Spatiotemporal patterns of phenology of the alien Phaeophyceae Sargassum muticum on the Atlantic coast of Morocco

1 Research Unit Phycology, Blue Biodiversity and Biotechnology, P3B-LB2VE, Department of Biology, Faculty of Sciences, Chouaib Doukkali University, El Jadida, Morocco. (SE) E-mail: elatouanisamir@gmail.com. ORCID-iD: https://orcid.org/0000-0001-5885-7419 (ZB) (Corresponding author) E-mail: Belattmania.z@ucd.ac.ma. ORCID-iD: https://orcid.org/0000-0003-1415-3625 (SK) E-mail: souk.kaidi@gmail.com. ORCID-iD: https://orcid.org/0000-0003-3016-8691 (AC) E-mail: chaouti@ucd.ac.ma. ORCID-iD: https://orcid.org/0000-0003-2840-7059 (AR) E-mail: abreani@yahoo.fr. ORCID-iD: https://orcid.org/0000-0002-9380-3595 (BS) E-mail: Sabour.b@ucd.ac.ma. ORCID-iD: https://orcid.org//0000-0002-5569-0980 2 CCMAR – Centre of Marine Sciences, University of Algarve, Faro, Portugal. (AHE) E-mail: aengelen@ualg.pt. ORCID-iD: https://orcid.org/0000-0002-9579-9606 (EAS) E-mail: eserrao@ualg.pt. ORCID-iD: https://orcid.org/0000-0003-1316-658X


INTRODUCTION
Sargassum muticum (Yendo) Fensholt (Yendo 1907) is regarded as one of the most aggressive introduced marine macroalgae (Boudouresque and Verlaque 2002). It is widely distributed in the Pacific Northwest from Kuri and Sakhalin Island in Russia to Haifong in southern China (Yamada 1956, Kang 1966, Yoshida 1983). S. muticum was observed outside its original range for the first time on the North American Pacific coast of British Columbia, Canada (Scagel 1956). Subsequently, it reached northern California (USA) (Abbott and Hollenberg 1976) and Mexico (Devinny 1978). The spread of this species continued towards the south along the west coast of Baja California (Espinoza 1990). In Europe, populations of S. muticum have been recorded in France (Critchley et al. 1983, Gruet 1983, the Netherlands (Prud'Homme and Nienhuis 1982) and the United Kingdom (Jones and Farnham 1973). S. muticum has also been reported in the Mediterranean (Critchley et al. 1983, Knoepffler-Péguy et al. 1985, Curiel et al. 1995. S. muticum is now present on most of the European Atlantic coasts and continues to spread in particular on the southern coasts of the Iberian Peninsula (Bermejo et al. 2012) and in the British Isles (Engelen et al. 2015). It has also reached the north Atlantic coast of Africa (Sabour et al. 2013).
The temporal and spatial variation of growth, density and reproduction of S. muticum populations in different regions have been described previously (e.g. Pedersen et al. 2005, Plouguerné et al. 2006, Baer and Stengel 2010. Generally, S. muticum length varies from 75 cm (De-Wreede 1978, Gorham and Lewey 1984, Espinoza 1990 to 150 cm (Critchley et al. 1987, Givernaud et al. 1991, Wernberg et al. 2001), but the length can exceed 2 m (e.g. 5 m: Gorham and Lewey 1984, 2.5-10 m: Karlsson and Loo 1999, Sabour et al. 2013, Belattmania et al. 2018. Several factors have been highlighted as potential determinants of ecological success, including, includ-ing air and water temperature, substrate type, hydrodynamic conditions and habitat typology. Previous studies have reported local variability in S. muticum densities depending on exposure (Viejo 1997), grazing pressure (Plouguerné et al. 2006) and seabed topography (Harries et al. 2007a, b). Growth and reproduction in S. muticum are supported by relatively warm water (up to 25°C) (Hales and Fletcher 1990), but high air temperatures and long spawning periods in rocky habitats have a negative impact on gamete release (Engelen et al. 2008). It has been reported that the development and morphological variation between S. muticum populations in different Irish coastal habitats is related to the degree of exposure to the waves (Baer and Stengel 2010). Given the quasi-synchronous colonization of these different habitats, these authors suggested that phenotypic plasticity was the most likely explanation for such variations.
The present work aims to investigate the spatiotemporal dynamics of the newly established populations of S. muticum in different habitats with different topographic profiles on the Atlantic coast of Morocco. This study is the first of its kind carried out on African coasts outside the boreal biogeographic regions and at the southernmost latitudes of the Mauritanian region. Furthermore, it will help fill the knowledge gap in data necessary for any trial of rational valorization, control or eradication.

Sampling sites
Three sampling sites located along the El Jadida shoreline (northwestern Atlantic coast of Morocco) with different topographic profiles favourable to the development of well settled stands of this alga were chosen (Fig. 1). Site S1, located at the southern limit of Deauville Beach (33°15'17.3"N 8°29'52.3"W), consists of a sandy beach with shallow intertidal soft bottoms and a few patches of hard substrates. The thalli of S. muticum are fixed on intertidal bedrock covered with sand. The alga forms a scattered stand with large submerged thalli that exceptionally reach 7 m. Site S2 (33°14'44.7"N 8°32'33.0"W) consists of a large intertidal platform (>200 m) of rocky substratum (bedrocks) with a noteworthy roughness and sheltered shallow tidal rockpools oriented northwestwards. These rockpools shelter a dense and extensive canopy of S. muticum populations protected against the strong wave action by artificial walls called bechkiras. Site S3 (33°13'55.8"N 8°33'24.8"W), located to the south of El Jadida, corresponds to a large subhorizontal rocky platform. At this site, S. muticum populations are limited to rocky intertidal pools with water mass depths varying from 0.5 to 1 m.

Field surveys and sampling design
At the three study sites, monthly samplings were carried out at low tides using 1 m 2 quadrats (1×1 m) random-ly positioned on a linear transect parallel to the shore and spaced 10 m from the middle intertidal zone. Density, length and maturity index of S. muticum were measured monthly from December 2012 to December 2014.
Density was considered as the total number of thalli (individuals) per sampled area (1 m 2 ). The length of thallus was measured from the fixation discs to the apical part of the longest primary lateral (Plouguerné et al. 2006). The maturity of S. muticum was regarded as the appearance of the receptacles on the tertiary branches. The maturity index indicates the ratio of the number of mature individuals to the total number of individuals.

Statistical analyses
Three-way ANOVA was applied to test for the effects of sampling site and time (month and year) on density, maturity index and length of thalli, considering site, month and year as fixed factors. When significant effects of these factors were found, the post hoc com-  parisons were based on Tukey's pairwise comparison test. Prior to ANOVA tests, the homogeneity of variances and normality were verified using the Kolmogorov-Smirnov test and the Levene test, respectively. When these parametric tests were not met, data were log(x+1)-transformed to remove heteroscedasticity (Underwood 1997). These statistical analyses were carried out in SPSS v 23.

RESULTS
The monitoring of S. muticum phenology showed that the thalli length varies significantly between sites and months, and between years (Table 1), except for May and July (Tukey post hoc comparison; p>0.05) and September and December (Tukey post hoc comparison; p>0.05). The longest thalli were detected at the Deauville Beach site (Fig. 2), with maximum lengths of 498.14±11.10 and 643.33±11.10 cm in June 2013 and July 2014, respectively. At site S2, the lengths of thalli reached the highest values of 188±3.73 cm in June 2014 (Fig. 2). However, the greatest alga length did not exceed 95±1.65 cm at site S3 (Fig. 2). The alga grows gradually from autumn, when primary branches rise from the perennial part, bearing large basal leaves whose role is probably to increase the photosynthetic surface. As primary lateral growth progresses, air vesicles and secondary branches begin to develop. The air vesicles keep the seaweed straight in the water column towards the surface. In early summer (June-July), the thalli elongation slows down before reaching maximum values (6 m). At the end of the summer (mid-August to September), thalli length plummets because the vegetative growth rate declines rapidly and lateral branches start to degenerate leaving only a discoid holdfast from which primary laterals regenerate during the following winter.
The density of S. muticum stands depicted significant spatiotemporal variations over two years, 2013 and 2014 (ANOVA, p<0.01), with no significant interaction effect of the factors site and year (Table 1). At   site S1, despite the heterogeneous spatial distribution of the alga, the density recorded in the winter and early spring of 2014 showed high average values ranging from 40 to 48 ind. m -2 before falling sharply (28 ind. m -2 ) in May 2014 (Fig. 3). In contrast to Deauville Beach (S1), at S2 and S3 the S. muticum stands in the rocky pools (Fig. 3) maintained high average densities from winter to early summer (23-39 ind. m -2 in 2013, 43-46 ind. m -2 in 2014), with significant differences between August and September and the cold months; Table 2), when the species naturally degenerates after the reproductive period. Spatiotemporal monitoring of S. muticum maturity showed that the maturity index varied significantly between sites and months (except for October, November, December, January and February, Tukey post hoc comparison; Table 2), with no significant differences between years (p>0.05; Table 1). The thalli at S1 began to develop receptacles on the tertiary branches from April (maturity index =0.39). However, at S2 and S3, the appearance of these reproductive organs was earlier, from March 2013 and 2014, during which the maturity rates varied from 0.11 to 0.28 depending on the site (Fig.  4). Apart from the delay in the appearance of receptacles at S1, the maturity gradually increased from early spring to reach a maximum value of 1 (corresponding to a maturity rate of 100%) in early summer, with water temperatures reaching 20°C (Fig. 4).

DISCUSSION
The seasonal variation in the length of S. muticum thalli on the Moroccan Atlantic coasts corresponds to the classical pattern often observed in the life cycle of S. muticum, with three characterizing stages: slow initial growth; fast growth and lateral elongation; and reproduction and degeneration (Wernberg-Møller et al. 1998). Some authors subdivide the growth rhythm of this species into two phases: one of moderate growth in autumn-winter with a slow increase in thallus length, and one of strong growth during the spring-summer period (Arenas et al. 1995).
On a global geographic scale, S. muticum has shown a latitudinal variation in the phenology of vegetative growth and plasticity in the niche of the reproductive period. In the paralysis zone of Limfjorden in Denmark, the species depicted rapid growth from May to July followed by a phase of senescence when maturity reached its maximum (Wernberg-Møller et al. 1998). The same growth pattern has been demonstrated in Canada (DeWreede 1978), the United Kingdom (Jephson andGray 1977, Gorham andLewey 1984), the Netherlands (Critchley et al. 1987), France (Givernaud et al. 1991) and Spain (Arenas et al. 1995), although there are some latitudinal differences in the timing of the growth cycle, probably due to environmental stimuli (Norton and Deysher 1989, Hales and Fletcher 1990, Arenas et al. 1995. In the south of England, no dormancy period has been recorded since the growth of a generation begins before the lateral branches of the previous year are completely decomposed (Jephson and Gray 1977). Hwang and Dring (2002) highlighted the role of the photoperiod on the elongation of the seedlings of S. muticum. They demonstrated a back-up of growth starting under short photoperiods (8.16) corresponding to winter conditions. Furthermore, Uchida et al. (1991) reported that the lengthening of branches is promoted by short day conditions. In southern Europe, the winter dormancy period is not always obvious, however. Vegetative growth is high from winter to early summer, resulting in maximum lengths exceeding 4 metres (Sfriso and Facca 2013). On the Atlantic coasts of Morocco, S. muticum does not seem to exhibit clear winter dormancy but nevertheless shows a marked seasonality in terms of growth and reproduction, with maxima during the spring-summer period.
The spatial variation in thalli length detected in the present study seems to be linked to the prevailing typology and hydrodynamism caracterizing each sampling site: at site S1, rocky bed covered by sand, is an open environment with fairly significant depths continuously immersed and semi-protected by the harbour jetty, promotes the elongation of the thalli, which undergo their highest vegetative growth from winter to early summer. However, shallow rockpools at both S2 and S3 limit the length and the apical growth of S. muticum thalli, which are frequently truncated at the ends of their primary axes on the surface of the pool under wave action. This promotes lateral branching. The same finding was previously reported by Chamberlain et al. (1979). According to Lann et al. (2012), S. muticum thalli at sheltered, sandy-bed sites are three times longer than those at exposed, bedrock sites. This variation in the thalli length of S. muticum as a function of the biotope has also been demonstrated in other studies (Andrew and Viejo 1998a, b, Engelen et al. 2005, Plouguerné et al. 2006. It has been reported that the length of S. muticum on the Normandy coasts (France) depends mainly on the depth of water available in the pools and the bathymetric level (Givernaud et al. 1991). In its native region of southeastern Asia, S. muticum is considered to be a scarce species among the local macroalgal flora, reaching a maximum of 1 to 2 metres in length. However, outside its native range, the species is invasive and can forms dense stands with thalli length up to 16 meters (Critchley et al. 1983).
If the thalli length is rather favoured by the depth of the water mass, the density of S. muticum stands seems to be mainly a function of the substrate nature (at sites S2 and S3, with rocky substrata, the alga provided the highest densities), a finding corroborating the conclusions of Givernaud et al. (1991) on the coasts of Basse-Normandie in France. Additionally, Harries et al. (2007a, b) reported that the density of the species decreases as depth increases and, thus, light decreases. As for most algal species inhabiting the horizons of the intertidal zone, the density of S. muticum varies according to the bathymetric level, rarely emerging as it is not very resistant to desiccation and abundant, especially in pools of middle and lower levels (Givernaud et al. 1991, Engelen et al. 2015. At site S1, where the thalli are immersed in a column of water continuously exceeding 1 m, the low luminosities could be added to the substrate nature to explain the early decrease in the density of the species from early spring. At sites S2 and S3, S. muticum is abundant, particularly in shallow rockpools that are sufficiently lit in the middle and low tidal zones. The species almost disappears from the high tidal and lowest low-tide zones. In addition to the substrate nature and the bathymetry, some authors (e.g. Staehr et al. 2000, Plouguerné et al. 2006, Thomsen et al. 2006 have raised the role of hydrodynamics in the settlement of this species. Le Lann (2009) demonstrated that the species density is higher at a rocky exposed site than at a sandy and sheltered site. Seasonal changes in water movement could be the main factor influencing the density of Sargassaceae populations (Plouguerné et al. 2006). For S. muticum, the density can vary significantly over short periods because of high recruitment events in the autumn and the rapid growth of individuals in spring (Thomsen et al. 2006). Generally, the density of S. muticum is dependent on the substrate type and hydrodynamics. This macroalgal species acclimates more easily at rocky and semi-exposed sites than at sites with a sandy, protected substrate with low water mass circulation.
The slight shift in the maturity timing of S. muticum at site S1 is probably linked to the typology and hydrodynamic conditions in comparison with those at sites S2 and S3. In these two habitats, the primary axes are continually truncated under the wave action. This promotes the branching and lateral growth of secondary and tertiary branches and, consequently, the earlier appearance of the receptacles. Similar results have been made by Gaylord et al. (1994), Viejo et al. (1995) and Le Lann (2009), who reported that the laterals of individuals living on exposed zones are often torn by waves or swells when they reach a critical length. This also agrees with the results previously discussed for the elongation of thalli at the Deauville Beach site (S1), which seems to take precedence over branching and thus results in maximum length of 5 to 6.4 m, whereas at sites S2 and S3 S. muticum hardly exceeds maxima of 1.2 to 1.9 m and 0.9 m, respectively.
The period of the reproductive cycle of S. muticum varies among regions according to the geographical latitude (Engelen et al. 2015). Along the west coast of North America, the reproduction season extends from June to September. However, in areas further south, the reproduction period tends to start earlier and last longer (Norton and Deysher 1989). On the northwest coast of Baja California, Mexico, fertile individuals may be present throughout the year, with a peak of maturity in spring and summer (Aguilar-Rosas and Machado-Galindo 1990). On the European coasts, the mature thalli of S. muticum can be observed in early spring, in summer and until early autumn. In northern Europe, the annual life cycle of S. muticum includes the following phases: initial growth in spring, elongation in early summer, reproduction in summer and degeneration in autumn (Wernberg-Møller et al. 1998). In Denmark, however, the number of mature individuals is high in July and August. In Ireland, receptacle development typically begins in June and the breeding period is short and is limited to August (Baer and Stengel 2010). In southern England, S. muticum reaches reproductive maturity between July and September (Jephson and Gray 1977). On the Brittany coast of France, mature individuals are observed until the autumn (Le Lann et al. 2012). In southern Europe, thalli of S. muticum develop reproductive receptacles from winter to early summer. On the north coast of Portugal, the breeding period can last from April to August, while in the south, the maturity phase of the cycle lasts from January to September depending on the location (Engelen et al. 2008). All of these studies therefore suggest a latitudinal dependence in the phenology of S. muticum. The maturity period is increasingly precocious and spread out as one moves from norther towards southern latitudes. These differences in reproductive phenology are generally linked to the geographic variation of environmental variables rather than to physiological differences in populations themselves at their different latitudes.
Although higher water temperatures generally lead to faster development of receptacles and early reproduction of the alga (Norton and Deysher 1989), this statement is not valid for all populations of S. muticum. For example, Aguilar-Rosas and Machado- Galindo (1990) reported that the peak of the reproductive period on the coasts of Baja California occurs much later than expected, though mature reproductive individuals are present throughout the year. This delay has been attributed to the upwelling effect along these coasts (Aguilar-Rosas and Machado-Galindo 1990). In addition to the water temperature, the length of days is important in the timing of S. muticum reproduction. Despite these significant latitudinal variations in the maturity period of S. muticum, sensu stricto reproduction is not simultaneous for all thalli. On the Moroccan coast, the increase in the maturity index from early spring indeed reveals a gradual maturation of populations to reach their maximum values at the middle of summer (July-August), when receptacles on tertiary branches become mature and reveal on their surface germinating oogonia and zygotes.
In view of the data obtained through this phenological monitoring, the harvest of S. muticum should be carried out in May-June, a period corresponding to the maximum growth of thalli with largely completed sexual reproduction, which ensures a sustainable valorization. Otherwise, for an invasion control approach, the species should be harvested from March to April, when populations are partially mature.
In conclusion, this study, highlighted various aspects of the dynamics of Sargassum muticum populations on the Atlantic coast of Morocco. By following two annual cycles at three sites with different topographic profiles, the results showed that this species has a spatiotemporal phenotypic plasticity favourable to installation and invasion. The life cycle of S. muticum on the Moroccan coastline has two distinct growth phases: slower growth in winter and faster growth phase in spring─early summer. The species then exhibits great elongation and branching activity during the spring-summer period, with a maximum thallus length of 1 to more than 5 m depending on the type of habitat. The rocky platform site covered with continuously submerged sand (S1) favours thalli elongation, whereas at rocky sites exposed to more marked hydrodynamic forces (S2 and S3), the maximum length of the alga is determined by the water body depth in pools. If the length of fronds is favoured by the depth of the water body, the density of the stands seems to be mainly a function of the substrate nature. Generally, S. muticum is more easily acclimated in shallow rockpools that are sufficiently lit and semi-exposed to waves in the middle and low tidal zones than at sites with sandy and protected substrates with low water circulation.