Introduction
Over the last decades, an increasing number of studies have considered the patterns of gametogenesis, sexual reproduction, and fertilization of seaweeds by means of an integrated biological and ecological approach (e.g., Brawley and Johnson, 1992; Santelices, 2002). Several experimental and field observations allowed to characterize gametogenesis, gamete release, and subsequent fertilization of broadcasting species as a tightly synchronized and environmentally controlled process (Santelices, 2002). Certainly, a remarkable seasonality in different phases of sexual reproduction (especially gametogenesis and spawning) results from a variety of environmental factors that satisfy physiological requirements for reproduction or from the use of environmental cues to coordinate reproduction (Lüning and Tom Dieck, 1989). However, detailed information in the literature is still scarce (Holmes and Brodie, 2004). Indeed, studies that simultaneously examine the effects of several environmental factors on sexual reproduction, in particular on gametogenesis and spawning, appear to be quite difficult. Nevertheless, they are necessary to delineate the conditions that must be simultaneously met for successful sexual reproduction, especially for alien species (Brawley and Johnson, 1992). Focusing on alien invasive species, knowledge of life cycle progression transcends pure academic interest (Liu et al., 2017), as establishing the occurrence of their sexual reproduction events has important environmental management implications, both for gametes and zygote dispersal and the potential to generate more invasive genotypes (Ellstrand and Schierenbeck, 2000, 2006).
The genus Caulerpa (Chlorophyta: Bryopsidales) is a group of marine green siphonous algae distributed in tropical and subtropical regions (Meinesz and Boudouresque, 1996; Ratana-Arporn and Chirapart, 2006), some of which have recently seriously invaded the Mediterranean Sea (Klein and Verlaque, 2008). The Caulerpa genus includes about 85 species (Guiry and Guiry, 2020), and it is considered one of the most widespread, conspicuous, and abundant groups of seaweeds worldwide (Silva, 2003). Species belonging to this genus are multinucleated (coenocyte) and colonial, and they have a modular organization. They are all characterized by the lack of cellulose in their cell walls (Silva, 1992), and they present great phenotypic plasticity and morphological variability that arise in several morphological forms and variants (Belleza and Liao, 2007). In the last few decades, the genus has attracted considerable research interest, because Caulerpa taxifolia (Vahl) C. Agardh and Caulerpa cylindracea Sonder have significantly expanded their range of distribution in the basin where they are currently considered as highly invasive (Verlaque et al., 2003; Piazzi et al., 2005; Klein and Verlaque, 2008). They represent an important threat to the diversity of benthic coastal ecosystems (Boudouresque and Verlaque, 2002), interfering with native species (Piazzi et al., 2001a; Piazzi and Cinelli, 2003) and modifying benthic assemblages (Argyrou et al., 1999).
Besides vegetative dispersion (Žuljević and Antolić, 2000) that represents the main dispersal mode for these species (Ceccherelli and Cinelli, 1999; Ceccherelli and Piazzi, 2001; Ceccherelli et al., 2002), Panayotidis and Žuljević (2001) suggested that such widespread and rapid spread is also probably supported by successful sexual reproduction. Indeed, sexual reproduction is common in the life cycle of the Caulerpa species, and it has been documented in populations from both tropical and temperate waters (Goldstein and Morrall, 1970; Enomoto and Ohba, 1987; Ohba et al., 1992; Clifton, 1997; Clifton and Clifton, 1999). While some authors claimed that the thallus found in nature has a haplodiplobiontic life cycle with heteromorphic alternation of generations (Goldstein and Morrall, 1970; Kapraun and Nguyen, 1994), other authors suggested that a diploid or haploid life cycle with only one generation is present (Ishihara et al., 1981; Ohba et al., 1992; Benzie et al., 2000). Anyway, several articles describe the thallus as monoecious, producing and releasing simultaneously both gametes and dying after gamete release (e.g., Žuljević and Antolić, 2000).
Several observations on sexual reproduction and gamete release by the Caulerpa species were reported for Caribbean reefs (e.g., Clifton, 1997; Clifton and Clifton, 1999), and indicated that sexual reproduction can also play an important role in the dynamics of these algae. Nevertheless, very scarce data on sexual reproduction and gamete release for the Caulerpa species in the Mediterranean Sea are currently present in the literature (Varela-Alvarez et al., 2012). In particular, with regard to two of the most abundant species in the basin, C. prolifera and C. taxifolia, only male or female gametes were observed (Meinesz, 1979; Žuljević and Antolić, 2000). Finally, considering C. cylindracea that is currently the most invasive Caulerpa species in the Mediterranean, even if there is a general agreement that C. cylindracea also reproduces sexually (e.g., Flagella et al., 2008; Klein and Verlaque, 2008; Katsanevakis et al., 2010), the entire lifecycle has not been described, and specific data for the Thyrrenian Sea are not present in the literature yet. Indeed, only two articles describing gametogenesis and spawning in the Mediterranean strain of this species are currently available in the literature. The first referred to the Adriatic Sea and the observation of only female gametes is reported (Žuljević et al., 2012). On the contrary, both male and female gametes were observed as well as planozygotes in the Aegean Sea, but only for a particular variant of the species [Caulerpa racemosa var. occidentalis (J. Agardh) Børgesen] (Panayotidis and Žuljević, 2001).
In this article, gamete release by C. cylindracea in the Thyrrenian Sea (Sardinia, Western Mediterranean) was investigated for the first time, focusing on the main environmental factors triggering the gametogenesis and spawning of the species. To this purpose, a combination of field and laboratory experiments was conducted in 2018–2019. First, a 2-year mensurative experiment was performed in the field during summer to individuate for the first time and, eventually, quantify the occurrence of C. cylindracea gametogenesis and spawning events, linking them to environmental conditions. Furthermore, based on the results of the field work, a complementary laboratory experiment was performed to directly test, under controlled conditions, the effect of the abiotic factors that appeared to play a key role in gamete release processes of the species in the area.
Discussion
This study presents the first report of gametogenesis and spawning by C. cylindracea in the Thyrrenian Sea and provides useful information on the environmental factors triggering such processes. First of all, several gametogenesis events were observed in Tavolara Punta Coda Cavallo MPA during the period of the study, and two gamete release events also occurred, even if no zygotes were observed, as described in few previous studies on the species (Panayotidis and Žuljević, 2001; Žuljević et al., 2012).
Despite both the observed morphology of FT and the general pattern of gametogenesis of C. cylindracea appearing to be quite similar to that of other conspecific species (e.g., Miyake and Kunieda, 1937; Clifton, 1997; Phillips, 2009), some relevant differences also emerged.
With regard to timing, in Tavolara Punta Coda Cavallo MPA, the spawning events appeared to be shorter (10 min) than those described by Miyake and Kunieda (1937) (1 h), while their duration was quite similar to those reported by both Panayotidis and Žuljević (2001) and Žuljević et al. (2012) for other parts of the Mediterranean basin (5−10 min). These results suggest that the duration of spawning events can be quite variable, as already observed for other algae (Pearson and Serrao, 2006).
Moreover, according to Clifton (1997), generally, algae with external fertilization, like Bryopsidales, are characterized by a highly synchronized reproductive pattern, with tens to thousands of conspecific algal thalli (at least 3.5% of the population) becoming simultaneously fertile in the same small area overnight and several spawning events occurring together. Analyzing the data obtained during the field experiment, a different situation was instead recorded. The mean percentage of simultaneously observed FT in the 300 cm2 considered in each site exceed 50% (max. 63.64%) during only 6 of the 120 observations and, in most cases, only one or two FT were, on the whole, counted, with less than 1% of the population fertile. Moreover, only two single spawning events were overall observed. A quite different situation was, instead, observed under controlled conditions, where in some cases FT/TT ratio exceeded 90% and RT/FT ratio exceeded 100%. The lower rates of both FT and releasing events observed during the field study, in comparison to both the laboratory ones and those recorded by Clifton (1997) for reef environments, can be explained considering that the observations of Clifton (1997) referred to the tropical waters of which C. cylindracea is native and not to the Mediterranean, where the environmental conditions are certainly different and so, presumably, different cues act (tidal, photoperiod, lunar phases, etc.). According to Pearson et al. (1998), indeed, environmental cues should be taken into account among the most important triggers playing a key role in coordinating gametogenesis. Considering that the most energy-consuming phase of sexual reproduction is gametogenesis (Shinkawa et al., 2019), it is plausible to suppose that gamete production and release happen when the conditions are favorable in order not to waste energy.
According to Brawley and Johnson (1992), in particular, synchronicity can act by interfering with both gametogenesis and spawning in different ways, ensuring that mature gametes are available for synchronous release when favorable conditions occur (Pearson et al., 2004). On the contrary, Yund (2000) observed that, under non-ideal conditions, environmental cues usually do not act, with the consequence that no synchronous release will occur; in this case, only few FT and spawns are recorded. Therefore, despite the remarkable capability of C. cylindracea to adapt in response to the main ecological factors (Verlaque et al., 2000), it can be assumed that the quite low number of fertile and RT observed during the field study is due to the loss of cues regulating and coordinating sexual reproductive events.
The data from both the field and controlled condition experiments revealed remarkable differences in fertile and RT rates in relation to the environmental factors. These results suggest that also for C. cylindracea, gametogenesis and spawning are strictly related to environmental conditions, as already observed for several quite similar species (Clifton, 1997), and that successful reproduction usually depends on a delicate juxtaposition of different conditions (Santelices, 2002). Environmental factors directly operate to complete algal lifecycles (growth, reproduction, spore germination, and again growth) (Agrawal, 2012) and gametogenesis, and the other phases of sexual reproduction, which appeared to occur only when the main environmental factors (temperature, salinity, photoperiod, main nutrients, etc.) satisfy the physiological requirements for reproduction (Lüning and Tom Dieck, 1989).
Although during observations several environmental factors were considered as determinants for gamete production and release, no differences were observed for light and nutrient availability, as well as for salinity among the study areas and sampling periods. On the contrary, some interesting results were overall obtained considering seawater temperature and water motion. In particular, significant differences in FT/TT ratio were recorded among areas and periods with different hydrodynamics and seawater temperatures, respectively; furthermore, for the former, a clear negative correlation with the ratio of FT was highlighted from the analysis of data obtained under controlled conditions both for FT/TT and RT/FT. These results are in accordance with those of other studies that demonstrated the importance of water motion and temperature in triggering gametogenesis and spawning for other similar species (Giese and Kanatani, 1987; Clifton and Clifton, 1999), and they suggest that such factors must be considered when investigating C. cylindracea sexual reproduction.
With regard to temperature, in the field, a significantly higher FT/TT ratio was observed in the second part of August and in September, when the mean seawater temperature was about 24°C, while in the first half of summer, when the temperature was higher, a lower number of FT were observed. Furthermore, the results of the laboratory experiment highlighted a similar trend, as the FT/TT ratio was significantly higher in the aquaria with a temperature of 24°C, while remarkably lower ratios were, instead, observed at both 18 and 30°C. These results are in accordance with those of other studies on macro and microalgae (e.g., Henry, 1988; Dieck, 1992; Kalita and Tytlianov, 2003; Agrawal, 2012), and they suggest that C. cylindracea must be included in the list of green algae characterized by a very narrow range of temperatures favorable for sexual reproduction. For most algae, indeed, the temperature range that allows for reproduction is considerably limited compared to that for vegetative growth, and they may reproduce asexually or sexually at different, also quite small, temperature ranges (League and Greulach, 1955).
Anyway, considering the few data available in the literature for the species, some differences can be noticed. A slightly higher gametogenesis temperature was, indeed, described both by Panayotidis and Žuljević (2001), with FT primarily observed at 26°C, and Žuljević et al. (2012) with a peak of FT recorded at 25°C, even if they asserted that on the whole C. racemosa in the Adriatic Sea becomes fertile when the seawater temperature is between 20 and 25°C.
Some quite interesting information can also be obtained by analyzing the correlation between FT/TT and RT/FT ratios and water motion, which resulted negative. Higher ratios, indeed, were observed in the aquaria where no water motion occurred. A similar pattern was also observed in the field, where the FT/TT ratio appeared to be related to a stimulating effect of calm condition for gametogenesis. These results suggest that a stimulatory effect of calm conditions seems to exist for gametogenesis and not only for gamete release, as already observed for other species of Bropsydales (Brawley et al., 1999; Speransky et al., 2000). According to Pearson et al. (1998), C. cylindracea seems to be among the algae that have the ability to perceive water motion via boundary layer bicarbonate levels and photosynthetic metabolism (Pearson and Brawley, 1996, 1998), and to consider it as a signal coordinating the different phases of its sexual reproduction.
Stressing the remarkable direct effects of wave forces affecting intertidal organisms, Denny (1995) asserted that they primarily included damage and, especially, displacement of unattached parts that usually occur for gametes floating in water more than adult individuals attached to the substratum. The hypothesis of gamete displacement and dilution is also confirmed by the significantly lower abundance of gametes in the water near the discharge tubes observed in aquaria with high water motion. Even if, in all the aquaria, gamete abundances appeared to be quite similar to those observed by Goldstein and Morrall (1970), for several species of the Caulerpa genus, significantly higher abundances were observed under no water motion conditions, especially when a temperature of 24°C were simulated.
From the obtained data, the effect of temperature is evident only in the optimal condition of water motion, thus allowing us to suppose that water motion is the main factor in C. cylindracea gametogenesis and spawning (as also confirmed by the differences in the Pearson correlation coefficient). Temperature, instead, seems to have, especially in some cases (e.g., 30°C when water motion is remarkable) a secondary additive effect. Actually, for some other seaweeds, gamete release is strictly restricted to calm intervals (Serrão et al., 1996), thereby preventing the gamete dilution predicted to occur by turbulent flow and water exchange (Pennington, 1985; Denny and Shibata, 1989; Levitan et al., 1992). Indeed, gamete production only under low water motion conditions represents an extremely valuable adaptation for an organism inhabiting the intertidal zone, as it allows to finalize gamete release, reducing dispersion. In this way, a large number of gametes appear to be available for gamete collision and fertilization, enhancing the probability of zygote formation.
These results also provide some interesting insights into the relationship between C. cylindracea diffusion and the climate changes that are interesting in the Mediterranean Sea (e.g., Adloff et al., 2015; Marbà et al., 2015). This basin is considered a miniature ocean highly affected by climate changes (Lejeusne et al., 2010). Furthermore, climatic models predict that this basin will be one of the regions that will experience the main effects of both the global warming trend and of an increase in extreme events significantly altering currents and water motion in it. Considering that the two main factors playing a key role in the gametogenesis and spawning of C. cylindracea appear to be precisely sea water temperature and motion, for which the species seems to have a narrow range of tolerance, it is plausible to hypothesize that ongoing climate changes can significantly reduce the chances of successful reproduction of the species in the basin. In case of not optimal environmental conditions, indeed, reproducing asexually could be more advantageous, as recently observed for other species (Endo et al., 2021). Therefore, a situation of increased water motion and temperature similar to one of those simulated during the laboratory experiment, especially in summer, when the vegetative growth of the species reaches its maximum (Piazzi et al., 2001b), will lead to a reduction in the number of FT and, consequently, in the probability of spawning events to occur.
A further observation that a favorable combination of several interacting factors must occur for FT production and gamete release (Serrao et al., 1999; Santelices, 2002) is represented by significantly lower FT/TT ratio that was observed during field observations when compared with both of those obtained under controlled conditions. Indeed, these data suggest that in the field, other factors presumably not acting in the aquaria played a role in such processes, as already observed for the Caulerpa genus during other studies (Phillips, 2009; Žuljević et al., 2012). Considering the other factors triggering gametogenesis and spawning for algae (e.g., extracellular organic substances, pollution, pH of the medium, osmotic stress, etc., according to Agrawal, 2012), an active role of pheromones and other inhibiting extracellular substances can be supposed for the above-mentioned processes. Pheromones are particularly important for the chemical communication between individuals preceding the sexual reproduction of algae (Maier, 1993). While such substances can be easily diluted in the water under natural conditions, at least when thallus abundance is not particularly high, their efficiency in aquaria is expected to be higher. Indeed, in the aquaria, gametogenesis and release can also be enhanced by the absence of extracellular organic substances produced by other algae that could have an inhibitory effect on the reproduction of the considered species, as observed by Wichard and Oertel (2010) and Lüning et al. (2000) for other seaweeds.
Finally, also considering the peculiar morphological features of the observed gametes, some interesting considerations can be made. All the gametes observed during the study had the same length (5.5 to 6.1 μm) and width (1. to 2.5 μm) and lacked the eyespot (Figure 3D). These results are in accordance with that of Goldstein and Morrall (1970) and to Žuljević et al. (2012) that observed that anisogamous gametes for the species with female and male differ for the size and the presence/absence of the eyespot. Therefore, all the observed gametes presumably were males while no female ones were observed, contrary to what was described by Žuljević et al. (2012) for the Adriatic Sea, who observed only larger female gametes with the eyespot. A quite similar result was instead obtained by Phillips (2009) for the congeneric species C. taxifolia, for which, even if a few female gametes were observed, a markedly male sex ratio was noticed. These results suggest that gender expression in Tavolara Punta Coda Cavallo MPA populations of C. cylindracea has deviated from the 1:1 male:female allocation, expected when sex is wholly genetically determined, as already observed for C. prolifera, for which either only male (Meinesz, 1979) or female gametes (Dostal, 1929) were described. Even if sex ratios in macroalgal species have been poorly studied (Holmes and Brodie, 2004), and the causal factors responsible for biased sex ratios are still almost unknown, these results can be explained by considering that Caulerpa populations can be composed of many male plants, few bisexual plants, and no female ones.
In conclusion, the obtained data suggest that C. cylindracea sexual reproduction occurs in the Thyrrenian Sea and that it should be taken into account when focusing on the invasion dynamics of the species. Moreover, as the gametogenesis and spawning of C. cylindracea are mainly regulated by water motion and temperature, such factors must be considered for the management of the species, considering that, as sexual reproduction occurs, new more invasive genotypes for the species could be produced in the Mediterranean, even if the ongoing climate changes must also be considered in the reproduction and diffusion dynamics of the species.