Canopy-forming algae of the order Fucales and Laminariales (Phaeophyceae) are among the most ecologically and socio-economically valuable marine species in temperate waters (Steneck et al., 2002; Smale et al., 2013; Bennett et al., 2015). They provide a structural and trophic framework that supports rich biodiversity by providing food, shelter, and habitat for other associated species (Bustamante et al., 2017; Teagle et al., 2017), and are responsible for nutrient cycling and CO₂ storage (Krause-Jensen and Duarte, 2016; Filbee-Dexter and Wernberg, 2020).

Macroalgal forests are undergoing major regressions worldwide due to a combination of multiple natural and anthropogenic sources of disturbance (Steneck et al., 2002; Strain et al., 2014; Mineur et al., 2015; Krumhansl et al., 2016). In recent years, an increasing number of studies have reported changes in the distribution and abundance of these macroalgal populations as a result of ocean warming and thermal anomalies (especially marine heat waves, MHWs) (e.g., Smale, 2020 and the references therein; Bevilacqua et al., 2019; Savonitto et al., 2021; Verdura et al., 2021). Populations at the edge of their range appear to be particularly affected by this trend (e.g., Viejo et al., 2011; Nicastro et al., 2013; Araújo et al., 2014; Álvarez-Losada et al., 2020; Gurgel et al., 2020). Thermal anomalies may affect the phenology and physiology of these species, impairing their performance, increasing their vulnerability to other stressors, and eventually leading to population declines and local extinction events (Wernberg et al., 2010, 2016; Gouvêa et al., 2017; de Bettignies et al., 2018). These events could also lead to changes in associated species and their interactions (Vergés et al., 2016; Wernberg et al., 2016; Provost et al., 2017), which may ultimately result in detrimental cascading effects on ecosystem functions and the resulting provision of goods and services (Smale et al., 2013; Vergés et al., 2014; Straub et al., 2019). To date, most works addressing the effects of heat stress on the physiology and biology of brown algae have been manipulative and laboratory-based, focusing on kelps, with the ‘sporophyte phase’ being the most studied (e.g., Wilson et al., 2015; Burdett et al., 2019; Nepper-Davidsen et al., 2019; Fernández et al., 2020; Hereward et al., 2020; Diehl et al., 2021; Umanzor et al., 2021). As for the Fucales, the genus Fucus is the most extensively studied, and works on adults predominate over those on early life stages (e.g., Strömgren, 1977; Pearson et al., 2009; Jueterbock et al., 2014; Nielsen et al., 2014; Graiff et al., 2015; Mota et al., 2015; Smolina et al., 2016; Rothäusler et al., 2018; Figueroa et al., 2019). Overall, the trend that emerges is a high sensitivity in the early life stages and a relative ability of adults to grow and survive over broader temperature ranges and to physiologically compensate for thermal stress.

In the Mediterranean Sea, macroalgal forests are dominated by Cystoseira sensu lato (s.l.) species (Fucales, Phaeophyceae). In recent decades, they have declined or become locally extinct due to anthropogenic pressure (e.g., Thibaut et al., 2005, 2015; Falace et al., 2010; Perkol-Finkel and Airoldi, 2010; Blanfuné et al., 2016). To date, there is little evidence of natural recovery of damaged Cystoseira populations (e.g., Munda, 2000; Iveša et al., 2016; Orlando-Bonaca and Rotter, 2018; Medrano et al., 2020), because once losses have occurred, recovery from nearby populations tends to be difficult due to the short dispersal of eggs/zygotes and low connectivity of populations (e.g., Soltan et al., 2001; Buonomo et al., 2017; Capdevila et al., 2018). There is evidence that thermal anomalies and warming can alter the reproductive phenology, germling growth, and viability of Cystoseira s.l. species (Celis-Plá et al., 2017; Capdevila et al., 2018; Savva et al., 2018; Bevilacqua et al., 2019; Cáliz et al., 2019; Mancuso et al., 2019; Savonitto et al., 2021; Verdura et al., 2021). As the Mediterranean Sea is warming faster than the oceans and thermal anomalies occur with increasing intensity, frequency, and duration (Diffenbaugh et al., 2007; Vargas-Yáñez et al., 2008; IPCC, 2019; Pastor et al., 2020; Pisano et al., 2020), examining the response of Cystoseira s.l. species to temperature may provide useful insights into their potential future fate under global warming.

The present study focuses on Ericaria giacconei Serio et G. Furnari (= Cystoseira hyblaea Giaccone), a species endemic to the Sicily Channel (Central Mediterranean Sea) that lives in the intertidal and upper sublittoral at depths of 0.2–1.5 m on semi-exposed and exposed rocky shores. Maximum vegetative and reproductive development occurs in winter, from January to March, when mean seawater temperature at 1 m depth ranges from 14.7 to 16.1°C. This species was described at Punta D’Aliga (southern coast of Sicily, Italy) (Giaccone, 1985), where it is locally extinct (Cormaci et al., 2012). Its current range is fragmented and restricted to two localities: Cap Bon (Kelibia) along the Northern Tunisian coast (Bouafif et al., 2016) and Portopalo di Capo Passero (Isola delle Correnti) in Southern Italy (present study). Its disappearance from the type locality, its limited range, and the fact that it lives in shallow waters raise concerns about the possible fate of E. giacconei in the current warming regime. This species, like other Cystoseira s.l. species, is listed in some international agreements (e.g., Barcelona Convention, Directive 92/43/EEC), but these are not legally binding.

The objective of this study is to determine the thermal tolerance of both early developmental stages and adults of E. giacconei. Adult photosynthetic efficiency and egg release, zygote settlement, and embryo development were examined at five temperature treatments from 12 to 28°C. Evidence for the likely response of this species to projected climate change is provided, together with a thorough discussion on its conservation status. Another outcome of this work is the embryology of E. giacconei, which has never been described before.

After acclimation, the adult primary branches of E. giacconei had F_v/F_m values ranging from 0.606 to 0.768, attesting the viability and good physiological status of the photosynthetic apparatus of the samples.

F_v/F_m values were stable throughout the experiment, although slight but significant changes were observed as a function of temperature and time (Figure 2A, Table 1, and Supplementary Table 1). Specifically, at 12°C F_v/F_m statistically increased over time by 5.3% (Supplementary Table 1). The interaction between temperature and time had a significant effect on F₀ and F_m (Table 1 and Supplementary Table 1); from t₁ to t₃, both parameters were stable at 18 and 24°C, whereas F₀ significantly decreased in samples at 12 and 15°C and F_m at 15 and 28°C (Figures 2B,C and Supplementary Table 1). PI_abs was only affected by temperature (Table 1 and Supplementary Table 1): it was highest at 28°C and gradually decreased from 24 to 15°C, with the lowest values at 12°C at t₃ (Table 1, Figure 2D, and Supplementary Table 1).

Ericaria giacconei has branched pigmented antheridia and ovoid oospheres retained in the conceptacle (Supplementary Figure 2). The following embryological traits were observed: the first and second division of the zygote are parallel to each other and the third division is perpendicular to the previous ones. The embryo development takes place directly on the substratum: four primary rhizoids are formed fixing it (Supplementary Figure 3).

The RP did not differ significantly among thermal treatments, making them comparable at the beginning of the experiment (Table 2 and Figure 3A).

No significant effects of temperature were detected on RE (Figure 3B and Table 2). In contrast, temperature significantly affected SE (Figure 3C and Table 2). Specifically, SE at 28°C was lower than all other treatments. RE (Figure 3B) and SE (Figure 3C) showed a bell-shaped response to temperature, and the quadratic model fitted to the data explained 51 and 61% of the variability for RE and SE, respectively (Table 3).

PERMANOVA on embryo status revealed a significant Tr × Ti interaction (Table 4), indicating that temporal patterns of embryonic development differed significantly between temperature treatments. These differences were evident in the nMDS ordination of Tr × Ti centroids (Figure 4). The centroids of 12 and 15°C clustered alongside those of 18 and 24°C, the latter also showing marked separation between 20 and 44–92 h AF. These differences were mainly due to the fact that at 20 h AF a higher percentage of eggs, zygotes, or two-celled embryos were found in the treatments at 18 and 24°C than in those at 12 and 15°C (Figure 4A). In contrast, multicellular embryos or rhizoids were found in the treatments at 12 and 15°C in each time interval (Figure 4B), suggesting that the development rate was faster at lower temperatures. In addition, embryo mortality was consistently higher at 18 and 24°C than at 12 and 15°C, with the highest percentage of dead embryos recorded at 24°C (Figure 4C).

Climate change, coupled with multiple anthropogenic and natural stressors occurring in coastal ecosystems, poses a major threat to the long-term survival of marine forests. From this perspective, studying species vulnerability to temperature stress can provide relevant insights that can be used to make more robust and integrated predictions for marine forest conservation and management.

In our experiment, adults of E. giacconei were not negatively affected by temperatures, indicating an expected ability to acclimatize to a wide range of temperatures typical of the Mediterranean Sea and especially the intertidal zone. All temperatures to which thalli were exposed had a statistically significant, but not physiologically relevant effect on F_v/F_m (max increase +5% at 12°C, from 0.659 to 0.694; max decrease −1% at 28°C, from 0.731 to 0.725), which remained generally steady and within the range of values indicative of a healthy PSII, i.e., >0.6 (e.g., Celis-Plá et al., 2014; Smolina et al., 2016; Falace et al., 2018b; Savva et al., 2018; Cáliz et al., 2019; Verdura et al., 2021). In contrast, temperatures above 18°C caused an almost equal decrease in F₀ and F_m (Figures 2B,C). Photosystem II is considered the most heat-sensitive component of the photosynthetic apparatus, especially at the level of the oxygen-evolving complex (Oukarroum et al., 2016). Impairment of this component leads to a progressive decrease in electrons entering the electron transport chain from PSII until its complete inactivation (Allakhverdiev et al., 2008). Several parameters of the fast Chl_aF transients, such as the maximal and basal fluorescence (F_m and F₀) and the derived maximum quantum yield (F_v/F_m), are the most appropriate tools for detecting early effects of heat stress, as they have been shown to correlate with heat sensitivity/tolerance (Allakhverdiev et al., 2008). In particular, the increase in F₀ is closely related to the temperature at which PSII is inactivated (Yamane et al., 2000). For the aforementioned reasons, this could be interpreted as a transient adaptation of the photosynthetic apparatus to the temperature change rather than heat stress.

Several works reporting the effects of temperature on photosynthetic efficiency of brown algae have shown that adults are generally tolerant of temperature fluctuations. For instance, Chl_aF of E. selaginoides adults was not affected after exposure to temperatures up to 28°C for 15 days (Cáliz et al., 2019). Savva et al. (2018) reported that F_v/F_m of Cystoseira compressa exposed from 12 to 34°C maintained values close to the optimum in the range of 19.2–30.9°C. Similarly, Mancuso et al. (2019) observed an increase in F_v/F_m in the field up to 28°C when the algae were submerged, and a marked decrease during tidal emersion only when air temperature exceeded 28°C. Accordingly, populations of Fucus serratus from southern areas of North Atlantic showed a decrease in PI_abs only when temperatures ranged from 28 to 36°C (Jueterbock et al., 2014), although F. serratus is a cold-affine species. In our case, adults of E. giacconei showed higher PI_abs at the upper extreme of the tested temperature range (Figure 2D), suggesting that they have better PSII efficiency in warm seasons. Negative effects on Chl_aF parameters were observed in Fucus distichus only when thalli were exposed to temperatures 10–15^° above their optimum (Smolina et al., 2016), and in E. selaginoides when dissolved CO₂ and nutrients were also altered (Celis-Plá et al., 2017).

The tolerance of adult thalli of E. giacconei and the other intertidal Cystoseira s.l. species to temperatures up to 28°C might be related to an adaptation to the highly dynamic habitat they colonize. Indeed, the intertidal is characterized by large temperature fluctuations due to tidal cycles, especially during the warmer months. Notably, during summer tidal cycles, at the site where E. giacconei was sampled, these algae can experience temperatures ranging from 28°C (seawater temperature) at 1 m depth at high tide to 41°C (air temperature) at low tide within a few hours (Servizio Informativo Agrometereologico Siciliano, 1995; Clementi et al., 2019). In contrast, species that are not adapted to such extreme environmental changes might be more sensitive to temperature increases. For example, Verdura et al. (2021) reported that adults of the subtidal species Ericaria crinita showed a marked decrease in biomass, F_v/F_m, and C:N ratio during a 30-day period at 28°C. Similarly, Sato et al. (2020) observed a decrease in PSII efficiency in the subtidal kelp Saccharina sculpera maintained at temperatures ≥28°C, while the optimal range for the tested population was 22–24°C.

Despite the high tolerance of Cystoseira s.l. adults, especially of intertidal species, to temperature fluctuations, little is known about the possible effects on early developmental stages and developmental processes. Apart from the oldest embryological studies (e.g., Guern, 1962; Colombo et al., 1982; Gil-Rodríguez et al., 1988; Motta et al., 1988; Alongi et al., 1999), the embryogenesis of many Cystoseira s.l. species is still poorly known (Falace et al., 2018a; Savonitto et al., 2019). Based on reproductive traits and zygote division sequence, E. giacconei fits into the first embryological group described by Guern (1962), which includes most Cystoseira s.l. species (e.g., Ericaria mediterranea, Gongolaria elegans, and E. selaginoides).

Regarding the effect of seawater temperature on early developmental stages, we found that the eggs’ release efficiency did not vary significantly among the tested temperatures. However, greater exudate production was observed at higher temperatures (Supplementary Figure 4). Exudates, typically phlorotannins, are released by macroalgae under stress conditions (Sieburth and Jensen, 1969; Kroes, 1970; Abdala-Díaz et al., 2006). The settlement efficiency of the zygotes of E. giacconei increased from 12 to 18°C, but no statistically significant difference was found, then it started to decrease (24°C) and dropped significantly at 28°C. Remarkably, the extremely low settlement efficiency at 28°C was due to the fact that eggs and zygotes had undergone cell lysis and clustered together (Supplementary Figure 5).

The detrimental effect of heat was even more pronounced during germling development. Embryos were able to fully develop only at 12 and 15°C, while mortality increased sharply at 18°C and all germlings died at 28°C. The highest development rate observed at 15°C (highest percentage of embryos with rhizoids already after 20 h AF) suggests that this temperature represents the thermal optimum for reproduction and development of the early life stages. Actually, it corresponds to the mean seawater temperature during the winter months when the species reproduces.

To date, very few studies have investigated the potential effects of warming on the early life stages and in adults of Cystoseira s.l. species (e.g., Cáliz et al., 2019; Capdevila et al., 2019; Verdura et al., 2021). These studies focused specifically on the effects of high temperatures on the settlement and survival of recruits, showing that higher temperatures lead to embryo death. In particular, a tolerance threshold of 24°C was found in Ericaria zosteroides (as. C. zosteroides), a deep-sea species (Capdevila et al., 2019), and 28°C in Ericaria selaginoides (as C. tamariscifolia) (Cáliz et al., 2019) and Ericaria crinita (Verdura et al., 2021), two species from shallower waters. These results are only partially consistent with ours, as almost all germlings in this study failed to settle or survive at 28°C. However, in contrast to previous studies, we tested a broader temperature range and found that although E. giacconei is an intertidal to upper sublittoral species endemic to the southern Mediterranean, and thus hypothetically adapted to high temperatures, its thermal optimum is at much lower temperatures (12–15°C) than the other Cystoseira s.l. species examined.

Our findings suggest that E. giacconei is a stenothermic, cold-adapted macroalga that requires an extremely narrow range of low temperatures for embryonic development and survival. These results support the findings of Bouafif and Langar (2019) who, by modeling the potential spatial distribution of Cystoseira s.l. species in Tunisia, reported that E. giacconei occurs only in the colder waters of northern Tunisia. Sites where E. giacconei thrives could represent climatic refugia where the species still survives (e.g., Lourenço et al., 2016; Abelson et al., 2020; Verdura et al., 2021). The Sicilian Channel is characterized by a surface current called “Modified Atlantic Water” (MAW), forming two flows: one along the Sicilian shelf and the other off the Tunisian coast (Robinson et al., 1999; Béranger et al., 2004; Jouini et al., 2016). The complex bathymetry, as well as the water circulation, favor a semi-permanent upwelling regime, which is enhanced by local winds (e.g., Mistral) along the southern coast of Sicily. Therefore, the interplay of surface currents and upwelling provides lower sea surface temperatures along the coast (Raffa et al., 2017), but cannot prevent the occurrence of adverse climatic conditions.

Exceptionally high temperatures for several consecutive days during the reproductive season (e.g., Supplementary Figure 6) may actually lead to massive mortality of zygotes/embryos, thus defeating the reproductive efforts of the species. Furthermore, the negative effects of warming on recruitment could be exacerbated by other stressors that have been shown to negatively affect the early developmental stages of Cystoseira s.l., such as herbicides and pollutants (de Caralt et al., 2020).

From this point of view, the recruitment of new individuals seems to be the real bottleneck for the population dynamics of E. giacconei, as recruitment failures, if they occur over several years, can lead to lower population densities, ultimately affecting their long-term survival.

The stenothermic nature of the early life stages and the warmer sea areas that evenly surround the few localities with favorable conditions make this species a dotted endemism (Giaccone and Di Martino, 1996). Consequently, E. giacconei may become extinct if climate change continues with the current pattern. In the Sicilian Channel, several studies have already reported the disappearance of infralittoral stenoecious species of Cystoseira s.l. due to the increase in sea surface temperature and changes in deep circulation (Alongi et al., 2004; Catra et al., 2006; Serio et al., 2006), further evidence of the tropicalization process caused by climate change affecting the Mediterranean Sea (Boero et al., 2008; Furnari and Cormaci, 2009; Marbà et al., 2015).

Together with all Mediterranean species of Cystoseira s.l. (except C. compressa), E. giacconei is included in the “List of Threatened or Endangered Species” of Barcelona Convention (modified Annex II of the “Protocol on Specially Protected Areas and Biological Diversity”; United Nations Environment Agency, 2019; Verlaque et al., 2019), but its conservation status has not yet been defined by the IUCN (like the fucoid Sargassum, see Thibaut et al., 2016). In our opinion, E. giacconei should be included in the IUCN Red List of Species (International Union for Conservation of Nature, 2021) and classified as Critically Endangered due to its limited distribution and high vulnerability. As a conservation strategy, the climate refugia that ensure the persistence of E. giacconei should receive the highest level of protection.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

AF, MT, and GA conceived the ideas and designed the methodology. GM and GA collected samples in the field. AF, GS, and MS performed the experiments in aquaria. FC and SB performed the statistical analysis. AF led the writing of the manuscript. AF, GM, GS, MS, FC, SB, and MT contributed significantly to the draft of the manuscript and approved the submitted version. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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