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Temperature and irradiance effects on growth and photosynthesis of Caulerpa (Chlorophyta) species from the eastern Mediterranean
Aquatic Botany 104 (2013) 106–110
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Aquatic Botany journal homepage: www.elsevier.com/locate/aquabot
Temperature and irradiance effects on growth and photosynthesis of Caulerpa (Chlorophyta) species from the eastern Mediterranean Shimrit Ukabi a,b,∗ , Zvy Dubinsky a , Yosef Steinberger a , Alvaro Israel b a b
The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel Israel Oceanographic & Limnological Research, Ltd., The National Institute of Oceanography, P.O. Box 8030, Tel Shikmona, Haifa 31080, Israel
a r t i c l e
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Article history: Received 2 June 2011 Received in revised form 28 August 2012 Accepted 30 August 2012 Available online 10 September 2012 Keywords: Caulerpa species Mediterranean Temperature Irradiance Photosynthesis
a b s t r a c t We evaluated the effects of temperature and irradiance on growth and photosynthetic activity of Caulerpa prolifera, C. mexicana and C. scalpelliformis, all common species in the eastern Israeli Mediterranean. The growth of these species was negative at 15 ◦ C but optimal at 23–26 ◦ C, averaging 16% at 23 ◦ C and 48% at 26 ◦ C. For all species, the effect of irradiance on growth was seen by a large number of buds (regeneration of new thalli from mother leaves), particularly at the high (120 mol photons m−2 s−1 ) experimental irradiance. The species most sensitive to high irradiance was C. scalpelliformis, for which growth was negative from 60 mol photons m−2 s−1 and above. Photosynthetic rates and photosynthetic parameters generally correlated with growth, irradiance, and temperature conditions found in the natural environments for all three species. © 2012 Elsevier B.V. All rights reserved.
1. Introduction The genus Caulerpa is widespread and common in the intertidal and subtidal zones of tropical and subtropical regions (Taylor, 1960; Luning, 1990). From the ca. 75 species described worldwide, six, C. prolifera, C. mexicana, C. scalpelliformis, C. ollivieri, C. racemosa, and C. taxifolia, have been described for the Mediterranean Sea (Meinesz, 1980; Einav, 1998; UNEP, 1999; Einav, 2007). From these, only three species, C. prolifera (Forsskål) J.V. Lamouroux, C. mexicana Sonder ex Kützing, and C. scalpelliformis (R. Brown ex Turner) C. Agardh, were reported to be found along the Israeli Mediterranean coast in recent years (Einav, 2007; Einav and Israel, 2007). Caulerpa species have been intensively investigated due to their unique siphonous thallus composed of a single multinucleate cell, their value as food for humans (Paul and Fenical, 1987; Meyer and Paul, 1992; Critchley and Ohno, 1998), and the invasive properties of some of the species (Ceccherelli et al., 2002; Cevik et al., 2007). Caulerpa taxifolia is one remarkable example underlying the importance of ecophysiological responses of the genus to environmental factors, such as temperature and irradiance. Komatsu et al. (1997) found a strong correlation between growth capacity and prevailing temperatures and irradiances for C. taxifolia
∗ Corresponding author at: The Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel. Tel.: +972 54 4499618; fax: +972 3 9388947. E-mail addresses: Shimrit [email protected], [email protected] (S. Ukabi). 0304-3770/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aquabot.2012.08.007
cultured under controlled conditions. The upper range of temperature for positive growth was 31.5–32.5 ◦ C and the lower range was 9–10 ◦ C. The algae could survive below 10–12 ◦ C and new stolons and fronds developed at 15 and 17.5 ◦ C, respectively. These experiments also showed that optimal irradiances were between 88 and 338 mol photons m−2 s−1 (14 h light:10 h dark). Ruitton et al. (2005) reported significant seasonal fluctuations in growth for C. racemosa, also known to be an invasive species, and found that the highest growth occurred in fall and the minimum growth occurred from winter to early spring. Several other studies have addressed the combined responses of photosynthesis and growth of Caulerpa to environmental variables (Ceccherelli and Cinelli, 1997; ColladoVides and Robledo, 1999; Smith and Walters, 1999; Piazzi and Ceccherelli, 2002; Raniello et al., 2004; Montefalcone et al., 2007). Terrados and Ros (1992) reported that on a seasonal basis, photosynthesis and respiration rates of C. prolifera increased linearly with water temperature (10–30 ◦ C). This species also maintained positive net photosynthesis at 10 ◦ C during winter. Caulerpa species from the eastern Mediterranean have only been investigated regarding their abundance and distribution patterns (Rayss and Edelstein, 1960; Lundberg, 1986; Einav, 1993, 1998), with no approach to their ecophysiological adaptations to natural environments. To the best of our knowledge, only two published investigations on Caulerpa from the Israeli Mediterranean have addressed the growth and development of C. prolifera under cultivation (Levi and Friedlander, 2004; Friedlander et al., 2006). Consequently, in this study, we aimed to evaluate the effect of temperature and irradiance on the growth and photosynthetic
S. Ukabi et al. / Aquatic Botany 104 (2013) 106–110
activity of the three recently validated species of Caulerpa, C. prolifera (Forsskål) J.V. Lamouroux, C. mexicana Sonder ex Kützing, and C. scalpelliformis (R. Brown ex Turner) C. Agardh. Based on the reported phenotypic plasticity of the genus (Verlaque et al., 2003; Einav and Israel, 2007; Guiry and Guiry, 2011), we hypothesized that there would be distinct responses of photosynthesis and growth among species that will likely reflect the growth conditions in nature. 2. Materials and methods 2.1. Algal collection and acclimatization About 50 untouched thalli and their rhizoids of C. prolifera (Forsskål) J.V. Lamouroux, C. mexicana Sonder ex Kützing, C. scalpelliformis (R. Brown ex Turner) C. Agardh, were collected from the intertidal zone near the Israel Oceanographic and Limnological Research (IOLR) Institute, Haifa, Israel. The specimens were cleaned and the rhizoids buried in the first 2–4 cm of a 5-cm sea sand layer, in three separate 20-L glass aquaria filled with seawater collected at the study site. The tanks were kept in a growth room at 24 ◦ C with a continuous seawater flow and aeration at an average of 17 mol photons m−2 s−1 (12 h/12 h, measured with a LICOR 250A light meter), following Levi and Friedlander (2004). These stocked algae were kept at these conditions during 4 weeks in order to standardize acclimation before the initiation of the experiments. 2.2. Assessing the effect of temperature Single thalli (n = 5–7) attached to their respective rhizoids were taken from the stocks, gently dried from excessive water and weighed. The length of these thalli and their photosynthetic rates were also measured (see below) at this time. Then, the algae were replanted in four experimental glass tanks (27 cm × 27 cm × 20 cm each) set at 15◦ , 23◦ , 26◦ , and 30 ◦ C using water baths. Irradiance was kept constant for all four tanks at 17 mol photons m−2 s−1 (12 h/12 h). Growth was estimated after 21 days by measuring both the changes in total biomass (rhizoids + thalli) and the thallus length. Growth rates were assumed linear and were expressed as relative growth on a week basis (RGR), as follows: RGR = Wf − Wi /Wi /w × 100; in which Wf = final fresh weight or thallus length, Wi = initial fresh weight or thallus length, w = experiment length in weeks. Similarly, photosynthetic rates were also evaluated following this 21-day period. In order to verify possible regrowth of damaged thalli at the extreme tested temperatures (i.e., 15 and 30 ◦ C), the temperatures were adjusted to 26 ◦ C for an additional period of 21 days. The tanks were then checked for the presence of newly developing Caulerpa leaves, together with measurements of their respective lengths. 2.3. Assessing the effect of irradiance The effect of irradiance on growth rates and photosynthesis was determined by replanting stocked in four black plastic tanks (32 cm × 25 cm × 7 cm each), in a way similar to that used for experiments aimed at measuring temperature effects. The tanks were set at 1.5, 10, 60, or 120 mol photons m−2 s−1 (measured as for the above experiments) by covering them with neutral plastic nets at a temperature of 26 ◦ C. The thalli were grown for 21 days and their growth monitored by measuring biomass increments and thallus surface area. Changes in thallus area were determined by photographing the algae before and after the 3-week experimental period, and the photos (reflecting changes in area) analyzed with the Image Tool program (UTHSCSA Image Tool Version 3.0 Script Language Reference Manual (Manual revision 5)). Photosynthetic
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responses were determined before and after the experimental period as described below. 2.4. Determination of photosynthesis Photosynthetic quantum yields (Fv /Fm ) were determined using a pulse amplitude modulated (PAM) fluorometer (Diving-PAM, Walz, Germany). Thalli from the experimental tanks were placed in the dark for 20 min using leaf clips. After the initial fluorescence (Fo ) measurement, thalli were exposed to a single saturating light pulse of 5000 mol photons m−2 s−1 for 0.8 s in order to drive the maximum fluorescence (Fm ). The optimal quantum yield was then calculated as (Fm − Fo )/Fm , where (Fm − Fo ) corresponds to the variable fluorescence Fv . 2.5. Photosynthesis vs. irradiance curves Photosynthesis and respiration rates in response to irradiance (PI curves) were measured with a Clark-type oxygen electrode (Yellow Springs 5331 type), similar to Dubinsky et al. (1987). Irradiance levels (measured with a LI-COR 1000 Quantum Sensor, LI-COR, Nebraska) applied to the algal sample were provided by low-noise illuminator 97-41-52 (Cole Parmer Instrument Company, Chicago). Before each experiment, samples were dark-adapted in order to oxidize plastoquinone (PQ) pool and avoid a time lag in photosynthesis (Moberg et al., 1997). In addition, samples were bubbled with a stream of N2 in order to reduce the initial oxygen concentration to ca. 80% saturation, inserted into a 15 mL cuvette, and allowed to equilibrate with the chamber temperature, set at 24 ◦ C. Next, dark respiration was measured for 120 s followed by measurements of net photosynthetic rates while the cuvette was illuminated with a sequence of 10 irradiances, each illumination period lasting 120 s. The various light intensities were achieved by different filters mounted in front of the incubation chamber window. Photosynthetic rate was calculated from the linear portion of the slope during the light periods. PI curves were fitted using the following equation: Hyperbolic tangent equation :
P(E) = Pmax
eE/EK − e−E/EK eE/EK + e−E/EK
Photosynthetic parameters (alpha, ˛; Pmax , Ik , and LCP) were calculated using programs developed by Ben-Zion and Dubinsky (1988) and Platt and Jassby (1976). 2.6. Statistical analysis All data were subjected to analysis of variance using an SAS model [two-way ANOVA (SAS, 1988)]. The experiments were randomized block designed as to minimize the effects of nuisance factors. Differences obtained at a level of p < 0.05 were considered significant. Duncan’s and Tukey’s multiple range tests were used to evaluate differences between separate means (Sokal and Rohlf, 1969). 3. Results After 7 days of acclimation at the various temperatures, thalli of all three Caulerpa species deteriorated at 15 ◦ C, and completely disappeared by day 21 (Fig. 1). Following a recovery period of 21 days, only C. prolifera recovered on day 21, as witnessed by new leaves emerging from the stolon buried in the sandy substrate. At 23 and 26 ◦ C, new thallus clones (new “buds” appearing on old Caulerpa leaves) showed at a range of 3–7 buds per mother leaf, and were taken into consideration to estimate growth. Hence, the thalli relative growth rates as based on length measurements at
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Fig. 1. (A) Relative growth rate (RGR %) based on thallus length of C. prolifera, C. mexicana and C. scalpelliformis grown at 15, 23, 26 and 30 ◦ C during 21 days (n = 8). (B) Photosynthetic yields based on fluorescence responses (Fv /Fm ) determined under the same conditions (n = 3).
23 ◦ C was positive for two species, with 15% and 16% thallus elongation (p < 0.05) for C. prolifera and C. mexicana, respectively, while no significant changes were observed for C. scalpelliformis. The trend of positive growth continued at 26 ◦ C with thallus elongation of 56%, 41%, and 46% for C. prolifera, C. mexicana, and C. scalpelliformis, respectively (p < 0.05, Fig. 1A). At 30 ◦ C, thalli of all three species experienced decreases in length and, therefore, in their growth capacity (Fig. 1A). C. prolifera and C. scalpelliformis exhibited negative growth at an average of 10%, while C. mexicana exhibited negative growth of 53% after the 21-day experimental period (p < 0.001, Fig. 1A). At 15 ◦ C, the initial photosynthetic yields were similar for C. prolifera and C. scalpelliformis, and significantly lower for C. mexicana (p < 0.001, Fig. 1B). No measurements followed since the thalli deteriorated completely at this temperature (see above). After the 21-day experimental period, the photosynthetic yields were similar for the three species at the optimal temperatures of 23 and 26 ◦ C (Fig. 1B). A significantly higher tolerance to high temperature was found at 30 ◦ C for C. scalpelliformis since the photosynthetic yield in this species decrease only by 50% as compared to an average of 75% for C. prolifera and C. mexicana (p < 0.001, Fig. 1B). Out of the four different experimental irradiances (1.5, 10, 60, and 120 mol photons m−2 s−1 ) at 120 mol photons m−2 s−1 the initial leaf of C. prolifera did not elongate much but rather developed a large number of new buds. C. mexicana showed the same pattern while C. scalpelliformis fully disappeared at this irradiance (Fig. 2A). With increasing irradiance, the growth of C. prolifera ranged from 170 to 113% and that of C. mexicana ranged from 186 to 40% (Fig. 2A). A very similar pattern of growth was observed when growth was calculated on a fresh weight basis (Fig. 2B). Maximal net photosynthesis was similar for C. prolifera and C. mexicana, both being photosynthetically more tolerant than C. scalpelliformis to high irradiance (Fig. 2C). As determined from PI equations, Caulerpa in this study exhibited net photosynthesis
Fig. 2. (A) Relative growth rate (RGR %) based on thallus length of C. prolifera, C. mexicana and C. scalpelliformis grown at 1.5, 10, 60, and 120 mol photons m−2 s−1 during the 21 days (n = 4). (B) Relative growth rate (RGR %) based on thallus weight determined under the same conditions (n = 4). (C) Photosynthetic yields based on fluorescence responses (Fv /Fm ) determined under the same conditions (n = 4).
(PN ) that was significantly different (p < 0.01) between each of the species. Grading photosynthetic capacity, C. scalpelliformis exhibited the highest photosynthetic rates, followed by C. prolifera and C. mexicana (Fig. 3 and Table 1). C. scalpelliformis exhibited the highest ˛ and Ik (p < 0.05) while C. mexicana showed the highest respiration rates.
Fig. 3. Net photosynthetic rates (PN) (mol O2 mol−1 chl a min1 ) as a function of irradiance for C. prolifera, C. mexicana and C. scalpelliformis measured at 24 ◦ C.
S. Ukabi et al. / Aquatic Botany 104 (2013) 106–110 Table 1 Photosynthetic parameters determined from PI curves at 24 ◦ C for C. prolifera, C. mexicana and C. scalpelliformis. ˛: initial slope; Pmax : light saturated net photosynthetic rate; Ik : irradiance needed to reach maximal photosynthetic rates (mol photons−1 ); LCP: compensation irradiance (net photosynthetic rate = dark respiration rate, mol photons m−2 s−1 ); r2 : correlation coefficient values for PI curves. Photosynthetic parameters
C. prolifera C. mexicana C. scalpelliformis
˛
Pmax
Ik
LCP
r2
0.0256 0.0160 0.0935
0.497 0.381 0.510
1.94 2.39 5.46
5.02 1.06 0.83
0.586 0.377 0.742
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Acknowledgements We thank S. Koren and Y. Schnytzer for technical assistance and A. Yamshon and E. Shaham for assistance with the experiments. Thanks to Dr. D. Iluz for sharing his knowledge and for his helpful explanations, and to Ms. S. Victor for her useful language comments. We gratefully acknowledge the painstaking and friendly assistance of Dr. J. Vermaat in reviewing the manuscript, which resulted in a greatly improved final version. This research is part of the Ph.D. Dissertation of S.U., and was conducted at Bar-Ilan University.
References 4. Discussion From the three species of Caulerpa investigated, C. prolifera was the most tolerant to extreme experimental temperatures largely behaving as a eurythermal species that adjusts to the annual course of water temperature and irradiance (Terrados and Ros, 1992; Friedlander et al., 2006). In the eastern Mediterranean, C. prolifera further encounters in nature low levels of total N and P (less than 5 and 2 M, respectively), low irradiance (30 mol photons m−2 s−1 ), and moderate temperatures (22–28 ◦ C). Under these conditions, growth rates can be 30–130% per week, with the highest growth observed at low irradiance and high temperature (Friedlander et al., 2006). Tolerance to high temperatures is also effective in C. scalpelliformis, for which optimal conditions for growth and bud formation (i.e., new thalli or “leaf” regeneration from the main leaf) occurred at 30 ◦ C, similar to results shown by Ertan et al. (1998). Hence, it appears logical that C. scalpelliformis is very active photosynthetically, more than the other two Caulerpa species, at high temperature. Also, the growth of C. prolifera took place in the form of buds developing from older leaves, rather than length expansion of these leaves. C. scalpelliformis had the fastest responses to low irradiances and showed the highest photosynthetic capacity, as corroborated by its photosynthetic parameters. C. mexicana acclimated well to high light at the expense of relatively high respiration rates. Furthermore, an expected correlation was found between photosynthesis and growth rates in all species at least under the experimental conditions imposed in the present study. During algal collection, we observed that C. scalpelliformis was found only in rims or under rocky platforms in which light was very dim (Rilov and Galil, 2009), while C. mexicana was more clearly exposed to high irradiances within the intertidal. Thus, experimental responses coincided with the habitats normally occupied by these Caulerpa species in their natural environments. In a parallel study on the seasonal ecological distribution of Caulerpa species along the Israeli Mediterranean shore (Ukabi et al., 2012), it was found that these same Caulerpa species achieve maximal biomass during summer time, while only short stolons with sparse upright axes predominate during winter time. Most ecological studies on Caulerpa in the Mediterranean have focused on C. taxifolia, apparently due to its invasive nature substantiated by high flexibility and ability to grow under a wide range of temperatures and irradiances (Uchimura et al., 2000; Ljiljana et al., 2006). Within a time frame of about 10 years, C. taxifolia spread in the Mediterranean, affecting six countries and more than 13,000 ha of sea bottom (Meinesz, 2001). In conclusion, our study points to C. prolifera as the species most tolerant to irradiance, as shown by Häder et al. (1997), followed by C. mexicana. All three species responded positively to a temperature range of 23–26 ◦ C, with a relative advantage of C. scalpelliformis at 30 ◦ C. Temperatures of 15 ◦ C and below are inhibitory of aboveground growth.
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Contents lists available at SciVerse ScienceDirect
Aquatic Botany journal homepage: www.elsevier.com/locate/aquabot
Temperature and irradiance effects on growth and photosynthesis of Caulerpa (Chlorophyta) species from the eastern Mediterranean Shimrit Ukabi a,b,∗ , Zvy Dubinsky a , Yosef Steinberger a , Alvaro Israel b a b
The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel Israel Oceanographic & Limnological Research, Ltd., The National Institute of Oceanography, P.O. Box 8030, Tel Shikmona, Haifa 31080, Israel
a r t i c l e
i n f o
Article history: Received 2 June 2011 Received in revised form 28 August 2012 Accepted 30 August 2012 Available online 10 September 2012 Keywords: Caulerpa species Mediterranean Temperature Irradiance Photosynthesis
a b s t r a c t We evaluated the effects of temperature and irradiance on growth and photosynthetic activity of Caulerpa prolifera, C. mexicana and C. scalpelliformis, all common species in the eastern Israeli Mediterranean. The growth of these species was negative at 15 ◦ C but optimal at 23–26 ◦ C, averaging 16% at 23 ◦ C and 48% at 26 ◦ C. For all species, the effect of irradiance on growth was seen by a large number of buds (regeneration of new thalli from mother leaves), particularly at the high (120 mol photons m−2 s−1 ) experimental irradiance. The species most sensitive to high irradiance was C. scalpelliformis, for which growth was negative from 60 mol photons m−2 s−1 and above. Photosynthetic rates and photosynthetic parameters generally correlated with growth, irradiance, and temperature conditions found in the natural environments for all three species. © 2012 Elsevier B.V. All rights reserved.
1. Introduction The genus Caulerpa is widespread and common in the intertidal and subtidal zones of tropical and subtropical regions (Taylor, 1960; Luning, 1990). From the ca. 75 species described worldwide, six, C. prolifera, C. mexicana, C. scalpelliformis, C. ollivieri, C. racemosa, and C. taxifolia, have been described for the Mediterranean Sea (Meinesz, 1980; Einav, 1998; UNEP, 1999; Einav, 2007). From these, only three species, C. prolifera (Forsskål) J.V. Lamouroux, C. mexicana Sonder ex Kützing, and C. scalpelliformis (R. Brown ex Turner) C. Agardh, were reported to be found along the Israeli Mediterranean coast in recent years (Einav, 2007; Einav and Israel, 2007). Caulerpa species have been intensively investigated due to their unique siphonous thallus composed of a single multinucleate cell, their value as food for humans (Paul and Fenical, 1987; Meyer and Paul, 1992; Critchley and Ohno, 1998), and the invasive properties of some of the species (Ceccherelli et al., 2002; Cevik et al., 2007). Caulerpa taxifolia is one remarkable example underlying the importance of ecophysiological responses of the genus to environmental factors, such as temperature and irradiance. Komatsu et al. (1997) found a strong correlation between growth capacity and prevailing temperatures and irradiances for C. taxifolia
∗ Corresponding author at: The Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel. Tel.: +972 54 4499618; fax: +972 3 9388947. E-mail addresses: Shimrit [email protected], [email protected] (S. Ukabi). 0304-3770/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aquabot.2012.08.007
cultured under controlled conditions. The upper range of temperature for positive growth was 31.5–32.5 ◦ C and the lower range was 9–10 ◦ C. The algae could survive below 10–12 ◦ C and new stolons and fronds developed at 15 and 17.5 ◦ C, respectively. These experiments also showed that optimal irradiances were between 88 and 338 mol photons m−2 s−1 (14 h light:10 h dark). Ruitton et al. (2005) reported significant seasonal fluctuations in growth for C. racemosa, also known to be an invasive species, and found that the highest growth occurred in fall and the minimum growth occurred from winter to early spring. Several other studies have addressed the combined responses of photosynthesis and growth of Caulerpa to environmental variables (Ceccherelli and Cinelli, 1997; ColladoVides and Robledo, 1999; Smith and Walters, 1999; Piazzi and Ceccherelli, 2002; Raniello et al., 2004; Montefalcone et al., 2007). Terrados and Ros (1992) reported that on a seasonal basis, photosynthesis and respiration rates of C. prolifera increased linearly with water temperature (10–30 ◦ C). This species also maintained positive net photosynthesis at 10 ◦ C during winter. Caulerpa species from the eastern Mediterranean have only been investigated regarding their abundance and distribution patterns (Rayss and Edelstein, 1960; Lundberg, 1986; Einav, 1993, 1998), with no approach to their ecophysiological adaptations to natural environments. To the best of our knowledge, only two published investigations on Caulerpa from the Israeli Mediterranean have addressed the growth and development of C. prolifera under cultivation (Levi and Friedlander, 2004; Friedlander et al., 2006). Consequently, in this study, we aimed to evaluate the effect of temperature and irradiance on the growth and photosynthetic
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activity of the three recently validated species of Caulerpa, C. prolifera (Forsskål) J.V. Lamouroux, C. mexicana Sonder ex Kützing, and C. scalpelliformis (R. Brown ex Turner) C. Agardh. Based on the reported phenotypic plasticity of the genus (Verlaque et al., 2003; Einav and Israel, 2007; Guiry and Guiry, 2011), we hypothesized that there would be distinct responses of photosynthesis and growth among species that will likely reflect the growth conditions in nature. 2. Materials and methods 2.1. Algal collection and acclimatization About 50 untouched thalli and their rhizoids of C. prolifera (Forsskål) J.V. Lamouroux, C. mexicana Sonder ex Kützing, C. scalpelliformis (R. Brown ex Turner) C. Agardh, were collected from the intertidal zone near the Israel Oceanographic and Limnological Research (IOLR) Institute, Haifa, Israel. The specimens were cleaned and the rhizoids buried in the first 2–4 cm of a 5-cm sea sand layer, in three separate 20-L glass aquaria filled with seawater collected at the study site. The tanks were kept in a growth room at 24 ◦ C with a continuous seawater flow and aeration at an average of 17 mol photons m−2 s−1 (12 h/12 h, measured with a LICOR 250A light meter), following Levi and Friedlander (2004). These stocked algae were kept at these conditions during 4 weeks in order to standardize acclimation before the initiation of the experiments. 2.2. Assessing the effect of temperature Single thalli (n = 5–7) attached to their respective rhizoids were taken from the stocks, gently dried from excessive water and weighed. The length of these thalli and their photosynthetic rates were also measured (see below) at this time. Then, the algae were replanted in four experimental glass tanks (27 cm × 27 cm × 20 cm each) set at 15◦ , 23◦ , 26◦ , and 30 ◦ C using water baths. Irradiance was kept constant for all four tanks at 17 mol photons m−2 s−1 (12 h/12 h). Growth was estimated after 21 days by measuring both the changes in total biomass (rhizoids + thalli) and the thallus length. Growth rates were assumed linear and were expressed as relative growth on a week basis (RGR), as follows: RGR = Wf − Wi /Wi /w × 100; in which Wf = final fresh weight or thallus length, Wi = initial fresh weight or thallus length, w = experiment length in weeks. Similarly, photosynthetic rates were also evaluated following this 21-day period. In order to verify possible regrowth of damaged thalli at the extreme tested temperatures (i.e., 15 and 30 ◦ C), the temperatures were adjusted to 26 ◦ C for an additional period of 21 days. The tanks were then checked for the presence of newly developing Caulerpa leaves, together with measurements of their respective lengths. 2.3. Assessing the effect of irradiance The effect of irradiance on growth rates and photosynthesis was determined by replanting stocked in four black plastic tanks (32 cm × 25 cm × 7 cm each), in a way similar to that used for experiments aimed at measuring temperature effects. The tanks were set at 1.5, 10, 60, or 120 mol photons m−2 s−1 (measured as for the above experiments) by covering them with neutral plastic nets at a temperature of 26 ◦ C. The thalli were grown for 21 days and their growth monitored by measuring biomass increments and thallus surface area. Changes in thallus area were determined by photographing the algae before and after the 3-week experimental period, and the photos (reflecting changes in area) analyzed with the Image Tool program (UTHSCSA Image Tool Version 3.0 Script Language Reference Manual (Manual revision 5)). Photosynthetic
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responses were determined before and after the experimental period as described below. 2.4. Determination of photosynthesis Photosynthetic quantum yields (Fv /Fm ) were determined using a pulse amplitude modulated (PAM) fluorometer (Diving-PAM, Walz, Germany). Thalli from the experimental tanks were placed in the dark for 20 min using leaf clips. After the initial fluorescence (Fo ) measurement, thalli were exposed to a single saturating light pulse of 5000 mol photons m−2 s−1 for 0.8 s in order to drive the maximum fluorescence (Fm ). The optimal quantum yield was then calculated as (Fm − Fo )/Fm , where (Fm − Fo ) corresponds to the variable fluorescence Fv . 2.5. Photosynthesis vs. irradiance curves Photosynthesis and respiration rates in response to irradiance (PI curves) were measured with a Clark-type oxygen electrode (Yellow Springs 5331 type), similar to Dubinsky et al. (1987). Irradiance levels (measured with a LI-COR 1000 Quantum Sensor, LI-COR, Nebraska) applied to the algal sample were provided by low-noise illuminator 97-41-52 (Cole Parmer Instrument Company, Chicago). Before each experiment, samples were dark-adapted in order to oxidize plastoquinone (PQ) pool and avoid a time lag in photosynthesis (Moberg et al., 1997). In addition, samples were bubbled with a stream of N2 in order to reduce the initial oxygen concentration to ca. 80% saturation, inserted into a 15 mL cuvette, and allowed to equilibrate with the chamber temperature, set at 24 ◦ C. Next, dark respiration was measured for 120 s followed by measurements of net photosynthetic rates while the cuvette was illuminated with a sequence of 10 irradiances, each illumination period lasting 120 s. The various light intensities were achieved by different filters mounted in front of the incubation chamber window. Photosynthetic rate was calculated from the linear portion of the slope during the light periods. PI curves were fitted using the following equation: Hyperbolic tangent equation :
P(E) = Pmax
eE/EK − e−E/EK eE/EK + e−E/EK
Photosynthetic parameters (alpha, ˛; Pmax , Ik , and LCP) were calculated using programs developed by Ben-Zion and Dubinsky (1988) and Platt and Jassby (1976). 2.6. Statistical analysis All data were subjected to analysis of variance using an SAS model [two-way ANOVA (SAS, 1988)]. The experiments were randomized block designed as to minimize the effects of nuisance factors. Differences obtained at a level of p < 0.05 were considered significant. Duncan’s and Tukey’s multiple range tests were used to evaluate differences between separate means (Sokal and Rohlf, 1969). 3. Results After 7 days of acclimation at the various temperatures, thalli of all three Caulerpa species deteriorated at 15 ◦ C, and completely disappeared by day 21 (Fig. 1). Following a recovery period of 21 days, only C. prolifera recovered on day 21, as witnessed by new leaves emerging from the stolon buried in the sandy substrate. At 23 and 26 ◦ C, new thallus clones (new “buds” appearing on old Caulerpa leaves) showed at a range of 3–7 buds per mother leaf, and were taken into consideration to estimate growth. Hence, the thalli relative growth rates as based on length measurements at
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Fig. 1. (A) Relative growth rate (RGR %) based on thallus length of C. prolifera, C. mexicana and C. scalpelliformis grown at 15, 23, 26 and 30 ◦ C during 21 days (n = 8). (B) Photosynthetic yields based on fluorescence responses (Fv /Fm ) determined under the same conditions (n = 3).
23 ◦ C was positive for two species, with 15% and 16% thallus elongation (p < 0.05) for C. prolifera and C. mexicana, respectively, while no significant changes were observed for C. scalpelliformis. The trend of positive growth continued at 26 ◦ C with thallus elongation of 56%, 41%, and 46% for C. prolifera, C. mexicana, and C. scalpelliformis, respectively (p < 0.05, Fig. 1A). At 30 ◦ C, thalli of all three species experienced decreases in length and, therefore, in their growth capacity (Fig. 1A). C. prolifera and C. scalpelliformis exhibited negative growth at an average of 10%, while C. mexicana exhibited negative growth of 53% after the 21-day experimental period (p < 0.001, Fig. 1A). At 15 ◦ C, the initial photosynthetic yields were similar for C. prolifera and C. scalpelliformis, and significantly lower for C. mexicana (p < 0.001, Fig. 1B). No measurements followed since the thalli deteriorated completely at this temperature (see above). After the 21-day experimental period, the photosynthetic yields were similar for the three species at the optimal temperatures of 23 and 26 ◦ C (Fig. 1B). A significantly higher tolerance to high temperature was found at 30 ◦ C for C. scalpelliformis since the photosynthetic yield in this species decrease only by 50% as compared to an average of 75% for C. prolifera and C. mexicana (p < 0.001, Fig. 1B). Out of the four different experimental irradiances (1.5, 10, 60, and 120 mol photons m−2 s−1 ) at 120 mol photons m−2 s−1 the initial leaf of C. prolifera did not elongate much but rather developed a large number of new buds. C. mexicana showed the same pattern while C. scalpelliformis fully disappeared at this irradiance (Fig. 2A). With increasing irradiance, the growth of C. prolifera ranged from 170 to 113% and that of C. mexicana ranged from 186 to 40% (Fig. 2A). A very similar pattern of growth was observed when growth was calculated on a fresh weight basis (Fig. 2B). Maximal net photosynthesis was similar for C. prolifera and C. mexicana, both being photosynthetically more tolerant than C. scalpelliformis to high irradiance (Fig. 2C). As determined from PI equations, Caulerpa in this study exhibited net photosynthesis
Fig. 2. (A) Relative growth rate (RGR %) based on thallus length of C. prolifera, C. mexicana and C. scalpelliformis grown at 1.5, 10, 60, and 120 mol photons m−2 s−1 during the 21 days (n = 4). (B) Relative growth rate (RGR %) based on thallus weight determined under the same conditions (n = 4). (C) Photosynthetic yields based on fluorescence responses (Fv /Fm ) determined under the same conditions (n = 4).
(PN ) that was significantly different (p < 0.01) between each of the species. Grading photosynthetic capacity, C. scalpelliformis exhibited the highest photosynthetic rates, followed by C. prolifera and C. mexicana (Fig. 3 and Table 1). C. scalpelliformis exhibited the highest ˛ and Ik (p < 0.05) while C. mexicana showed the highest respiration rates.
Fig. 3. Net photosynthetic rates (PN) (mol O2 mol−1 chl a min1 ) as a function of irradiance for C. prolifera, C. mexicana and C. scalpelliformis measured at 24 ◦ C.
S. Ukabi et al. / Aquatic Botany 104 (2013) 106–110 Table 1 Photosynthetic parameters determined from PI curves at 24 ◦ C for C. prolifera, C. mexicana and C. scalpelliformis. ˛: initial slope; Pmax : light saturated net photosynthetic rate; Ik : irradiance needed to reach maximal photosynthetic rates (mol photons−1 ); LCP: compensation irradiance (net photosynthetic rate = dark respiration rate, mol photons m−2 s−1 ); r2 : correlation coefficient values for PI curves. Photosynthetic parameters
C. prolifera C. mexicana C. scalpelliformis
˛
Pmax
Ik
LCP
r2
0.0256 0.0160 0.0935
0.497 0.381 0.510
1.94 2.39 5.46
5.02 1.06 0.83
0.586 0.377 0.742
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Acknowledgements We thank S. Koren and Y. Schnytzer for technical assistance and A. Yamshon and E. Shaham for assistance with the experiments. Thanks to Dr. D. Iluz for sharing his knowledge and for his helpful explanations, and to Ms. S. Victor for her useful language comments. We gratefully acknowledge the painstaking and friendly assistance of Dr. J. Vermaat in reviewing the manuscript, which resulted in a greatly improved final version. This research is part of the Ph.D. Dissertation of S.U., and was conducted at Bar-Ilan University.
References 4. Discussion From the three species of Caulerpa investigated, C. prolifera was the most tolerant to extreme experimental temperatures largely behaving as a eurythermal species that adjusts to the annual course of water temperature and irradiance (Terrados and Ros, 1992; Friedlander et al., 2006). In the eastern Mediterranean, C. prolifera further encounters in nature low levels of total N and P (less than 5 and 2 M, respectively), low irradiance (30 mol photons m−2 s−1 ), and moderate temperatures (22–28 ◦ C). Under these conditions, growth rates can be 30–130% per week, with the highest growth observed at low irradiance and high temperature (Friedlander et al., 2006). Tolerance to high temperatures is also effective in C. scalpelliformis, for which optimal conditions for growth and bud formation (i.e., new thalli or “leaf” regeneration from the main leaf) occurred at 30 ◦ C, similar to results shown by Ertan et al. (1998). Hence, it appears logical that C. scalpelliformis is very active photosynthetically, more than the other two Caulerpa species, at high temperature. Also, the growth of C. prolifera took place in the form of buds developing from older leaves, rather than length expansion of these leaves. C. scalpelliformis had the fastest responses to low irradiances and showed the highest photosynthetic capacity, as corroborated by its photosynthetic parameters. C. mexicana acclimated well to high light at the expense of relatively high respiration rates. Furthermore, an expected correlation was found between photosynthesis and growth rates in all species at least under the experimental conditions imposed in the present study. During algal collection, we observed that C. scalpelliformis was found only in rims or under rocky platforms in which light was very dim (Rilov and Galil, 2009), while C. mexicana was more clearly exposed to high irradiances within the intertidal. Thus, experimental responses coincided with the habitats normally occupied by these Caulerpa species in their natural environments. In a parallel study on the seasonal ecological distribution of Caulerpa species along the Israeli Mediterranean shore (Ukabi et al., 2012), it was found that these same Caulerpa species achieve maximal biomass during summer time, while only short stolons with sparse upright axes predominate during winter time. Most ecological studies on Caulerpa in the Mediterranean have focused on C. taxifolia, apparently due to its invasive nature substantiated by high flexibility and ability to grow under a wide range of temperatures and irradiances (Uchimura et al., 2000; Ljiljana et al., 2006). Within a time frame of about 10 years, C. taxifolia spread in the Mediterranean, affecting six countries and more than 13,000 ha of sea bottom (Meinesz, 2001). In conclusion, our study points to C. prolifera as the species most tolerant to irradiance, as shown by Häder et al. (1997), followed by C. mexicana. All three species responded positively to a temperature range of 23–26 ◦ C, with a relative advantage of C. scalpelliformis at 30 ◦ C. Temperatures of 15 ◦ C and below are inhibitory of aboveground growth.
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