance over time in tropical rhodophytes (Glogau Stevens, 1992; Beach et al., 2000; Dailer, 2006), with each examining a single species. Researchers have generally evaluated the presence, importance or magnitude of photoacclimation but have rarely considered the speed at which it occurs, especially when compared to other species; this is the first comparison of photoacclimation time-course of two species of algae grown under the same
conditions. This study tests the hypothesis that an invasive alga acclimates faster to changes to exposure in solar irradiance than a native congener. To this end, two congeners with similar local distributions, habitat, and morphology were reciprocally transplanted in an experimental mesocosm, and several physiological parameters measured in vivo. We predicted that the invasive species, G. salicornia, would react more rapidly than the native species, G. coronopifolia, to both increases and decreases in light quantity. 2. Methods 2.1. Collection of experimental materials Non-reproductive, representative G. salicornia and G. coronopifolia plants were collected at the seaward end of Paiko Lagoon, on O‘ahu’s south shore and at Kualoa Beach on O‘ahu’s windward shore over the span of eight months, from May to December of 2011 (Table S1). Additional samples of G. salicornia were collected from Kaimana Beach on the south shore, and of G. coronopifolia from windward Malaekahana Beach. Samples were selected for sun- or shade-acclimated pigmentation, i.e. paler or darker, respectively. Species determinations for each population were made in the field using morphological characters, and in the laboratory using primers and methods after Sherwood and Presting (2007) for the universal plastid amplicon (UPA) marker, part of the plastid 23S rDNA. 2.2. Reciprocal transplants in outdoor culture Between May and November of 2011, reciprocal transplants were performed in outdoor culture in five trials. Each trial consisted of two replicates for each of four treatments (sun to shade with its corresponding shade control and shade to sun with its corresponding sun control) of each of two species. Trials were conducted with ten replicate plants per treatment for a total of 80 individuals for the entire experiment. Photoacclimation was tracked and its effects measured every two days via PAM fluorometry, in vivo spectrophotometry and wet weight, in that order. All measurements were made on plants in the same order and at the same time, on each day. Each sample was maintained in a separate aerated, filtered seawater-filled 5 l aquaria set in a mesocosm – outdoor water baths on the balcony of the St. John Plant Science Building, University of Hawai‘i at Mānoa. Seawater was collected adjacent to the Makai Pier, near the easternmost tip of O‘ahu, and enriched with 10 μM NaNO3 and 2 μM PO4, to avoid nutrient limitation (Beach, 1996). This enriched seawater was replaced every other day. Water temperature of the cultures was maintained between 25 and 30 °C, measured with HOBO pendant loggers (Onset Computer Corporation, Pocasset, MA). Salinity was monitored periodically with a Reichert temperature compensated refractometer and adjusted to 34‰ with additions of distilled water. Salinity varied between 34 and 38‰ during the duration of the experiment. Water baths were shielded from rain with transparent polymethylmethacrylate sheet roofs. Samples of both species were initially acclimated to either full sunlight, with daily maxima ranging from ca. 1500–3300 μmol m−2 s−1 PAR (mean = 2450 μmol m−2 s−1 PAR), or shade (covered in three layers of medium grade shade-cloth), resulting in daily maxima ranging from ca. 70–180 μmol m−2 s−1 PAR (mean = 115 μmol m−2 s−1 PAR). These irradiances closely approximated those reported for G. salicornia field conditions in Beach et al. (1997). This acclimation period lasted for 11–17 days or until relative uniformity of plant color was achieved among samples. PAR was continuously monitored with LI−COR LI-193 underwater spherical quantum sensors placed in aquaria amongst, but separate from, the samples. Mean irradiance was sampled at 15-min intervals and stored in LI−COR LI-1000 data loggers. After the initial acclimation period, samples from each irradiance treatment were 2
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1) rETRmax as the product of PAR, ETR-Factor, PPS2/PPPS and Y(II), where ETR-Factor is the ratio of photons absorbed by photosynthetic pigments to incident photons, PPS2/PPPS is the ratio of photons absorbed by PSII to photons absorbed by photosynthetic pigments, and Y(II), the effective photochemical quantum yield of PSII and 2) Ek as ETRmax/α, where α is the initial slope of the RLC. 2.4. In vivo absorbance analyses An in vivo absorbance spectrum (400–750 nm) was acquired from each sample immediately after each fluorometric measurement. Spectra were obtained using a Shimadzu UV Vis-2101 spectrophotometer with a 150 mm integrating sphere following Beach et al. (1997). Specimens were analyzed by placing a non-overlapping layer of tissue between two white cards with a mask window cut of 1.0 cm by 0.7 cm. Spectra were normalized to Chl a maxima at ∼678 nm after spectra acquisition after Smith and Alberte (1994); these pigment-to-chlorophyll ratios act as proxies for pigment concentrations in living tissue that could otherwise only be obtained via destructive solvent extraction. The specific absorbance maxima of accessory pigment-to-chlorophyll ratios that were selected for analysis were allophycocyanin (APC) at 649:678 nm, phycocyanin (PC) at 625:678 nm, phycoerythrin (PE) at 568:678, a combination of carotenoid and phycoerythrin (Car/PE) at 495:678 nm, and a combination of carotenoid and Chl a (Car/Chl) at 440:678 nm (Fig. 2). These pigment identities are based on published absorbance maxima (Haxo and Blinks, 1950; Smith and Alberte, 1994; Beach et al., 1997, 2000).
Fig. 1. Change in the ratio of 568:678 nm (PE) absorbance thru time for replicates of Gracilaria coronopifolia samples transferred from sun to shade environment (solid line). Samples were considered acclimated when parameter values ceased to be significantly different than control values (dashed line). *P < 0·05; **P < 0·01; ***P < 0·001. Mean ± SE, n = 10.
placed in their correspondingly alternate irradiance regimes, i.e. full sunlight (high light or HL) acclimated samples were placed in shaded (low light or LL) water baths and vice versa. 2.3. PAM fluorometry
2.5. Growth
PAM fluorometry parameters were measured using a Walz JUNIORPAM fluorometer every two days starting from the day that the samples were transplanted. Photosynthetic parameters were assessed by exposing the second or third node from the tip (ca. 3–6 cm from the tip for G. salicornia samples and 2–4 cm for G. coronopifolia) on each specimen to the fluorometer’s fiber optic cable, which was secured with a Walzsupplied magnetic leaf clip. Rapid light curves (RLC) were initiated immediately, without “dark-acclimation”, and generated by exposing tissue to a series of saturation pulses applied after 10 s exposures to actinic irradiance of increasing exposure ranging from 25 to 420 μmol m−2 s−1 PAR in LL acclimated samples and from 66 to 820 μmol m−2 s−1 PAR in HL acclimated samples (following Beer and Axelsson, 2004). Different PAR ranges were required for HL and LL acclimated samples as lower level PAR exposure in HL samples resulted in an incomplete RLC, while PAR pulses higher than 420 resulted in a depressed effective quantum ((Fm′-F)/Fm′) yield below a reliable threshold (as per Beer and Axelsson, 2004) in LL samples. The photosynthetic parameters rETRmax (relative maximum electron transport rate), and Ek (light saturation index) were obtained from RLC data with Wincontrol-3 software (Heinz Walz GmbH, Effeltrich, Germany). Wincontrol-3 calculates
Growth was measured by obtaining wet weight immediately after each spectrum was obtained. Tissue were blotted to remove excess water and specimens were weighed on a Sartorius analytical balance Model TE214S + 0.0001 gm. Mean relative growth rates (RGR) were measured using the method detailed by Hunt (1982): RGR = (lnWt2 -lnWt1)/(t2 -t1), where W is the total plant wet mass in grams and t is time in days. 2.6. Data analyses Two-way mixed ANOVA, with time as the within-subjects factor and light environment as the between-subjects factor, was used to determine whether treatment and control groups differed over time for photosynthetic parameters, pigment to chlorophyll ratios and growth rates using SPSS version 25 (IBM). When significant interactive effects occurred, a priori multiple comparisons with the False Discovery Rate correction (Benjamini and Hochberg, 1995) were conducted to determine how many days were needed for photosynthetic and pigment
Fig. 2. in vivo absorbance spectra of sun-acclimated (grey lines) and shade acclimated (black lines) G. salicornia (triangles) and G. coronopifolia (circles). All spectra normalized to chlorophyll a absorbance peak at 678 nm. n = 10. 3
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parameters of treatments to reach corresponding control values, and to determine differences between growth rates between treatments and controls.
Table 1 Summary of independent-samples t-tests of sun to shade treatments. n = 10. Values represent number of days samples of each species took to go from a HL state to a LL state for each parameter. For full graphical presentation of all data here summarized, see Figure S1.
3. Results Native G. coronopifolia did not perform as well as invasive G. salicornia in mesocosm culture; measuring days did not extend beyond 14 days for G. coronopifolia, in contrast to G. salicornia which was viable and measured for 16–20 days. One control and two treatment G. coronopifolia plants lost substantial mass in shaded culture by the end of their respective trials, though not before photoacclimation had been achieved, while the other samples maintained their vigor. G. salicornia plants responded more favorably to culture, with only one LL control sample losing substantial mass, although fragmentation often occurred as a result of dissolution between segments. The texture of plant bodies for both species became more flexible as they acclimated to high light and more turgid as the plants acclimated to low light. Treatments in this study took on the general visual appearance of their corresponding control, with regard to apparent pigmentation, with the exception of LL-HL samples of G. salicornia, which took on a light brown pigmentation with yellow branch tips rather than the yellow to orange exhibited by HL control samples and by sun-exposed plants in the field. Mixed ANOVA results revealed statistically significant interactions at the 0.05 level between light environment and time between each treatment and control pairing for both species across all PAM and pigment parameters except RGR, where only LL-HL G. salicornia demonstrated a difference (Table S2). Likewise, main effects for light environment were statistically significant between each treatment and control pairing for both species across all PAM and pigment parameters, reflecting the initial planned differences between control and treatment for each test. Main effects for time were statistically significant between each treatment and control pairing for both species across all PAM and pigment parameters, with three exceptions: G. salicornia shade to sun for PE and for Car/Chl, and for G. coronopifolia sun to shade for Car/ PE. Full acclimation for each parameter was considered to have been achieved at the first time when treatment values of each parameter ceased to be statistically different from corresponding control values (e.g. Fig. 1). The number of days required for this condition to be met varied by parameter, by treatment, and by species. Many treatment values that achieved acclimation did not remain static, but deviated from control values after first acclimating to the new light environment (Table S3). Similarly, parameter values for most control groups did not remain static, particularly for those samples exposed to prolonged periods of intense sunlight. Sun-acclimated samples exhibited high absorbance values at both carotenoid-associated absorbance maxima as well as reduced absorbance values at all phycobilin-specific maxima relative to shade-acclimated plants of both species (Fig. 2). Conversely, phycobilin absorbance values increased and the carotenoid absorbance decreased for shade acclimated plants.
G. coronopifolia G. salicornia
rETRmax
Ek
APC
PC
PE
Car/PE
Car/Chl
4 2
2 4
8 10
8 12
6 4
2 12
4 4
reached control levels faster in G. salicornia, while PC and PE response times were the same for both species, and Car/PE absorbance maxima adjusted faster in G. salicornia. Comparison of time course rates between species were indeterminable at the Car/Chl a absorbance maximum for G. coronopifolia as treatment values never reached those of controls; instead, both treatment and control increased as this maximum at about the same pace over the course of the experiment (Table 2). G. coronopifolia did not fully acclimate as judged by changes in the Car/Chl a maximum because treatment and control values reacted to their light environment at a similar rate, e.g. carotenoid values in the HL control never stabilized, but instead increased about as fast as carotenoid values in the LL-HL treatment. A similar phenomenon of increasing Car/Chl a absorbance in both treatment and control affected G. salicornia LL-HL results, but due to increased variance in the response of that species for that parameter, control and treatment ceased being statistically significant at day 6. RGRs were not significantly different between treatment and controls for G. coronopifolia. G. salicornia were significantly different between treatment and controls (Table S2), but differences did not emerge until after four days, with the treatment maintaining an increased mean RGR 51.72 % (±1.51 % SE) higher than that of the control for the duration of the experiment (Fig. 3). 4. Discussion This study compared rates of photoacclimation between two closely related, often co-occuring rhodophytes, one an invasive species, the other an increasingly uncommon native. Our goal was to better understand whether faster physiological responses to changes in ambient light can explain the relative success of the invader. This turned out not to be the case for these two Gracilaria species, though the results suggest that G. salicornia possesses other advantageous photoacclimatory characteristics not shared by its native congener. Feng et al. (2007a) compared several species of invasive and non-invasive embryophytes, determining that invasive plants could acclimate to a broader range of irradiances. Yamashita et al. (2000) found that an invasive tree recovered from a sudden increase in light faster than non-invasives. This study, the first to compare acclimation rates between native and invasive species of algae, found that while the invasive acclimated readily to a broader range of irradiance than did the native, it did not adjust faster to changing irradiance than the native plant; if judged by the overall rate of acclimation, i.e. the time at which all processes have finished acclimating, the invasive was slightly faster in adjusting to increased light, while the native was somewhat faster in adjusting to shade. However, the overall time is a simplistic approach to evaluating acclimation time-course. Photoacclimation is comprised of a series of complex processes, which past studies have shown often happen at different rates. Lightharvesting, photoprotection, and photosynthesis in red algae are processes requiring different suites of pigments that in turn require different resources to operate (Beach and Smith, 1996; Schubert et al., 2006; Zubia et al., 2014). These processes are physically separated in the rhodophyte chloroplast with carotenoids associated exclusively with Photosystem I, phycobilins exclusively with Photosystem II, and chlorophyll present within both (Gantt and Cunningham, 2001).
3.1. Sun to shade acclimation Pairwise comparisons indicated G. coronopifolia acclimated more rapidly than did G. salicornia, with the following exceptions: for both rETRmax and PE measurements, G. salicornia adjusted faster than G. coronopifolia, while Car/Chl a acclimated at the same time (Table 1). RGRs were not significantly different between HL-LL treatment and controls for either species. (Fig. 3) 3.2. Shade to sun acclimation Results of this treatment were more mixed: pairwise comparisons indicated rETRmax adjusted faster to increased light in G. coronopifolia, Ek adjusted at the same time in both species, APC treatment values 4
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Fig. 3. Relative growth rates (wet weight) in treatments (solid lines) and controls (dashed lines). a: G. salicornia LL → HL; b: G. salicornia HL → LL; c: G. coronopifolia LL → HL; G. coronopifolia HL → LL. Mean ± SE, n = 10.
pigments have adjusted (Anabaena variabilis in Collins and Boylen, 1982). In this study, no process could be said to be faster than another in a general sense, as response time varied across treatments and by species. Added to the question of which specific processes are relatively faster is whether shade acclimation is faster or slower than sun acclimation. Of the few studies that have measured both sun and shade acclimation, most concluded that these two processes were completed in the same amount of time (Collins and Boylen, 1982; Henley and Ramus, 1989; Sims and Pearcy, 1990; Glogau Stevens, 1992). Dailer (2006) reported contemporaneous acclimation for both directions, with the above notable exception. Beach et al. (2000) reported faster phycobilin acclimation for LL-HL than for HL-LL and Prézelin and Matlick (1980) found that the dinoflagellate Glenodinium sp. acclimated to low light within 12 h but took “several days” to acclimate to high light. In the present study, overall shade acclimation for G. coronopifolia took 10 days for sun acclimation and 8 days for shade acclimation, while the invasive G. salicornia took 8 days for sun acclimation and as long as 12 days for shade acclimation. Challenges in determining relative rates of photoacclimation are compounded by a general lack of agreement on how acclimation is should be assessed. A variety of acclimation rates have been reported for various species; however, comparing these values may not be appropriate, due to the variety in study design and methodology, the inconsistency of parameters assessed, and the lack of an agreed-upon delineation for the end-point of photoacclimation. The time course cited in a study is usually the overall rate, but in other instances a range of times is offered; as a result, the time course can be dependent on the number or types of processes measured. In early studies, and in some more recent ones only tangentially concerned with acclimation,
Table 2 Summary of independent-samples t-tests of shade to sun treatments. n = 10. Values represent number of days samples of each species took to go from a LL state to a HL state for each parameter. * indicates a failure to detect acclimation. For full graphical presentation of all data here summarized, see Figure S2.
G. coronopifolia G. salicornia
rETRmax
Ek
APC
PC
PE
Car/PE
Car/Chl
6 8
4 4
10 8
8 8
8 8
6 8
* 6
Although adjustment of pigment content can change in response to changing ambient irradiance, differential changes between pigment classes are not necessarily cotemporal. Changes in Chl a have been reported to stabilize before phycobilins (G. tikvahiae in Lapointe, 1981; Anacystis nidulans in Lönneborg et al., 1985) or after, as in G. chilensis (Glogau Stevens, 1992). Carotenoid levels have been reported to reach a state of acclimation before Ch a (G. tenuistipitata in Carnicas et al., 1999) or at the same time as Chl a (Collins and Boylen, 1982) in Anabaena variabilis, and faster than (G. tenuistipitata in Carnicas et al., 1999) or slower than (Ahnfeltiopsis concinna in Beach et al., 2000) phycobilins. Dailer (2006) found that rates of phycobilin acclimation to low light were twice that of carotenoids and Chl a in the summer compared to the winter in Eucheuma denticulatum, while rates for acclimation to high light for photosynthesis, phycobilins, carotenoids, and Chl a were all cotemporal regardless of season. Additionally, rates of changes in pigmentation, including chlorophyll, often appear to have little relationship to acclimation rates of photosynthesis, which often occurs well before that of pigmentation (Gonyaulax polyedra in Prézelin and Matlick, 1983; Anacystis nidulans in Lönneborg et al., 1985; Ahnfeltiopsis concinna Beach et al., 2000), but can also occur well after 5
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investigators often decided that samples were acclimated based on convenience, for example, after a preset amount of time had passed. Determining the importance of photoacclimation time course may require more consistency between studies. Although G. salicornia’s rates of change in photosynthetic responses do not appear to confer any advantage relative to G. coronopifolia, G. salicornia does respond positively to increases in light in a way that the native species does not. Transfers from shade to sun were not significantly different than controls for G. coronopifolia, but growth of G. salicornia treatments were over 50 % higher under the same conditions; this response may have implications for cases where disturbance forces changes in light regime. For example, G. salicornia mats experience wave action and other mechanical disturbance that often leads to breakage and the formation of canopy gaps and tumbleweed-like fragments. Attachment points left behind after breakage have the potential to regrow, while fragments that break off may settle and fuse in rocky crevices or other hard substrates (Smith et al., 2004), often in orientations inverted from their previous state (pers. obs.). Both exposed attachment points and reoriented fragments may present shadeacclimated tissue to a higher light environment. The results of this study suggest that this sudden increase in light may be followed by substantial increase in growth, similar to a study by Laing et al. (1989) that found the same phenomenon in G. chilensis. Opportunistic responses to gap formation have enabled other invasive plants to succeed at the expense of less photosynthetically plastic species (Yamashita et al., 2000; Liu et al., 2016); the tendency for shade-acclimated G. salicornia to experience rapid growth when introduced to high light levels may at least partially explain its success. The ability of G. salicornia to withstand prolonged shading may be related to its success at vegetative reproduction. In culture, shade-acclimated tissue is especially turgid and breakable relative to sun-acclimated tissue, and though this difference has not been evaluated in the literature in situ, this breakability may contribute to dispersal via fragmentation for which this species is known (Smith et al., 2004). As a result of prolonged light limitation, G. salicornia in this study often dissolved at the nodes, spontaneously fragmenting, where G. coronopifolia dissolved in indeterminate places. Although these fragments were not reintroduced to increased light, it is conceivable that they would regenerate; in this way, light limitation might be used to advantage for this species.
K. Hamel performed the experiments, analyzed the data, and wrote the manuscript. C.M. Smith provided supplies, equipment and editorial advice. Declarations of interest None. Acknowledgements The authors are indebted to Dr Amy Carlile and the Alison Sherwood Lab, University of Hawaiʻi for molecular identification of G. coronopifolia, to Ms. Nikolara Jansons, University of Hawaiʻi for field assistance, and to Dr. Julie Rose, NOAA Fisheries for consultation on statistical approach. Finally, the authors would like to acknowledge the thoughtful effort and constructive suggestions of the reviewers. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.aquabot.2020.103210. References Abbott, I.A., 1999. Marine Red Algae of the Hawaiian Islands. Bishop Museum Press, Honolulu. Amato, D., Smith, C., Duarte, T., 2018. Submarine groundwater discharge differentially modifies photosynthesis, growth, and morphology for two contrasting species of Gracilaria (Rhodophyta). Hydrology 5 (4), 65. https://doi.org/10.3390/ hydrology5040065. Anton, A., Geraldi, N.R., Lovelock, C.E., Apostolaki, E.T., Bennett, S., Cebrian, J., KrauseJensen, D., Marbà, N., Martinetto, P., Pandolfi, J.M., Santana-Garcon, J., 2019. Global ecological impacts of marine exotic species. Nat. Ecol. Evol. 3 (5), 787–800. https://doi.org/10.1038/s41559-019-0851-0. Beach, K.S., 1996. Photoadaptive Strategies of Hawaiian Macroalgae. PhD Dissertation. Botany Department, University of Hawai‘i at Mānoa, Honolulu. Beach, K.S., Smith, C.M., 1996. Ecophysiology of tropical rhodophytes I: microscale acclimation in pigmentation. J. Phycol. 32, 701–710. https://doi.org/10.1111/j.00223646.1996.00701.x. Beach, K.S., Borgeas, H.B., Nishimura, N.J., Smith, C.M., 1997. In vivo absorbance spectra and UV-absorbing compounds in tropical reef macroalgae. Coral Reefs 16, 21–28. https://doi.org/10.1007/s003380050055. Beach, K.S., Smith, C.M., Okano, R., 2000. Experimental analysis of rhodophyte photoacclimation to PAR and UV-Radiation using in vivo absorbance spectroscopy. Bot. Mar. 43, 525–536. https://doi.org/10.1515/BOT.2000.052. Beer, B., Axelsson, L., 2004. Limitations in the use of PAM fluorometry for measuring photosynthetic rates of macroalgae at high irradiances. Eur. J. Phycol. 39, 1–7. https://doi.org/10.1080/0967026032000157138. Benjamini, Y., Hochberg, Y., 1995. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc. Series B Stat. Methodol. 57 (1), 289–300. https://doi.org/10.1111/j.2517-6161.1995.tb02031.x. Carnicas, E., Jiménez, C., Niell, F.X., 1999. Effects of changes of irradiance on the pigment composition of Gracilaria tenuistipitata var. Liui Zhang et Xia. J. Photochem. Photobiol. B 50 (2–3), 149–158. https://doi.org/10.1016/S1011-1344(99)00086-X. Collins, C.D., Boylen, C.W., 1982. Ecological consequences of long-term exposure of Anabaena variabilis (Cyanophyceae) to shifts in environmental factors. Appl. Environ. Microbiol. 44, 141–148. Cunningham, F.X., Vonshak, A., Gantt, E., 1992. Photoacclimation in the red alga Porphyridium cruentum. Plant Physiol. 100, 1142–1149. https://doi.org/10.1104/pp. 100.3.1142. Dailer, M., 2006. Photoecological Strategies Influencing the Invasive Success of Marine Macrophytes Euchuema Denticulatum on Hawaiian Coral Reefs. Master’s Thesis. Botany Department, University of Hawai‘i, Honolulu. Falkowski, P.G., LaRoche, J., 1991. Acclimation to spectral irradiance in algae. J. Phycol. 27, 8–14. Feng, Y.L., Wang, J.F., Sang, W.G., 2007a. Irradiance acclimation, capture ability, and efficiency in invasive and non-invasive alien plant species. Photosynthetica 45, 245–253. https://doi.org/10.1007/s11099-007-0040-2. Feng, Y.L., Wang, J.F., Sang, W.G., 2007b. Biomass allocation, morphology and photosynthesis of invasive and noninvasive exotic species grown at four irradiance levels. Oecologia 31, 40–47. https://doi.org/10.1016/j.actao.2006.03.009. Fisher, T., Minnaard, J., Dubinsky, Z., 1996. Photoacclimation in the marine alga Nannochloropsis sp. (Eustigmatophyte): a kinetic study. J. Plankton Res. 18, 1797–1818. https://doi.org/10.1093/plankt/18.10.1797. Gantt, E., 1990. Pigmentation and photoacclimation. In: Cole, K.M., Sheath, R.G. (Eds.), Biology of the Red Algae. Cambridge University Press, Cambridge, U.K, pp. 203–220. Gantt, E., Cunningham, F.X., 2001. Algal pigments. Encyclopedia of Life Sciences. John Wiley, Oxford, UK. https://doi.org/10.1038/npg.els.0000323.
5. Conclusion The invasive potential of G. salicornia has been attributed to its ability to sequester nutrients (Larned, 1998), benefit from low herbivory, asexual reproduction via fragmentation, and broad tolerance to environmental conditions (Smith et al., 2004; Amato et al., 2018), including to a wide range of irradiance regimes (Beach et al., 1997). This study reinforces the notion that G. salicornia possesses broad tolerance to solar irradiance, thriving both in direct tropical sunlight and to long periods of deep shade. Moreover, G. salicornia’s tendency for rapid growth when transitioning from shade to sun may be of advantage following disturbances that create gaps in this species’ unique matforming morphology. In light of these findings, it seems unlikely that relative rates of photoacclimation alone can account for differential success of native and invasive species, though additional research along these lines might demonstrate otherwise. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Contributors K. Hamel and C.M. Smith conceived and designed the experiments. 6
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