Epibiont hydroids on beachcast Sargassum in the Mexican Caribbean

Massive accumulations of pelagic species of Sargassum have generated recent social, economic and ecological problems along Caribbean shores. In the Mexican Caribbean, these events have prompted the study of diverse biological and ecological aspects of these macroalgae. However, studies on their associated biota, including Hydrozoa, remain scarce. This research provides important species observations in an area where data is lacking. The occurrence and percent cover of hydroids on Sargassum thalli collected on the beach at Puerto Morelos, Quintana Roo, Mexico from April 2018 to March 2019 was studied. Three pelagic species and morphotypes of Sargassum from this area were analyzed: Sargassum fluitans III, S. natans I and S. natans VIII, as well as a benthic species, S. polyceratium var. ovatum. A total of 14 taxa of hydroids, belonging to the superorders “Anthoathecata” and Leptothecata, were identified. In our study, more hydroid taxa were observed on axes of the different species of Sargassum than on leaves or aerocysts. In general, the greatest species richness of hydroids was observed from February to April. Results show that live hydrozoans attached to pelagic Sargassum are transported into the area. This should be considered in future management measures that address the recurring coastal abundance of Sargassum and its associated biota in the Caribbean region.

In this study, species of pelagic Sargassum stranding in Puerto Morelos (Mexican Caribbean) were analyzed to determine: (1) the species richness of epibiont hydroids; (2) the monthly/seasonal occurrence and percent coverage of hydroids on algae; (3) any differences in the presence of hydroids among axes, leaves and aerocysts of the algal thalli; (4) differences in composition of hydroids between Sargassum species and their morphotypes.

MATERIALS AND METHODS
Thalli of fresh pelagic Sargassum were collected on the beach at Puerto Morelos (Mexican Caribbean) (20.84 N-86.87 W) from April 2018 to March 2019 (Fig. 1A). Puerto Morelos is a small fishing and tourist village located on the northern part of an extensive barrier-fringing reef tract that extends from Belize to the Yucatan Strait. In the forereef zone, the most conspicuous components of the biota are gorgonians, small hemispherical coral heads and macroalgae (Ruíz-Rentería, Van Tussenbroek & Jordán-Dahlgren, 1998). The climate in the region has three characteristic seasons: warm and dry (March-May), winter storm with occasional short showers (November-February) and rainy (June-October) (Huntington, 1912;Schmitter-Soto et al., 2002). During the period of our study (2018-2019), temperature and rainfall data (values expressed as average) followed the same seasonal pattern: warm and dry (28.08 C; 69.73 mm), winter storm (25.39 C; 75.89 mm) and rainy (29.59 C; 131.69 mm) (CONAGUA, 2018;. Each month, samples were taken from algal strandings on the beach, which remained wet by contact with waves. From each morphotype of Sargassum, a specimen with visible sessile epibionts was collected from the crest of the mounds of Sargassum (Fig. 1B). A total of 12 specimens were used to describe the occurrence and percent cover of hydroids on each Sargassum species and morphotypes. Thalli and epibionts were fixed in 96% ethanol. Algae in the samples were identified according to Parr (1939), Taylor (1960), Littler & Littler (2000) and Amaral-Zettler et al. (2017). Additionally, floating thalli of a local species of benthic Sargassum, incidentally detected in May and October 2018, were examined for epibiont hydroids.
In the laboratory, collected thalli were examined, and presence of hydrozoans in their axes, leaves and aerocysts was recorded. Hydroid coverage on thalli of both pelagic and benthic species of Sargassum was estimated following Cunha & Jacobucci (2010). The hydroid cover on each thallus (hydrorhiza of hydroids in contact with thallus) was measured by placing the alga between two clear rectangular acrylic plates, divided into 1 × 1 cm squares. When necessary, the thallus was cut into pieces to avoid the superposition of axes, leaves and aerocysts. Hydroid cover was estimated by counting the number of square divisions occupied by macroalgae with and without hydroids on both sides of the plates (Fig. 2). Hydroids were identified to species level using morphological criteria in descriptions from the taxonomic literature (Calder, 1988(Calder, , 1991bSchuchert, 2012). Nomenclature used here generally follows that of the World Register of Marine Species (WoRMS, 2019) and Oliveira et al. (2016).
Non-parametric statistical analyses were performed to determine differences in species composition and percent cover of epibiont hydroids between pelagic Sargassum species and morphotypes using the Bray-Curtis similarity index. Species from the benthic alga were excluded from the statistical analyses because only two months of samples were available for study. Data matrices were standardized and transformed with square root; subsequently, a non-metric multidimensional scaling (nMDS) analysis was performed. To assess the graphical relationships plotted by nMDS analysis, we considered Clarke (1993), who provided an indicator of stress value: excellent graphical representation (<0.05), good ordination (<0.1), usable representation (<0.2), and a possible misinterpretation (>0.2). A similarity percentage (SIMPER) analysis was also performed to identify representative hydroid species of the different Sargassum species, and significant differences were evaluated using one-way analysis of similarity (ANOSIM) at a 0.1% significance level (Clarke & Warwick, 2001). All analyses were performed with the software PRIMER v6 (Clarke & Gorley, 2006). Voucher specimens were deposited in the collection Cnidarians of the Gulf of Mexico and Mexican Caribbean "Lourdes Segura" (Faculty of Science, Multidisciplinary Teaching and Research Unit, Sisal, Yucatán, Mexico) under the code YUC-CC-254-11 from 001561 to 001569. The best preserved epibiont hydroids were selected for vouchering, with one of each species. Each specimen was deposited together with its fragment of Sargassum. These vouchers were indicated with a superindex in each of the taxa determined at the species level in Table 2 (indicated only by the six last numbers of the code).

RESULTS
Thalli of the specimens of Sargassum collected during the study corresponded to descriptions of the pelagic species and morphotypes of Sargassum fluitans III (n = 12; Figure 2 Scheme with details about methodology, with an example of the measure of hydroid cover found on the fronds of Sargassum. Adopted from the methodology described in Cunha & Jacobucci (2010).
Throughout the entire annual cycle, 14 epibiont taxa of hydroids were recorded. Nine of these were identified to species (Figs. 3 and 4), comprising seven genera, seven families, three orders and two superorders ("Anthoathecata", which is non-monophyletic and Leptothecata) ( Table 2). Due to damaged and insufficient morphological diagnostic characteristics in some specimens, three taxa were identified to genus (Halopteris, Clytia, Obelia), one to family (Plumulariidae) and one to suborder (Proboscoida). In addition to hydroids, bryozoans, mainly Jellyella tuberculata, and Polychaeta (serpulid worms) were observed on Sargassum thalli. Of the epibiont hydroid taxa associated with Sargassum thalli, 13 were observed on the axes, 12 on leaves and eight on aerocysts. Obelia dichotoma was found only on thalli axes, while Halopteris diaphana and Tridentata marginata were not found on thalli aerocysts (Table 2).
Two epibiont hydroid species were recorded on benthic S. polyceratium var. ovatum, which are new records for this algal species (Table 2). Tridentata marginata was present in May and October 2018, while T. turbinata was observed only in May 2018. The average cover of these two hydrozoans, only considering the two months in which S. polyceratium var. ovatum was collected, was 37.35%.
Over the year of study, four epibiont hydroid species were recorded on S. fluitans III, five on S. natans I and five on S. natans VIII (Table 2). We found that Aglaophenia latecarinata thrives year-round, followed by C. noliformis, which was recorded 11 of 12 months (in all seasons). Clytia hemisphaerica was recorded only in the month of October    (rainy season) (Fig. 5). Reproductive structures were noted in C. hemisphaerica and C. noliformis (September, October and December), during the transition from the rainy season to winter storm season. Combining monthly data on hydroids from pelagic morphotypes of Sargassum during the study, highest overall species richness of hydroids was recorded in April (six species), followed by February and March (five species), followed by a marked decrease in May (one species), the end of the warm and dry season (Fig. 5).
The annual average cover of hydrozoans on S. fluitans III, S. natans I and S. natans VIII were 42.67%, 28.17% and 67.97%, respectively. Clytia noliformis and A. latecarinata were the predominant species, accounting for 36.63% on S. fluitans III and 62.24% on S. natans VIII. Notably, A. latecarinata and C. hemisphaerica were recorded exclusively on S. natans VIII (Table 2). Throughout the year, the percentage cover of hydrozoan epibionts on species of pelagic Sargassum was higher in the winter storm season (52.80%) ( Fig. 6). Further seasonal breakdown shows that the percentage occupation of epibiont hydroids on structures of pelagic Sargassum species was highest (95.24%) on the axes during the winter storm season and lowest (47.06%) on the aerocysts during the warm and dry season. The nMDS ordination displays the spatial distribution of the monthly pelagic Sargassum species and morphotypes samples and a clear separation into three groups of epibiont hydroids. The stress value obtained with the ordination was 0.03 (Fig. 7). These groupings were significantly different based on the ANOSIM (global R = 0.78; P < 0.001) analysis. When compared by pairs, those with significant differences between them were S. natans VIII and S. fluitans III (R = 1; P < 0.001) and S. natans VIII and S. natans I (R = 0.88; P < 0.001), while no significant differences were found between S. natans I and S. fluitans III (R = 0.33; P > 0.001).
The SIMPER analysis showed that hydroids present on S. fluitans III had 89.32% similarity, while those on S. natans I showed a similarity of 34.79%, and those on S. natans VIII 89.26%. The hydroid A. latecarinata makes the most important contribution to the similarity of the hydroid assemblage (99.85%) on S. natans VIII, while variations in the percentage cover of hydroids of C. noliformis characterized assemblages on S. fluitans III (99.85%) and S. natans I (54.49%). Zanclea alba also makes an important contribution (33.21%) to the similarity in S. natans I assemblages (Table 3).

DISCUSSION
This study examined the species richness, occurrence, percentage cover and variations in composition of epibiont hydroids occurring on species of Sargassum. Of those studied, 92.86% were thecate hydroid taxa (Table 2). All taxa of hydroids identified during this study have been recorded earlier in the Gulf of Mexico (Calder & Cairns, 2009;Mendoza-Becerril, Simões & Genzano, 2018). However, these taxa cannot be considered as new records for the study area, because the local benthic hydroid species are unknown, and records here are of hydroids on drifting substrates. As suggested by Putman et al. (2018), pelagic Sargassum is transported through the open ocean and Caribbean Sea following several pathways until it reaches the Caribbean coast of Mexico.  The two hydroids occurring on benthic S. polyceratium var. ovatum, a species forming part of the local flora of Puerto Morelos (Díaz-Martín & Espinoza-Avalos, 2000), were not observed on the pelagic species and morphotypes of Sargassum species; this suggests that they may also be part of the local hydroid fauna. However, these hydroids have been recorded from pelagic (Stachowicz & Lindquist, 1997) and benthic species (Table 2). These hydroid species are new records from S. polyceratium var. ovatum. Nevertheless, it will be necessary to perform systematic surveys of both pelagic and benthic species of Sargassum to confirm if these hydroid species are exclusively found on S. polyceratium var. ovatum in the region.
The number of epibiont species found on beachcast Sargassum in Puerto Morelos (from two to five hydrozoan species per macroalgal species and morphotypes) is relatively low compared with other studies of the same algal genus. Nishihira (1965) recorded eight hydrozoan species on Sargassum hemiphyllum (Turner) C. Agardh from Japan, while Calder (1995) reported 10 species for S. fluitans III and eight species for S. natans in Bermuda. Moreover, Cunha & Jacobucci (2010) found 14 hydroid species on S. cymosum from Brazil. From all species of hydroids recorded on Sargassum here and in other cited studies, three are most frequent: A. latecarinata, C. noliformis and O. dichotoma (Table 2). Parr (1939) and Govindarajan et al. (2019) mentioned that A. latecarinata is often found on S. fluitans III and S. natans VIII.
We observed more hydroid taxa growth on axes compared with leaves and aerocysts. This comparison is the first at structural level of thallus, since other studies describe the variation in hydroid colonization along the thallus. For example, Fletcher & Day (1983) mentioned that the abundance of hydroids decreases distally in brown macroalgae Ecklonia radiata (C. Agardh) J. Agardh. Faucci & Boero (2000) reported 86% abundance of hydroids on the basal part of brown macroalgae Cystoseira spp., and Fraschetti et al. (2006) found significant differences in growth among thallus regions (basal, medium or distal parts), with the highest number of taxa often recorded on the basal portion of Cystoseira. This variation has been attributed to competitive interactions between the component species within the community or interactions between the epifauna and the host algae (Seed, 1986), growth rate of the epibiont and host (Rackley, 1974) and exposure to mechanical stress (e.g., abrasion, friction, water movement) (Faucci & Boero, 2000).
Our data no suggest seasonal variations in epibiont hydroid species richness, but were noted a variation in terms of percentage cover. Cunha & Jacobucci (2010) recorded seasonal variations, which tend to be higher during the Brazilian summer. Such seasonal variations have been attributed to fluctuations of biotic and abiotic factors (c.f. Cunha & Jacobucci, 2010;Cunha, Maruyama & Jacobucci, 2018), including macroalgal growth periods and/or hydroid reproductive strategies. Nonetheless, to clarify and contribute to a better understanding of any seasonal variation, supplementary studies are essential, both of the epibionts and their living substrates, and the types of specific interactions between them.
The three groups of epibiont hydroids characteristic of the pelagic Sargassum species and morphotypes had an excellent graphical representation and a strong relationship between epibiont-substrate (stress value = 0.03; global R = 0.78; P < 0.001). Differences in hydroid species composition on species and morphotypes of Sargassum may be associated with algal morphology and/or the substances they produce (Nishihira, 1967(Nishihira, , 1968, as well as the growth patterns of Sargassum and its epibionts (Burkenroad in Parr (1939)). Similar differences have been found in previous studies of floating Sargassum masses in the Sargasso Sea (Parr, 1939;Niermann, 1986;Calder, 1995). However, the hydroid taxa reported here are not exclusively found in Sargassum, as these hydroids have also been recorded on floating objects, rocks and other benthic macroalgae (Calder, 1995;Oliveira & Marques, 2007;Oliveira & Marques, 2011).
Finally, it should be noted that while hydroids on beachcast Sargassum were often deteriorated, it was possible to find colony parts with intact coenosarc in thecate species. Deterioration may be due to being fixed to a mobile and flexible substrate, to the mechanical stresses related to the action of currents before reaching land, to abrasion by surf and sand particles as they strand on beaches, and to exposure to air and terrestrial weather once ashore. Whatever the reason for the deterioration, and as suggested by Riedl (1966) and Gili et al. (1998), only hydroids with chitinous exoskeletons, including hydrothecae (superorder Leptothecata) usually survive such adverse environmental conditions. In other studies on hydroids of pelagic Sargassum, leptothecate taxa dominate, with percentages above 80% (Calder, 1995;Martinelli-Filho, Morais & Aviz, 2016;Mendoza-Becerril, Simões & Genzano, 2018). Given the regenerative potential of hydroids, attachment of colonies on autochthonous substrates can occur. Moreover, nematocysts were observed in collected material, indicating that hydroids maintain their stinging capacity and the ability to cause painful rashes and erythematous papules, as has observed in other species of Hydrozoa (Rifkin, Fenner & Williamson, 1993;Marques, Haddad & Migotto, 2002).
Inasmuch as reports on major strandings of Sargassum in the Caribbean region are relatively recent, advance knowledge of these events beyond the adverse effects is needed (c.f. Hinds et al., 2016;Dutch Carribean Nature Alliance, 2019). The impacts of epibionts (e.g., hydroids) on seaweeds need to be assessed along with the phenomenon of golden tides. Awareness of the impact of toxic sessile and motile biota on humans will be needed as recommendations for the use and management of beachcast Sargassum are promulgated.

CONCLUSIONS
The coastal area of the Mexican Caribbean has recently experienced massive incursions of brown seaweeds. The epibiont fauna of these phyophyceans is entirely unknown here, and its origin has not been determined, whether local or transported from other regions along with their substrates. This study provides the first report of epibiont hydroid taxa on certain species and morphotypes of beachcast pelagic Sargassum. Moreover, data are provided on their occurrence and percent coverage, and distribution among axes, leaves and aerocysts of the algal thalli. The composition and occurrence of hydroids changes monthly and varies between Sargassum species and their morphotypes.