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Comparative morpho-physiological analysis between Ciona robusta and Ciona savignyi
Journal of Experimental Marine Biology and Ecology 485 (2016) 83–87
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Journal of Experimental Marine Biology and Ecology journal homepage: www.elsevier.com/locate/jembe
Comparative morpho-physiological analysis between Ciona robusta and Ciona savignyi Andrea Tarallo a,⁎, Mitshuaru Yagi b, Shin Oikawa c, Claudio Agnisola d, Giuseppe D'Onofrio a a
Dept. of Biology and Evolution of Marine Organisms, Stazione Zoologica Anton Dohrn, Villa Comunale, 80121 Naples, Italy Faculty of Fisheries, Nagasaki University, 1-14 Bunkyo, Nagasaki 852-8521, Japan c Fishery Research Laboratory, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka 811-3304, Japan d Dept. of Biology, University of Naples Federico II, Complesso Universitario di Monte SantAngelo, Edificio 7, Via Cinthia, 80126 Naples, Italy b
a r t i c l e
i n f o
Article history: Received 18 April 2016 Received in revised form 9 September 2016 Accepted 12 September 2016 Available online xxxx Keywords: Sea squirt Ascidians Metabolic rate Morphophysiology Tunic Ecology
a b s t r a c t The seasquirt Ciona robusta and its co-generic Ciona savignyi, are two long-divergent species, sharing the same habitat and competing for the same spaces and resources. Their very similar morphology has been responsible for the misidentification of the two organisms, and, consequently, for the underestimation of their geographic distributions and new areas of co-occurrence. In spite of the large amount of knowledge built up by developmental biologists, few data are available regarding the morpho-physiology of the two sea squirts. The comparison of morphological and physiological features carried out in the present study (i.e. length-body weight correlation, tunic/organ ratio, tissue-specific retained water and metabolic rate), highlighted slight, but significant, differences strongly supporting the hypothesis of different ecological strategies for the two species. More precisely, C. savignyi invests more energy in growing faster and taller, likely to improve the quality and concentration of the filtered food, and sustains a faster metabolic/growth rate. Conversely, C. robusta invests more into tunic thickness, reducing the risk of predation, even if this likely means a slower metabolic/growth rate. In addition it was noted that in both species, the tunic absorbs more water than the other tissues, a peculiarity that may allow them to better counteract sudden fluctuations in environmental salinity. © 2016 Elsevier B.V. All rights reserved.
1. Introduction In the last decade, different research groups have shown that the species Ciona intestinalis is actually a complex of genetically differentiated types (Caputi et al., 2007; Iannelli et al., 2007; Nydam and Harrison, 2010; Suzuki et al., 2005; Zhan et al., 2010). Very recently (Brunetti et al., 2015), the “types” formerly denoted as A and B, were ascribed respectively to two distinct species: Ciona robusta Hoshino and Tokioka, 1967 and C. intestinalis (Linnaeus, 1767). On the basis of the different morphological features observed at the larval stage, Pennati et al. (2015) confirmed the new classification. Due to their very close morphological features, C. robusta, C. intestinalis, and Ciona savignyi Herdman, 1882 were wrongly considered for a long time to be the same species (Hoshino and Nishikawa, 1985; Lambert and Lambert, 1998; Smith et al., 2010), in spite of the fact that C. robusta and C. intestinalis were estimated to diverge approximately 20 Mya (Suzuki et al., 2005), while C. robusta and C. savignyi were estimated to diverge 180 Mya (Berná et al., 2009). Attention was focused on the comparison of C. robusta vs. C. savignyi, since numerous reports stressed that the two species show overlapping ⁎ Corresponding author. E-mail addresses: [email protected], [email protected] (A. Tarallo).
http://dx.doi.org/10.1016/j.jembe.2016.09.001 0022-0981/© 2016 Elsevier B.V. All rights reserved.
distribution areas, competing for space and resources: southern California (Byrd and Lambert, 2000; Lambert and Lambert, 2003), New Zealand (Smith et al., 2010), the Korean peninsula (Taekjun and Sook, 2014), and Tokyo Bay (M. Yoshikuni, personal communication). Moreover, along the California coast, C. savignyi has been reported to replace C. robusta (Lambert, 2007; Lambert and Lambert, 1998; Lambert and Lambert, 2003). Despite the interest of the scientific community towards sea squirts, few data are available about the physiology of these organisms. Early studies examined oxygen consumption in C. intestinalis embryos and adults (Holter and Zeuthen, 1944; Jørgensen, 1952). The measurements performed by Jørgensen were carried out according to the Winkler methodology, estimating a consumption rate of about 0.8 ml O2/h. Markus and Lambert (1983), although corroborating the results of Jørgensen by using the same methodology (0.82 ml O2/g dry weight/ h), stated that former data “were reported as relative values and not as weight-specific rates”. It should be noted that, while Markus and Lambert measured oxygen consumption on animals sampled in California, thus almost surely belonging to the new classified species C. robusta (ex C. intestinalis type A), the specimens used by Jørgensen were collected from Woods Hole (Massachusetts), thus most probably C. intestinalis (ex “type B”). As far as we know, no metabolic studies are available in the current literature for C. savignyi. Thus, in this study, morpho-
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physiological parameters, i.e. body size, tunic/organ dry weight ratio, tissue water retention, as well as the routine oxygen consumption rate as proxy for routine metabolic rate, were measured for adults of C. savignyi and compared to those of C. robusta. 2. Materials and methods 2.1. Specimens The seasquirt Ciona robusta was provided by Kyoto University (Kyoto, Japan). The adult individuals were obtained from in vitro fertilization of wild gametes, which after settlement on Petri dishes, were reared in the field in Maizuru Bay by Maizuru Fisheries Research Station (Nagahama, Maizuru-shi, Kyoto, Japan). The co-generic Ciona savignyi was collected in two different areas: Tokyo Bay, by Tokyo University (Tokyo, Japan), and Sugashima Bay, by Nagoya University (Nagoya, Japan). Individuals were classified on the basis of their morphological characters according to Smith et al. (2010) and Sato et al. (2012). The specimens reared in Maizuru Bay were identified as C. robusta since adults showed the siphon tubercles, as well as the peculiar pigmentation of the tip of the vas deferens. The specimens collected in Tokyo Bay were identified as C. savignyi, because of the lack of pigmentation on the vas deferens and the presence of marked orange pigmentation of the inhalant siphon. The animals were shipped to Kyushu University Fishery Research Laboratory (Fukutsu, Fukuoka, Japan) with minimal temperature rise during transportation and transferred to 50-l aquaria with running filtered seawater and continuous aeration. In the case of the reared C. robusta, the animals were manually removed from the Petri dishes. Individuals attached to each other were separated, and epibionts on the tunic surface were removed by tweezers. Water temperature (ranging from 16.5 °C to 18.0 °C) and salinity (on average 33.5 ppt) were checked twice a week. The animals were supplied once a day in the early morning with 10 ml of commercial algae mix (Shellfish Diet 1800, Reed Mariculture Inc., USA) and 5 ml of a 50 × 106 cells/ml commercial solution of Chaetoceros calcitrans (Higashimaru-marinetech PLC, Japan). During feeding, the water flow was stopped for 3 to 4 h to allow the animals to filter enough algae. The tanks were siphoned every two days and checked for dead individuals. After 6 days of acclimation to the laboratory conditions specimens were moved into an experimental tank. Temperature was maintained at 17 °C by a cooling/ heating system, and animals were left to fast for 48 h prior to the experiments. The water in the experimental tanks was completely replaced weekly. All experimental procedures were approved by a Kyushu University committee and conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals of Japan. 2.2. Respirometry & morphometric measurements The semi-closed method of Yagi and Oikawa (2014) was used to determine the effective oxygen rate consumption, as a proxy for basal metabolic rate (MR), in both species. Rate of oxygen consumption in resting condition was measured using oxygen electrode probes. To control for background respiration, a bottle that received water flowing out of the respiration chamber was used as a blank chamber. The bottle was sealed at the beginning of the oxygen consumption experiment, placed in the water-bath of the respiration chamber during the experiment, and the oxygen concentration determined at the end of the experiment (C0). Specimens were introduced into the respiration chambers (250 ml each) and left undisturbed to acclimate to the new conditions under a constant air-saturated water flow from 1 h up to 1.5 h after their siphons opened and extended. The chambers were then positioned in closed flow tanks with fine-controlled temperature (T = 17 °C). Closing time ranged from 45 min up to 3 h, depending on the size of the animals. Upon opening, two volumes of 50 ml were sampled from the respiration
chamber and the oxygen concentration was determined via a DO electrode (C1 and C2). The oxygen consumption was calculated as: C1 þ C2 O2 cons ¼ C 0 − chamber volume 2 Values of (C1 − C2) ≥ 0.1 ppm were discarded. The final data set consisted of 58 measurements for C. robusta and 44 for C. savignyi. After the respirometry experiments, the total length at maximum extension (BL) of each individual was measured. The tunic and the internal organs were dissected, rinsed in distilled water, drained with absorbent paper, and weighed separately to determine the wet weight (wW). Then all tissues were dried in a drying oven for at least 48 h at 60 °C to determine the dry weight (dW). The water retention of the separated tissues, as well as whole body water retention of the two species, were calculated as the percentage of wW to which the total dW has been subtracted (i.e. the evaporated water during the drying period). The mass specific MR was calculated as follows: Mass specific MR ¼
O2 cons Closing time whole body wW ðor dWÞ
2.3. Statistical analysis 2.3.1. Morphometrics To compare the interspecific morphological relationships between C. robusta and C. savignyi, known allometric equation models were applied. In particular, regarding the relationship of whole body wW (or dW) vs. BL, we referred to the power equation Y = aXb proposed by Carver et al. (2003) for C. intestinalis, where Y = whole body wW (or dW), X = BL. The equations were log-log linearized and fitted with the least squares method. Each distribution was checked for the normality of residuals (Shapiro-Wilk test, α b 0.01). The F-test was used to evaluate the best fit model: i) the model in which one curve fits all data sets (p-value N 0.05), or ii) the model in which each species is fitted by a different curve (p-value b 0.05). Regarding the tunic wW (or dW) vs. organ wW (or dW) relationship, it has been shown to be linear (Carver et al., 2006), following the equation Y = a + bX, where Y = weight of the tunic and X = weight of the organs. The r2 and p-value were also calculated (α b 0.05). Each distribution was checked for the normality of residuals and the F-test was used to evaluate the best-fit model. Regarding the tunic/organ ratio (expressed as grams of tunic divided by grams of organs), the statistical significance of the differences were assessed by the Mann-Whitney test. When multiple comparisons were performed, the Bonferroni correction for multiple tests was applied. 2.3.2. Water retention The amount of retained water in the tissues was calculated as a fraction of the total wW (p). According to Bartlett (1947), for n number of observations near the higher limit, i.e. p near to one, the values were qffiffiffiffiffiffiffiffiffiffiffiffi 1 . The differences were transformed by applying Y ¼ 2sin−1 p− 2n assessed by the Mann-Whitney test. 2.3.3. Metabolic rate The relationship between body mass and MR has been massively studied (see Agutter and Wheatley, 2004 for a review), and is known to follow the equation Y = aXb, where Y = MR, and X = body mass (whole body wW, or dW regarding the data here analyzed). As already seen above, the obtained equations were log-log linearized, and fitted with the least squares method. Each distribution was checked for the
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normality of residuals (Shapiro-Wilk test, α b 0.01) prior that F-test was used to evaluate the best fit model. Regarding the mass specific MR (expressed as mg O2 × kg−1 × h−1) the statistical significance of the differences were assessed by the MannWhitney Test. All the statistical analyses were done in Prism 6 (GraphPad). 3. Results 3.1. Morphometric analyses The whole body wW of Ciona robusta and Ciona savignyi specimens ranged from 3.05 to 17.2 g and from 0.24 to 6.87 g, respectively. The corresponding BL ranged from 5.3 to 13.9 cm and from 2.2 to 8.9 cm, respectively. Unfortunately, in the present study, it was impossible to sample either bigger C. savignyi, or smaller C. robusta in order to increase the overlapping size range. Following Carver et al. (2003), who described the relationship between body mass and BL in C. intestinalis as a power function, the equations for the present data were whole body wW = 0.3068 × BL1.541 (r2 = 0.76, p-value b 0.001) for C. robusta, and = 0.1432 × BL1.779 (r2 = 0.74, p-value b 0.001) for C. savignyi (Fig. 1, panel A). In the same figure the equation obtained by Carver et al. (2003) for C. intestinalis is also indicated (Fig. 1, gray line). Pairwise comparison showed that differences among the three equations were all statistically significant (p-value b 0.01 by F-test Bonferroni corrected). The results were not affected if the wW was replaced by the corresponding dW values (Table 1). The relationship between the weights of the tunic and the organs has been described to be linear in C. intestinalis (Carver et al., 2006). Analyzing the same relationship for C. robusta and C. savignyi revealed a significant linear correlation between the two parameters, and the corresponding equations were: tunic wW = 0.011 + 2.027 × (organ wW) (r² = 0.72, p-value b 0.001) and tunic wW = 0.031 + 1.364 × (organ wW) (r² = 0.48, p-value b 0.001) for C. robusta and C. savignyi, respectively (Fig. 1, panel B). The tunic/organ ratio (i.e. the dW of the tunic divided by the dW of the organs) was calculated for each individual. Mean values were 2.5 for C. robusta and 1.3 for C. savignyi. The difference was statistically significant according to the Mann-Whitney test, p-value b 0.001 (Fig. 1, panel C). 3.2. Water retention Estimates of water retention were measured for the tunic and organs independently in each species. In C. robusta, the water retention for tunic and organs were 95.8% (S.D. 0.67) and 93.4% (S.D. 0.71), respectively. In C. savignyi the values were 95.6% (S.D. 0.83) and 93.3% (S.D. 0.72), respectively. In both species: i) the differences, in percentage of retained water, between tunic and organs were of the same order of magnitude (not significantly different); and ii) the water retention was significantly higher in the tunic than in the organs (pvalue b 0.001). Nevertheless, on average, the tunic of C. robusta tended to retain more water than that of C. savignyi (p-value b 0.001). The whole body water retention of the two species was also calculated. Mean values were 95.14% (S.D. 0.58) for C. robusta and 94.50% (S.D. 0.62) for C. savignyi. The differences, though significant according to the Mann-Whitney test (p-value b 0.001), are probably due to differences in size between the two samplings. 3.3. Oxygen consumption The oxygen consumption rate was measured by a Dissolved Oxygen probe as an indirect quantification of metabolic rate. Average dry mass specific MRs (388 mg O2 × kg−1 × h−1 and 576 mg O2 × kg−1 × h−1 for C. robusta and C. savignyi, respectively), were significantly different, p-value b 0.001. One would expect smaller animals to have a higher
Fig. 1. Panel A: Whole body wet weight (g) versus body length (cm) in Ciona robusta (n = 58) and Ciona savignyi (n = 44). Data from this work are compared with Ciona intestinalis from Carver et al. (2003). Panel B: Tunic wet weight (g) versus organs wet weight (g) in C. robusta (n = 58) and C. savignyi (n = 44). Data from this work are compared with C. intestinalis from Carver et al. (2006). Panel C: Distribution of tunic:organ wet weight ratio in C. robusta (n = 58) and C. savignyi (n = 44).
mass specific metabolic rate anyway, and the C. savignyi specimens used in this study were smaller than the C. robusta. This makes it very difficult to separate species differences from the effects of size differences. To avoid the mass dependence of the oxygen consumption rate, the linear log-log plot relationship between body mass and oxygen consumption was analyzed (Fig. 2). Ciona savignyi was described by the equation MR = 0.36BW0.22 (r2 = 0.64, p-value b 0.0001), while C. robusta by the equation MR = 0.92BW0.59 (r2 = 0.76, pvalue b 0.0001). The two logarithmic regressions were significantly different (p-value b 0.05, according to the F-test).
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Table 1 Coefficients of the body weight/body length equations calculated with wet and dry weight and the corresponding similarity test p-value.
Wet weight Allometric eq.
Dry weight Allometric eq.
C. robusta
C. savignyi
Extra sum of square
a b r2
0.3068 1.541 0.7597
0.1432 1.779 0.7414
b0.001
a b r2
0.01305 1.605 0.8006
0.009373 1.646 0.7379
b 0.001
4. Discussion The genus Ciona is widely studied in the evolutionary field, but relatively little is known about the comparative morpho-physiology of the species belonging to this group. In spite of an historical role as model organisms in life science (Gallo and Tosti, 2015; Liu et al., 2006; Satoh et al., 2003), only recently have peculiar morphological features been identified, leading to the inference of separate species, as in the case of Ciona intestinalis and Ciona robusta (Brunetti et al., 2015; Hoshino and Nishikawa, 1985; Pennati et al., 2015; Sato et al., 2012; Smith et al., 2010). Body wW and dW vs. body length (BL) scaling was analyzed first, and both Ciona robusta and Ciona savignyi were described by two power correlations, with exponents equal to 1.54 and 1.78, respectively (Fig. 1, panel A). The results were also compared with those published by Carver and colleagues (Carver et al., 2003; Carver et al., 2006), who first proposed a power dependence equation for body dW in terms of BL, i.e. dW = 0.000106(BL)2.38, most probably determined using C. intestinalis. According to the F-test, the body weight vs. BL distribution of the data among the three species could not be described by a single equation. In other words, the correlation turned out to be speciesspecific. More precisely, C. robusta showed a higher elevation than C. savignyi, thus confirming previous observations that C. savignyi is longer and more slender than C. robusta (Lambert, 2003). The differences between C. robusta and C. savignyi cannot be ascribed to different
Fig. 2. Whole individual oxygen consumption rate (MR) versus the body dry weight in Ciona robusta (n = 58) and Ciona savignyi (n = 44) in a linearized log-log graph.
whole-body water contents, since replacing the wW values by the corresponding body-mass dW values did not affect the results (Table 1). In the case of C. intestinalis, the growth of the tunic relative to the internal organs follows a linear relationship (Carver et al., 2006). The regression for C. robusta (slope 2.03) was steeper than that for C. savignyi (slope 1.36) (Fig. 1, panel B). These data indicate that the tunic of C. robusta grows faster than that of C. savignyi. The tunic of C. savignyi appeared to be softer than that of C. robusta, which is more chitinous and robust as described by Hoshino and Tokioka (1967). In particular, the average tunic/organ weight ratio in C. savignyi was half of that found in C. robusta (Fig. 1, panel C). In other words this means that in adult individuals of comparable body weight, the organ weight of C. savignyi should be twice that of C. robusta. In ascidians, tissues are highly permeated with water, accounting for ~95% of the whole body-weight for both C. savignyi and C. robusta. Interestingly, the amount of water retained by the tunic was significantly higher than that retained by the organs as whole. The difference was not species-specific; the gap in C. robusta (2.4%) was not significantly different to that of C. savignyi (2.2%). How to explain the peculiar behavior of the tunic? The tunic is a distinctive integumentary tissue, from which arose the name of the subphylum, i.e. Tunicata. It is basically made of celluloselike polysaccharides (De Leo et al., 1977), and has been considered to have a solely structural function. It is known to have a very high water content: 95–98% of the wet weight (Florkin and Scheer, 1974; this study). Part of the water derives from internal fluids, but most is “water of imbibition” (Florkin and Scheer, 1974). A possible explanation would be that the polysaccharides, thanks to their physical-chemical properties, confer to the tunic a higher water absorbing power than other tissues. Being the primary barrier between seawater and internal organs, the tunic may absorb more water during short-term seawater dilution, thus helping to maintain a correct ionic balance within the vital organs, or, at least, to reduce the osmotic shock. As a matter of fact, tunicates, indeed, cannot actively osmoregulate. In C. intestinalis it has been shown that, in order to counteract sudden changes in salinity, the animals try to avoid osmotic stress by closing their siphons (Shumway, 1978), probably a response common to all ascidians (Sims, 1984; Ukena et al., 2008). Nonetheless, highly tolerant species such as Ciona spp. can survive a wide range of salinities (12–40%), and withstand short periods of lower salinity (11‰) (Shenkar and Swalla, 2011). Thus the tunic, by attracting more water than the organs, could serve to allay environmental salinity fluctuations, in addition to its known structural function. The basal metabolic rate is considered the sum of all the biochemical reactions that take place within an organism. Here the rate of oxygen consumption has been measured as a proxy for metabolic rate (MR) in the adults of the two species, C. robusta and C. savignyi. Regarding C. robusta, only one previous measurement has been reported (Markus and Lambert, 1983). According to the authors, the mass specific MR per unit dry weight for C. robusta sampled in San Diego, California (referred to erroneously as C. intestinalis) was 377 mg O2 × kg−1 × h−1, in accordance with the average value presented here of 388 mg O2 × kg−1 × h−1. In terms of mass specific MR, the measurements for C. savignyi in the present study showed a higher rate, with an average value of 576 mg O2 × kg−1 × h−1. This difference could be, however, partially attributed to the different size ranges of our specimens. C. robusta was larger and heavier than C. savignyi, although both species were in the same stage of development, i.e. all the specimens were post-juveniles. Nevertheless, when the analyses was restricted to the specimens with comparable BL (from 5.5 up to 9 cm), C. savignyi still shows a higher mass specific MR (428 mg O2 × kg−1 × h−1, n = 12) than C. robusta (387 mg O2 × kg−1 × h−1, n = 38). The log-log allometric relationship between whole metabolic rate and body weight should be linear throughout almost the full range of sizes. In this respect, the log-log allometric relationship between oxygen consumption and body size was analyzed (Fig. 2). According to the F-test, C. savignyi and
A. Tarallo et al. / Journal of Experimental Marine Biology and Ecology 485 (2016) 83–87
C. robusta were described by two different equations. This allows a reasonable extrapolation of the C. robusta metabolic rate for lower body weights. The 95% confidence area of the mean for C. robusta and C. savignyi was plotted (Fig. 2, shadow area). Although only a few points (seven) from C. savignyi fall inside the confidence bands for C. robusta, the two areas did not overlap in the range of smaller body size. Thus the theoretical extrapolation suggested a higher MR for C. savignyi in the early growth phase. Although only a theoretical model, this hypothesis was supported by measurements on mass-specific water flow-rate in ascidians. Indeed, the pumping of seawater through the siphons is the major activity for sessile ascidians, and accounts for a large consumption of their total energy resources, as suggested by Sherrard and LaBarbera (2005a). These same authors compared the mass-specific volumetric flow rates of seawater ingestion through the siphons in adult ascidians. Within a comparable range of body mass (10 to 700 mg dry weight), the flow rate of C. savignyi was significantly higher (p-value b 0.001) than that of both C. intestinalis and C. robusta (referred to erroneously as C. intestinalis), thus supporting our inference that MR is likely higher in C. savignyi than C. robusta, also in a comparable size range. 5. Conclusion The overall picture derived from the morpho-physiological measurements conducted on the two closely related species, carries interesting implications for their ecology and interspecific interaction, and points to a new interesting functional role for the tunic, as well. In short, we observed that in both species the tunic retains more water than other tissues. This adaptation could be particularly useful for soft tunic tunicates in order to efficiently counteract temporary changes in salinity. Regarding interspecific differences, Ciona savignyi has a slender body form in which the organ volume occupies a major portion. On the contrary, Ciona robusta has a stiffer tunic twice as heavy as that of C. savignyi. This difference could reflect different ecological strategies, indeed C. robusta invests more in a stiffer and voluminous tunic. As suggested by previous authors, this strategy lowers the predation risk in ascidians (Sherrard and LaBarbera, 2005b), though stiffer tunics could hinder rapid expansion of the juvenile body (Sherrard and LaBarbera, 2005b). Differently, C. savignyi directs relatively more effort into height gain. The greater distance from the ground improves both the quality and the concentration of the ingested food, thus supporting potentially faster growth. Moreover, height also affects the position of the subject relative to other animals and macroalgae living in the vicinity, which may compete for food or block the siphons' flow (Sherrard and LaBarbera, 2005b). This explains the higher oxygen consumption predicted in C. savignyi and the higher seawater flow rate, an energycostly activity, especially in juveniles (Sherrard and LaBarbera, 2005b). Undeniably, the ecological interaction between C. savignyi and C. robusta needs further investigation. Our comparative report, stressing that C. savignyi could grow faster, primarily in the first phase of its life, and thus better exploit food resources, provides a background hypothesis to explain the observation that this species is replacing the indigenous C. robusta in new co-occurrence areas (Lambert, 2007; Lambert and Lambert, 2003). Acknowledgments Authors are grateful to Dr. C. Angelini, for helpful statistical advices, to Prof. M. Yoshikuni, for challenging discussions and assistance during the stage of AT at the Fishery Res. Lab. of Kyushu Univ. (Japan), and to Prof. G. Lambert and Dr. C. Carver, who provided insight and expertise that greatly improved the manuscript. Special thanks to the anonymous referees for valuable comments. AT has been supported by a SZN PhD fellowship.
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Persistence and differential distribution of nonindigenous ascidians in harbors of the Southern California Bight. Mar. Ecol. Prog. Ser. 259, 145–161. Liu, L., Xiang, J., Dong, B., Natarajan, P., Yu, K., Cai, N., 2006. Ciona intestinalis as an emerging model organism: its regeneration under controlled conditions and methodology for egg dechorionation. J. Zhejiang Univ. Sci. B 7, 467–474. Markus, J.a., Lambert, C.C., 1983. Urea and ammonia excretion by solitary ascidians. J. Exp. Mar. Biol. Ecol. 66, 1–10. Nydam, M.L., Harrison, R.G., 2010. Polymorphism and divergence within the ascidian genus Ciona. Mol. Phylogenet. Evol. 56, 718–726. Pennati, R., Ficetola, G.F., Brunetti, R., Caicci, F., Gasparini, F., Griggio, F., Sato, A., Stach, T., Kaul-Strehlow, S., Gissi, C., et al., 2015. Morphological differences between larvae of the Ciona intestinalis species complex: hints for a valid taxonomic definition of distinct species. PLoS One 10, e0122879. Sato, A., Satoh, N., Bishop, J.D.D., 2012. Field identification of “types” A and B of the ascidian Ciona intestinalis in a region of sympatry. Mar. Biol. 159, 1611–1619. Satoh, N., Satou, Y., Davidson, B., Levine, M., 2003. Ciona intestinalis: an emerging model for whole-genome analyses. Trends Genet. 19, 376–381. Shenkar, N., Swalla, B.J., 2011. Global diversity of Ascidiacea. PLoS One 6, e20657. Sherrard, K., LaBarbera, M., 2005a. Form and function in juvenile ascidians. II. Ontogenetic scaling of volumetric flow rates. Mar. Ecol. Prog. Ser. 287, 139–148. Sherrard, K.M., LaBarbera, M., 2005b. Form and function in juvenile ascidians. I. Implications of early juvenile morphologies for performance. Mar. Ecol. Prog. Ser. 287, 127–138. Shumway, S.E., 1978. Respiration, pumping activity and heart rate in Ciona intestinalis exposed to fluctuating salinities. Mar. Biol. 48, 235–242. Sims, L.L., 1984. Osmoregulatory capabilities of three macrosympatric stolidobranch ascidians, Styela clava Herdman, S. plicata (Lesueur), and S. montereyensis (Dall). J. Exp. Mar. Biol. Ecol. 82, 117–129. Smith, K.F., Cahill, P.L., Fidler, A.E., 2010. First record of the solitary ascidian Ciona savignyi Herdman, 1882 in the Southern Hemisphere. Aquat. Invasions 5, 363–368. Suzuki, M.M., Nishikawa, T., Bird, A., 2005. Genomic approaches reveal unexpected genetic divergence within Ciona intestinalis. J. Mol. Evol. 61, 627–635. Taekjun, L., Sook, S., 2014. Morphological and molecular identification of an introduced alien sea squirt (Tunicata: Ascidiacea) in Korea. 127, 284–297. Ukena, K., Iwakoshi-Ukena, E., Hikosaka, A., 2008. Unique form and osmoregulatory function of a neurohypophysial hormone in a urochordate. Endocrinology 149, 5254–5261. Yagi, M., Oikawa, S., 2014. Ontogenetic phase shifts in metabolism in a flounder Paralichthys olivaceus. Sci. Rep. 4, 7135. 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Journal of Experimental Marine Biology and Ecology journal homepage: www.elsevier.com/locate/jembe
Comparative morpho-physiological analysis between Ciona robusta and Ciona savignyi Andrea Tarallo a,⁎, Mitshuaru Yagi b, Shin Oikawa c, Claudio Agnisola d, Giuseppe D'Onofrio a a
Dept. of Biology and Evolution of Marine Organisms, Stazione Zoologica Anton Dohrn, Villa Comunale, 80121 Naples, Italy Faculty of Fisheries, Nagasaki University, 1-14 Bunkyo, Nagasaki 852-8521, Japan c Fishery Research Laboratory, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka 811-3304, Japan d Dept. of Biology, University of Naples Federico II, Complesso Universitario di Monte SantAngelo, Edificio 7, Via Cinthia, 80126 Naples, Italy b
a r t i c l e
i n f o
Article history: Received 18 April 2016 Received in revised form 9 September 2016 Accepted 12 September 2016 Available online xxxx Keywords: Sea squirt Ascidians Metabolic rate Morphophysiology Tunic Ecology
a b s t r a c t The seasquirt Ciona robusta and its co-generic Ciona savignyi, are two long-divergent species, sharing the same habitat and competing for the same spaces and resources. Their very similar morphology has been responsible for the misidentification of the two organisms, and, consequently, for the underestimation of their geographic distributions and new areas of co-occurrence. In spite of the large amount of knowledge built up by developmental biologists, few data are available regarding the morpho-physiology of the two sea squirts. The comparison of morphological and physiological features carried out in the present study (i.e. length-body weight correlation, tunic/organ ratio, tissue-specific retained water and metabolic rate), highlighted slight, but significant, differences strongly supporting the hypothesis of different ecological strategies for the two species. More precisely, C. savignyi invests more energy in growing faster and taller, likely to improve the quality and concentration of the filtered food, and sustains a faster metabolic/growth rate. Conversely, C. robusta invests more into tunic thickness, reducing the risk of predation, even if this likely means a slower metabolic/growth rate. In addition it was noted that in both species, the tunic absorbs more water than the other tissues, a peculiarity that may allow them to better counteract sudden fluctuations in environmental salinity. © 2016 Elsevier B.V. All rights reserved.
1. Introduction In the last decade, different research groups have shown that the species Ciona intestinalis is actually a complex of genetically differentiated types (Caputi et al., 2007; Iannelli et al., 2007; Nydam and Harrison, 2010; Suzuki et al., 2005; Zhan et al., 2010). Very recently (Brunetti et al., 2015), the “types” formerly denoted as A and B, were ascribed respectively to two distinct species: Ciona robusta Hoshino and Tokioka, 1967 and C. intestinalis (Linnaeus, 1767). On the basis of the different morphological features observed at the larval stage, Pennati et al. (2015) confirmed the new classification. Due to their very close morphological features, C. robusta, C. intestinalis, and Ciona savignyi Herdman, 1882 were wrongly considered for a long time to be the same species (Hoshino and Nishikawa, 1985; Lambert and Lambert, 1998; Smith et al., 2010), in spite of the fact that C. robusta and C. intestinalis were estimated to diverge approximately 20 Mya (Suzuki et al., 2005), while C. robusta and C. savignyi were estimated to diverge 180 Mya (Berná et al., 2009). Attention was focused on the comparison of C. robusta vs. C. savignyi, since numerous reports stressed that the two species show overlapping ⁎ Corresponding author. E-mail addresses: [email protected], [email protected] (A. Tarallo).
http://dx.doi.org/10.1016/j.jembe.2016.09.001 0022-0981/© 2016 Elsevier B.V. All rights reserved.
distribution areas, competing for space and resources: southern California (Byrd and Lambert, 2000; Lambert and Lambert, 2003), New Zealand (Smith et al., 2010), the Korean peninsula (Taekjun and Sook, 2014), and Tokyo Bay (M. Yoshikuni, personal communication). Moreover, along the California coast, C. savignyi has been reported to replace C. robusta (Lambert, 2007; Lambert and Lambert, 1998; Lambert and Lambert, 2003). Despite the interest of the scientific community towards sea squirts, few data are available about the physiology of these organisms. Early studies examined oxygen consumption in C. intestinalis embryos and adults (Holter and Zeuthen, 1944; Jørgensen, 1952). The measurements performed by Jørgensen were carried out according to the Winkler methodology, estimating a consumption rate of about 0.8 ml O2/h. Markus and Lambert (1983), although corroborating the results of Jørgensen by using the same methodology (0.82 ml O2/g dry weight/ h), stated that former data “were reported as relative values and not as weight-specific rates”. It should be noted that, while Markus and Lambert measured oxygen consumption on animals sampled in California, thus almost surely belonging to the new classified species C. robusta (ex C. intestinalis type A), the specimens used by Jørgensen were collected from Woods Hole (Massachusetts), thus most probably C. intestinalis (ex “type B”). As far as we know, no metabolic studies are available in the current literature for C. savignyi. Thus, in this study, morpho-
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physiological parameters, i.e. body size, tunic/organ dry weight ratio, tissue water retention, as well as the routine oxygen consumption rate as proxy for routine metabolic rate, were measured for adults of C. savignyi and compared to those of C. robusta. 2. Materials and methods 2.1. Specimens The seasquirt Ciona robusta was provided by Kyoto University (Kyoto, Japan). The adult individuals were obtained from in vitro fertilization of wild gametes, which after settlement on Petri dishes, were reared in the field in Maizuru Bay by Maizuru Fisheries Research Station (Nagahama, Maizuru-shi, Kyoto, Japan). The co-generic Ciona savignyi was collected in two different areas: Tokyo Bay, by Tokyo University (Tokyo, Japan), and Sugashima Bay, by Nagoya University (Nagoya, Japan). Individuals were classified on the basis of their morphological characters according to Smith et al. (2010) and Sato et al. (2012). The specimens reared in Maizuru Bay were identified as C. robusta since adults showed the siphon tubercles, as well as the peculiar pigmentation of the tip of the vas deferens. The specimens collected in Tokyo Bay were identified as C. savignyi, because of the lack of pigmentation on the vas deferens and the presence of marked orange pigmentation of the inhalant siphon. The animals were shipped to Kyushu University Fishery Research Laboratory (Fukutsu, Fukuoka, Japan) with minimal temperature rise during transportation and transferred to 50-l aquaria with running filtered seawater and continuous aeration. In the case of the reared C. robusta, the animals were manually removed from the Petri dishes. Individuals attached to each other were separated, and epibionts on the tunic surface were removed by tweezers. Water temperature (ranging from 16.5 °C to 18.0 °C) and salinity (on average 33.5 ppt) were checked twice a week. The animals were supplied once a day in the early morning with 10 ml of commercial algae mix (Shellfish Diet 1800, Reed Mariculture Inc., USA) and 5 ml of a 50 × 106 cells/ml commercial solution of Chaetoceros calcitrans (Higashimaru-marinetech PLC, Japan). During feeding, the water flow was stopped for 3 to 4 h to allow the animals to filter enough algae. The tanks were siphoned every two days and checked for dead individuals. After 6 days of acclimation to the laboratory conditions specimens were moved into an experimental tank. Temperature was maintained at 17 °C by a cooling/ heating system, and animals were left to fast for 48 h prior to the experiments. The water in the experimental tanks was completely replaced weekly. All experimental procedures were approved by a Kyushu University committee and conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals of Japan. 2.2. Respirometry & morphometric measurements The semi-closed method of Yagi and Oikawa (2014) was used to determine the effective oxygen rate consumption, as a proxy for basal metabolic rate (MR), in both species. Rate of oxygen consumption in resting condition was measured using oxygen electrode probes. To control for background respiration, a bottle that received water flowing out of the respiration chamber was used as a blank chamber. The bottle was sealed at the beginning of the oxygen consumption experiment, placed in the water-bath of the respiration chamber during the experiment, and the oxygen concentration determined at the end of the experiment (C0). Specimens were introduced into the respiration chambers (250 ml each) and left undisturbed to acclimate to the new conditions under a constant air-saturated water flow from 1 h up to 1.5 h after their siphons opened and extended. The chambers were then positioned in closed flow tanks with fine-controlled temperature (T = 17 °C). Closing time ranged from 45 min up to 3 h, depending on the size of the animals. Upon opening, two volumes of 50 ml were sampled from the respiration
chamber and the oxygen concentration was determined via a DO electrode (C1 and C2). The oxygen consumption was calculated as: C1 þ C2 O2 cons ¼ C 0 − chamber volume 2 Values of (C1 − C2) ≥ 0.1 ppm were discarded. The final data set consisted of 58 measurements for C. robusta and 44 for C. savignyi. After the respirometry experiments, the total length at maximum extension (BL) of each individual was measured. The tunic and the internal organs were dissected, rinsed in distilled water, drained with absorbent paper, and weighed separately to determine the wet weight (wW). Then all tissues were dried in a drying oven for at least 48 h at 60 °C to determine the dry weight (dW). The water retention of the separated tissues, as well as whole body water retention of the two species, were calculated as the percentage of wW to which the total dW has been subtracted (i.e. the evaporated water during the drying period). The mass specific MR was calculated as follows: Mass specific MR ¼
O2 cons Closing time whole body wW ðor dWÞ
2.3. Statistical analysis 2.3.1. Morphometrics To compare the interspecific morphological relationships between C. robusta and C. savignyi, known allometric equation models were applied. In particular, regarding the relationship of whole body wW (or dW) vs. BL, we referred to the power equation Y = aXb proposed by Carver et al. (2003) for C. intestinalis, where Y = whole body wW (or dW), X = BL. The equations were log-log linearized and fitted with the least squares method. Each distribution was checked for the normality of residuals (Shapiro-Wilk test, α b 0.01). The F-test was used to evaluate the best fit model: i) the model in which one curve fits all data sets (p-value N 0.05), or ii) the model in which each species is fitted by a different curve (p-value b 0.05). Regarding the tunic wW (or dW) vs. organ wW (or dW) relationship, it has been shown to be linear (Carver et al., 2006), following the equation Y = a + bX, where Y = weight of the tunic and X = weight of the organs. The r2 and p-value were also calculated (α b 0.05). Each distribution was checked for the normality of residuals and the F-test was used to evaluate the best-fit model. Regarding the tunic/organ ratio (expressed as grams of tunic divided by grams of organs), the statistical significance of the differences were assessed by the Mann-Whitney test. When multiple comparisons were performed, the Bonferroni correction for multiple tests was applied. 2.3.2. Water retention The amount of retained water in the tissues was calculated as a fraction of the total wW (p). According to Bartlett (1947), for n number of observations near the higher limit, i.e. p near to one, the values were qffiffiffiffiffiffiffiffiffiffiffiffi 1 . The differences were transformed by applying Y ¼ 2sin−1 p− 2n assessed by the Mann-Whitney test. 2.3.3. Metabolic rate The relationship between body mass and MR has been massively studied (see Agutter and Wheatley, 2004 for a review), and is known to follow the equation Y = aXb, where Y = MR, and X = body mass (whole body wW, or dW regarding the data here analyzed). As already seen above, the obtained equations were log-log linearized, and fitted with the least squares method. Each distribution was checked for the
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normality of residuals (Shapiro-Wilk test, α b 0.01) prior that F-test was used to evaluate the best fit model. Regarding the mass specific MR (expressed as mg O2 × kg−1 × h−1) the statistical significance of the differences were assessed by the MannWhitney Test. All the statistical analyses were done in Prism 6 (GraphPad). 3. Results 3.1. Morphometric analyses The whole body wW of Ciona robusta and Ciona savignyi specimens ranged from 3.05 to 17.2 g and from 0.24 to 6.87 g, respectively. The corresponding BL ranged from 5.3 to 13.9 cm and from 2.2 to 8.9 cm, respectively. Unfortunately, in the present study, it was impossible to sample either bigger C. savignyi, or smaller C. robusta in order to increase the overlapping size range. Following Carver et al. (2003), who described the relationship between body mass and BL in C. intestinalis as a power function, the equations for the present data were whole body wW = 0.3068 × BL1.541 (r2 = 0.76, p-value b 0.001) for C. robusta, and = 0.1432 × BL1.779 (r2 = 0.74, p-value b 0.001) for C. savignyi (Fig. 1, panel A). In the same figure the equation obtained by Carver et al. (2003) for C. intestinalis is also indicated (Fig. 1, gray line). Pairwise comparison showed that differences among the three equations were all statistically significant (p-value b 0.01 by F-test Bonferroni corrected). The results were not affected if the wW was replaced by the corresponding dW values (Table 1). The relationship between the weights of the tunic and the organs has been described to be linear in C. intestinalis (Carver et al., 2006). Analyzing the same relationship for C. robusta and C. savignyi revealed a significant linear correlation between the two parameters, and the corresponding equations were: tunic wW = 0.011 + 2.027 × (organ wW) (r² = 0.72, p-value b 0.001) and tunic wW = 0.031 + 1.364 × (organ wW) (r² = 0.48, p-value b 0.001) for C. robusta and C. savignyi, respectively (Fig. 1, panel B). The tunic/organ ratio (i.e. the dW of the tunic divided by the dW of the organs) was calculated for each individual. Mean values were 2.5 for C. robusta and 1.3 for C. savignyi. The difference was statistically significant according to the Mann-Whitney test, p-value b 0.001 (Fig. 1, panel C). 3.2. Water retention Estimates of water retention were measured for the tunic and organs independently in each species. In C. robusta, the water retention for tunic and organs were 95.8% (S.D. 0.67) and 93.4% (S.D. 0.71), respectively. In C. savignyi the values were 95.6% (S.D. 0.83) and 93.3% (S.D. 0.72), respectively. In both species: i) the differences, in percentage of retained water, between tunic and organs were of the same order of magnitude (not significantly different); and ii) the water retention was significantly higher in the tunic than in the organs (pvalue b 0.001). Nevertheless, on average, the tunic of C. robusta tended to retain more water than that of C. savignyi (p-value b 0.001). The whole body water retention of the two species was also calculated. Mean values were 95.14% (S.D. 0.58) for C. robusta and 94.50% (S.D. 0.62) for C. savignyi. The differences, though significant according to the Mann-Whitney test (p-value b 0.001), are probably due to differences in size between the two samplings. 3.3. Oxygen consumption The oxygen consumption rate was measured by a Dissolved Oxygen probe as an indirect quantification of metabolic rate. Average dry mass specific MRs (388 mg O2 × kg−1 × h−1 and 576 mg O2 × kg−1 × h−1 for C. robusta and C. savignyi, respectively), were significantly different, p-value b 0.001. One would expect smaller animals to have a higher
Fig. 1. Panel A: Whole body wet weight (g) versus body length (cm) in Ciona robusta (n = 58) and Ciona savignyi (n = 44). Data from this work are compared with Ciona intestinalis from Carver et al. (2003). Panel B: Tunic wet weight (g) versus organs wet weight (g) in C. robusta (n = 58) and C. savignyi (n = 44). Data from this work are compared with C. intestinalis from Carver et al. (2006). Panel C: Distribution of tunic:organ wet weight ratio in C. robusta (n = 58) and C. savignyi (n = 44).
mass specific metabolic rate anyway, and the C. savignyi specimens used in this study were smaller than the C. robusta. This makes it very difficult to separate species differences from the effects of size differences. To avoid the mass dependence of the oxygen consumption rate, the linear log-log plot relationship between body mass and oxygen consumption was analyzed (Fig. 2). Ciona savignyi was described by the equation MR = 0.36BW0.22 (r2 = 0.64, p-value b 0.0001), while C. robusta by the equation MR = 0.92BW0.59 (r2 = 0.76, pvalue b 0.0001). The two logarithmic regressions were significantly different (p-value b 0.05, according to the F-test).
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Table 1 Coefficients of the body weight/body length equations calculated with wet and dry weight and the corresponding similarity test p-value.
Wet weight Allometric eq.
Dry weight Allometric eq.
C. robusta
C. savignyi
Extra sum of square
a b r2
0.3068 1.541 0.7597
0.1432 1.779 0.7414
b0.001
a b r2
0.01305 1.605 0.8006
0.009373 1.646 0.7379
b 0.001
4. Discussion The genus Ciona is widely studied in the evolutionary field, but relatively little is known about the comparative morpho-physiology of the species belonging to this group. In spite of an historical role as model organisms in life science (Gallo and Tosti, 2015; Liu et al., 2006; Satoh et al., 2003), only recently have peculiar morphological features been identified, leading to the inference of separate species, as in the case of Ciona intestinalis and Ciona robusta (Brunetti et al., 2015; Hoshino and Nishikawa, 1985; Pennati et al., 2015; Sato et al., 2012; Smith et al., 2010). Body wW and dW vs. body length (BL) scaling was analyzed first, and both Ciona robusta and Ciona savignyi were described by two power correlations, with exponents equal to 1.54 and 1.78, respectively (Fig. 1, panel A). The results were also compared with those published by Carver and colleagues (Carver et al., 2003; Carver et al., 2006), who first proposed a power dependence equation for body dW in terms of BL, i.e. dW = 0.000106(BL)2.38, most probably determined using C. intestinalis. According to the F-test, the body weight vs. BL distribution of the data among the three species could not be described by a single equation. In other words, the correlation turned out to be speciesspecific. More precisely, C. robusta showed a higher elevation than C. savignyi, thus confirming previous observations that C. savignyi is longer and more slender than C. robusta (Lambert, 2003). The differences between C. robusta and C. savignyi cannot be ascribed to different
Fig. 2. Whole individual oxygen consumption rate (MR) versus the body dry weight in Ciona robusta (n = 58) and Ciona savignyi (n = 44) in a linearized log-log graph.
whole-body water contents, since replacing the wW values by the corresponding body-mass dW values did not affect the results (Table 1). In the case of C. intestinalis, the growth of the tunic relative to the internal organs follows a linear relationship (Carver et al., 2006). The regression for C. robusta (slope 2.03) was steeper than that for C. savignyi (slope 1.36) (Fig. 1, panel B). These data indicate that the tunic of C. robusta grows faster than that of C. savignyi. The tunic of C. savignyi appeared to be softer than that of C. robusta, which is more chitinous and robust as described by Hoshino and Tokioka (1967). In particular, the average tunic/organ weight ratio in C. savignyi was half of that found in C. robusta (Fig. 1, panel C). In other words this means that in adult individuals of comparable body weight, the organ weight of C. savignyi should be twice that of C. robusta. In ascidians, tissues are highly permeated with water, accounting for ~95% of the whole body-weight for both C. savignyi and C. robusta. Interestingly, the amount of water retained by the tunic was significantly higher than that retained by the organs as whole. The difference was not species-specific; the gap in C. robusta (2.4%) was not significantly different to that of C. savignyi (2.2%). How to explain the peculiar behavior of the tunic? The tunic is a distinctive integumentary tissue, from which arose the name of the subphylum, i.e. Tunicata. It is basically made of celluloselike polysaccharides (De Leo et al., 1977), and has been considered to have a solely structural function. It is known to have a very high water content: 95–98% of the wet weight (Florkin and Scheer, 1974; this study). Part of the water derives from internal fluids, but most is “water of imbibition” (Florkin and Scheer, 1974). A possible explanation would be that the polysaccharides, thanks to their physical-chemical properties, confer to the tunic a higher water absorbing power than other tissues. Being the primary barrier between seawater and internal organs, the tunic may absorb more water during short-term seawater dilution, thus helping to maintain a correct ionic balance within the vital organs, or, at least, to reduce the osmotic shock. As a matter of fact, tunicates, indeed, cannot actively osmoregulate. In C. intestinalis it has been shown that, in order to counteract sudden changes in salinity, the animals try to avoid osmotic stress by closing their siphons (Shumway, 1978), probably a response common to all ascidians (Sims, 1984; Ukena et al., 2008). Nonetheless, highly tolerant species such as Ciona spp. can survive a wide range of salinities (12–40%), and withstand short periods of lower salinity (11‰) (Shenkar and Swalla, 2011). Thus the tunic, by attracting more water than the organs, could serve to allay environmental salinity fluctuations, in addition to its known structural function. The basal metabolic rate is considered the sum of all the biochemical reactions that take place within an organism. Here the rate of oxygen consumption has been measured as a proxy for metabolic rate (MR) in the adults of the two species, C. robusta and C. savignyi. Regarding C. robusta, only one previous measurement has been reported (Markus and Lambert, 1983). According to the authors, the mass specific MR per unit dry weight for C. robusta sampled in San Diego, California (referred to erroneously as C. intestinalis) was 377 mg O2 × kg−1 × h−1, in accordance with the average value presented here of 388 mg O2 × kg−1 × h−1. In terms of mass specific MR, the measurements for C. savignyi in the present study showed a higher rate, with an average value of 576 mg O2 × kg−1 × h−1. This difference could be, however, partially attributed to the different size ranges of our specimens. C. robusta was larger and heavier than C. savignyi, although both species were in the same stage of development, i.e. all the specimens were post-juveniles. Nevertheless, when the analyses was restricted to the specimens with comparable BL (from 5.5 up to 9 cm), C. savignyi still shows a higher mass specific MR (428 mg O2 × kg−1 × h−1, n = 12) than C. robusta (387 mg O2 × kg−1 × h−1, n = 38). The log-log allometric relationship between whole metabolic rate and body weight should be linear throughout almost the full range of sizes. In this respect, the log-log allometric relationship between oxygen consumption and body size was analyzed (Fig. 2). According to the F-test, C. savignyi and
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C. robusta were described by two different equations. This allows a reasonable extrapolation of the C. robusta metabolic rate for lower body weights. The 95% confidence area of the mean for C. robusta and C. savignyi was plotted (Fig. 2, shadow area). Although only a few points (seven) from C. savignyi fall inside the confidence bands for C. robusta, the two areas did not overlap in the range of smaller body size. Thus the theoretical extrapolation suggested a higher MR for C. savignyi in the early growth phase. Although only a theoretical model, this hypothesis was supported by measurements on mass-specific water flow-rate in ascidians. Indeed, the pumping of seawater through the siphons is the major activity for sessile ascidians, and accounts for a large consumption of their total energy resources, as suggested by Sherrard and LaBarbera (2005a). These same authors compared the mass-specific volumetric flow rates of seawater ingestion through the siphons in adult ascidians. Within a comparable range of body mass (10 to 700 mg dry weight), the flow rate of C. savignyi was significantly higher (p-value b 0.001) than that of both C. intestinalis and C. robusta (referred to erroneously as C. intestinalis), thus supporting our inference that MR is likely higher in C. savignyi than C. robusta, also in a comparable size range. 5. Conclusion The overall picture derived from the morpho-physiological measurements conducted on the two closely related species, carries interesting implications for their ecology and interspecific interaction, and points to a new interesting functional role for the tunic, as well. In short, we observed that in both species the tunic retains more water than other tissues. This adaptation could be particularly useful for soft tunic tunicates in order to efficiently counteract temporary changes in salinity. Regarding interspecific differences, Ciona savignyi has a slender body form in which the organ volume occupies a major portion. On the contrary, Ciona robusta has a stiffer tunic twice as heavy as that of C. savignyi. This difference could reflect different ecological strategies, indeed C. robusta invests more in a stiffer and voluminous tunic. As suggested by previous authors, this strategy lowers the predation risk in ascidians (Sherrard and LaBarbera, 2005b), though stiffer tunics could hinder rapid expansion of the juvenile body (Sherrard and LaBarbera, 2005b). Differently, C. savignyi directs relatively more effort into height gain. The greater distance from the ground improves both the quality and the concentration of the ingested food, thus supporting potentially faster growth. Moreover, height also affects the position of the subject relative to other animals and macroalgae living in the vicinity, which may compete for food or block the siphons' flow (Sherrard and LaBarbera, 2005b). This explains the higher oxygen consumption predicted in C. savignyi and the higher seawater flow rate, an energycostly activity, especially in juveniles (Sherrard and LaBarbera, 2005b). Undeniably, the ecological interaction between C. savignyi and C. robusta needs further investigation. Our comparative report, stressing that C. savignyi could grow faster, primarily in the first phase of its life, and thus better exploit food resources, provides a background hypothesis to explain the observation that this species is replacing the indigenous C. robusta in new co-occurrence areas (Lambert, 2007; Lambert and Lambert, 2003). Acknowledgments Authors are grateful to Dr. C. Angelini, for helpful statistical advices, to Prof. M. Yoshikuni, for challenging discussions and assistance during the stage of AT at the Fishery Res. Lab. of Kyushu Univ. (Japan), and to Prof. G. Lambert and Dr. C. Carver, who provided insight and expertise that greatly improved the manuscript. Special thanks to the anonymous referees for valuable comments. AT has been supported by a SZN PhD fellowship.
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