New Records of Marine Macroalgae on the Caribbean on Coast of Costa Rica

The results obtained in this study highlight that further the macroalgae diversity in this region is even higher than what is reported to date. Here we present eight new records of marine macroalgae collected from the Caribbean coast of Costa Rica between March 2011 and May 2019. The morphological characteristics of Bryocladia cuspidata, Ceratodictyon variabile, Gracilaria hayi, Padina boergesenii, Zonaria tournefortii, Sargassum furcatum, Chaetomorpha aerea, and Bryopsis pennata var. secunda are described. The genera Bryocladia and Zonaria are reported for the first time for this region.


Introduction
The Caribbean coast of Costa Rica has an extension of approximately 215 km, ranging from Punta Castilla in the north to Punta Mona in the south. Its coastline is straighter and less rugged than the Pacific coast counterpart [1], providing a great diversity of marine ecosystems where macroalgal meadows form distinct habitats. The Atlantic coast is the more studied and the more diverse of the two coastlines. More than 350 species are reported in the Caribbean of Costa Rica, while for the Pacific coast have been reported around of 170 species of macroalgae, of which approximately 45 are shared with the Caribbean [2]. As ecosystem engineers and primary producers, macroalgae are key in structuring their local community and maintaining functional dynamics in coastal systems [3] [4] [5].
From a taxonomic standpoint, the first studies of macroalgae in the Caribbean coast of Costa Rica were done by [6]- [11], who reported the first species of benthic macroalgae for the region. Further systematic studies by [12] [13] [14] were not only limited to the description of species but also included data related to the ecology and distribution of macroalgae. Later, [15] described a new species within the order Gelidiales, while [16] [17] added several new records for this region, including species within the orders Cladophorales, Bryopsidales, Fucales, Ceramiales and Nemaliales. More recently, [18] described four new species in the genus Lobophora for Costa Rica, three being described for the Caribbean coast. It is noteworthy that the most comprehensive reports compiling the list of all macroalgae and cyanophytes species described for both the Pacific and Caribbean coast of Costa Rica to date are those by [19] [20]. Their publications included data from the collection sites, as well as information about the distribution of the species along both coasts.
In the past few decades, the Caribbean coast of Costa Rica, particularly in the vicinity of Puerto Viejo, Puerto Limón and Moín have been subjected to intense anthropogenic disturbances [21], such as increased urbanization, inappropriate wastewater management and an over-exploitation of marine resources [22]. Recent data obtained by [4] showed that urban development, particularly of jetties and restaurants built facing the beach, coupled with illegal sewage systems, are threatening benthic biodiversity significantly. Despite the ecological relevance of macroalgae and the increasing pressure to which they are exposed to, their diversity and interactions along the Caribbean coast of Costa Rica are still largely unexplored. The greatest sampling efforts have been conducted along the intertidal and subtidal areas within the Cahuita National Park [17], where the reef sites have been the most studied [19]. Other areas such as Puerto Limón, Moín, and Punta Mona have rarely been explored in terms of their algal diversity. Here we report and provide diagnostic characters of seven new macroalgal records collected from underexplored sites of the Caribbean coast of Costa Rica. We also provide comparisons between these new records and related taxa. This report strengthens our knowledge about the high marine biodiversity in Costa Rica.
Macroalgal sketches were created using a lucid camera attached to an Olympus SZH microscope. Sketches were edited using Photoshop CS3 (Adobe Systems). Voucher specimens were deposited in the Herbarium of the University of

Results
The eight species identified were classified within six families: Rhodomelaceae  Type locality: Panama, Galeta Point [27].
Notes: There were reproductive structures.
Habitat: plants of shallow water and from moderately sheltered locations.  Description: Thallus erect, reddish to reddish-brown, fleshy, 7.5 -14 cm high, attached to the substratum by a small discoid holdfast. Thalli flattened, strap-like, with a short basal stipe, up to 1 cm long, from which arise erect axes 4 -20 mm wide, irregularly branched. Specimens robust with dichotomies, or even polytomies, giving them a flabellate aspect. Some branches with cuneate base, 15 -25 mm wide. Margins smooth to slightly undulate. Apices rounded, lobed, bifurcate or sometimes cordate. In transverse section, base of thallus with up to 5 layers of ovate to quadrate cortical cells and up to 3 layers of large medullary cells, polygonal in shape to slightly flattened, with thickened walls. Tetrasporangia cruciately divided, scattered on both surfaces of thallus.
Remarks: Gracilariales have a wide geographical distribution, ranging from tropical to polar regions [26]. The order has about nine recognized genera, and their taxonomic history is controversial [27]. The genus Gracilaria Greville is the main source of agarose today [25] and is one of the most species-rich genera within the Rhodophyta. Species of Gracilaria that have a flat morphology are among the most taxonomically challenging taxa due to their phenotypic plasticity among all known species [28].  [29] [30] [31], and with the descriptions of G. cuneata made by [25] [32] [33].
The membranous texture and the crisped margins of the thallus shown by G. cuneata set it apart from G. hayi. On the other hand, Gracilaria cearensis has a thinner thallus than that of G. hayi, and the latter is more robust than G. galetensis and G. brasiliensis. The broad thallus, flabellate appearance, cordate axes, dichotomies in various branching orders, and the absence of marginal proliferations are useful characteristics for the identification of G. hayi.
The online information known up to now [24] restricts the distribution of G. hayi to Panama, Florida and North Carolina (US) and Brazil. It is very likely that the distribution of this species is much more widespread due to the similarities among flattened species, a fact that often contributes to erroneous identifications due to overlapping diagnostic characters. The identification of only one specimen or young plants can be difficult.  Remarks: Bryocladia F. Schmitz is a genus with only a few recognized species [24]. Bryocladia is close to Tayloriella; all branches in Tayloriella are said to be exogenously derived, whereas endogenous branchlets are characteristic of Bryocladia. Tayloriella includes four species that are distributed mainly in the Pacific Ocean.
Tayloriella abyssalis, which was described by [35], was later merged with Pterosiphonia hamata, with the current name of Savoiea hamata (Sinova) M.J. Wynne [36]. [37], who described Bryocladia borealis from Pacific Mexico, later transferred the species to Tayloriella, as T. borealis (E. Y Dawson) E. Y [38]. In all known species of Bryocladia the thallus is comprised of prostrate branches, attached by unicellular rhizoids, and erect branches, up to 10 cm long, having a percurrent axis with few secondary axes. All branches ecorticate, densely clothed with short, determinate, spine-like, exogenous polysiphonous branchlets, spirally arranged, and with up to 12 pericentral cells. [23] reported Bryocladia thyrsigera (J. Agardh) F. Schmitz and B. cuspidata for the subtropical Western Atlantic. Plants of B. thyrsigera arise from a creeping base and have main erect axes to 4 -8 cm tall. They may be somewhat entangled below, sparingly divided, with 10 -11 pericentral cells, ecorticate, above with numerous shorter branches as much as 2.5 cm long, bearing pinnate branchlets. These differences are verified at the regional level by [39] when comparing the two species for Venezuela.
The presence of Bryocladia cuspidata is new for the Central American region.
The species is very abundant in several localities of the southern Caribbean coast. This differs from the observations made by [40], who considered the species to be uncommon. It is known in the Caribbean region, Florida, Mexico, Cuba, Jamaica, Puerto Rico and other localities in the tropical and subtropical Western Atlantic [23].  Type locality: Madras, India [41].
Habitat: common on rocks in the intertidal zone and somewhat lower, particularly in crevices or on overhanging cliffs, often in rather exposed places. Found Remarks: At present, Ceratodictyon Zanardini is known to include nine species [23]. [42], relocated several species of the genus Gelidiopsis to Ceratodictyon, a genus of red algae in which the stem shape was originally described as reticulated and living in symbiosis with sponges.
By relying on the works of [8] [37] [43], [42] recognized the morphological similarities among the genera Gelidium and Gelidiopsis. Nonetheless, he was able to effectively identify Gelidiopsis variabile as a member of the genus Ceratodictyon.
The taxonomic distinction between Ceratodictyon variabile and C. planicaule (W.R Taylor) M.J. Wynne is controversial in the literature. Both specie s have already been considered synonyms by [42], but the differences observed by [44] [31] justify the maintenance of these two entities as separate species.
Ceratodictyon variabile differs from C. planicaule in its regular branching and cylindrical thallus all along its anatomy, attributes that were evident in the individuals collected at Playa Cocles and Playa Puerto Viejo. The presence or absence of anastomoses, as well as the width of the thallus and branching pattern, were useful characters for separating the species within the genus.
The presence of Ceratodictyon varibile was previously recorded in Central America: Belize [44], El Salvador [45], Nicaragua [45], and Panama [45]. However, this species is known up to now only from the Pacific coast of Costa Rica [45], although it has a wide distribution worldwide [23].  Meanwhile, individuals growing in insular intertidal zones show blades with non to slightly divided habits [48]. P. boergesenii is characterized by the presence of two to three layers of cells along the blade, one organized as a layer of medullary cells and two cortical layers. This characteristic construction differentiates the species from others from the tropical Western Atlantic recorded by [23]. According to [49], the character of separation between Padina boergesenii and P. gymnospora (Kützing) Sonder is based on the number of cell layers. Padina gymnospora presents three to six layers of cells, while P. boergesenii has three along the whole blade (10 [mc]) except the apex (10 [ac]). [48] considered both species to be conspecific, but observations on P. boergesenii by [48] allow us to consider this as a different species than P. gymnospora. Furthermore, [49]   Remarks: Of the 19 names recognized for the genus Zonaria C. Agardh [24], only Z. tournefortii has been recorded for the subtropical Western Atlantic [23]. The structure displayed by the thalli of Zonaria is the result of marginal growth and frequent tears. The morphology varies significantly depending on the age and environmental conditions, such as depth, swell, seasonality, and abrasion to which the thalli are exposed [58] [59]. [60],  Remarks: Currently, the genus Sargassum C. Agardh is recognized to include 358 species [24]. The traditional classification scheme has been based on polymorphic morphological characters (holdfast, axes, phylloids or "leaves", air vesicles and receptacles) is still accepted with some modifications [63]. Variations in the morphology of Sargassum have been clearly documented by several authors [64] [65], and recently proven using molecular techniques [66] [67]. Certain morphological characteristics such as leaves are considered of great utility for the recognition of species, with the shape, length, width, rigidity, margin and surface as the most relevant descriptors. The first four descriptors were considered by [8] [68] for the separation of species in the Western Atlantic. On the other hand, the margin of the leaves was considered to be an unreliable descriptor for species in the Philippines [69], although other authors considered this as a consistent trait to distinguish species in Brazil and Venezuela [64] [68]. Moreover, distinguishing the surface of the leaves either as simple or furrowed is another useful trait for the discrimination of species [8] [31] [40].
Of all the species recorded for Costa Rica [19], S. polyceratium Montagne exhibits the most variation in the morphology of its leaves. These findings are consistent with the results by [70] [71], who found 47 different morphotypes and significant seasonal variability in the morphology of the leaves. This character, however, is very consistent in the specimens of S. furcatum examined. Similar findings were described by [64]- [72].
Other species of Sargassum also show this variability in the diagnostic characters for the genus. S. furcatum, for example, was considered a form of S. vulgare by two different authors [73] [74]. The presence of receptacles is the most commonly used character in the taxonomy of the genus; however, it cannot be used to determine the species.
The size of cryptostomata is a characteristic of relative importance, where it should be considered to separate the specimens based on their state of maturity [64]. Moreover, the arrangement of cryptostomata is highly variable, with their size dependent on that of the leaves [65]. On the other hand, the presence of spines in the branches is a relevant character used for the identification of species in the Caribbean [31]. Also, the diameter and arrangement of the vesicles, although not a key character, helps to distinguish the species.
The spherical vesicles, occasionally hidden under the phylloids, are attributes observed in the S. furcatum. No air-bladders were observed in S. furcatum in specimens studied in the Gulf of Mexico by [75]. This is the first record in the Central America region, although the species is well distributed in the subtropical western Atlantic [23] and has also been recorded in the Philippines [24].  Remarks: At present, Chaetomorpha Kützing has 73 recognized species [24].
It is a cosmopolitan genus, with representatives that can be found in oceanic areas, estuaries and even freshwater. Morphologically it is formed by serial filaments, unbranched, and robust, either erect and attached with an elongate, thick-walled, basal cell or loose-lying. Growth is diffuse, producing cylindrical to barrel-shaped to rarely oval cells 20 -5000 um in diameter, generally consistent within species. One of the most significant characteristics in the delimitation of species within the genus Chaetomorpha is the thickness of the filaments, the stiffness of the thallus and the shape of the segments [76]. According to [77], Chaetomorpha aerea has been treated as a synonym of Chaetomorpha linum (O. F. Müller) Kützing by several authors [43] [78] [79]. [43], considered C. aerea and C. linum to be different forms of the same taxon, with the latter name taking precedence. Cytological studies [80] [81], either using morphological attributes [30] [82] [83] [84] or molecular techniques [85], confirmed these as distinct species. The main difference between these taxa is related to the habit. Chaetomorpha linum becomes entangled and is able to form green algal balls [86]. [8], considered that a very distinctive feature in C. linum is its ability to form "entangled masses …". In fact, the Atlantic coast of Costa Rica is listed as one of the localities visited by Taylor. On the other hand, Chaetomorpha aerea grows attached to the substrate using a basal disc and is not a free-form species as C. linum.
On the Atlantic coast of Costa Rica, C. aerea it is easily differentiated from other species of Chaetomorpha [19] because it has a straight basal cell that measures 200 to 1200 μm in length and presents quadratic to cylindrical cells as well as medium apical cells measuring 80 to 350 mm in diameter. This is a rare species for the area and has been collected only once. This coincides with the report by [31], who also considered C. aerea as a rare species for the Caribbean.
For Central America it has been reported only in Belize [44]. Nonetheless it was a worldwide distribution [24].  [87]. Initially there were only five species described by Lamouroux in 1809 [87]. Currently, a total of 58 species are recognized for the genus [24]. The external characters of morphology and anatomy, couple to reproductive aspects, are key for the identification of species of Bryopsis [88]. The records for Bryopsis for the Atlantic coast of Costa Rica [19] [20], include Bryopsis pennata Lamoroux and Bryopsis pennata var. leprieurii (Kützing) Collins & Hervey. Bryopsis pennata var. secunda differs from B. pennata because the first one presents mostly unilateral branchlets and the frond tends to be one-sided. On the other hand, Bryopsis pennata var. leprieurii has fewer branchlets, which are short, sometimes unilateral in discontinuous series, and with a naked axis.
However, given the phenotypic plasticity resulting from its changes environmental condition, the character states commonly used for the discrimination of entities at the specific level may be misleading [89]. This plasticity in the expression of characters is reinforced by the influence of environmental factors such as temperature and salinity [90] [91]. Although using characteristics of broad phenotypic plasticity may lead to incorrect identifications [92] [93], but are still the most broadly employed characters because reproductive and cytological descriptors are not enough to identify species within Bryopsis. This is the first report of this variety for Central America. However, Bryopsis pennata var. secunda is widespread and can be found in remote regions even within Sub-Antarctic islands [24].

Discussion
The descriptions for the new records provided in this study contribute to increasing the understanding of floristic diversity of the Caribbean coast of Costa Rica, particularly from its southernmost limit. To date, the number of species described for the country reaches a total of 377, coinciding with the predictions of [19] [20], who expected the diversity of macroalgae to be higher than what they reported. It should be noted that some new occurrences here described correspond to very conspicuous macroalgae of the families Dictyotaceae and Sargassaceae, which make a considerable contribution to biomass [94].
This study confirms that the area ranging from Cahuita to Manzanillo is the most studied so far in the Caribbean coast of Costa Rica. Still, with these new records, the phycological studies and overall records for the Caribbean remain limited. In part, this reflects the lack of sampling in subtidal areas as well as the costs associated with the logistics to work in the Caribbean coast. The area ranging from Puerto Viejo to Punta Castilla remains the most underexplored coastal region in Costa Rica. It is likely that sampling efforts could lead to increasing even more the diversity of macroalgae, and even, other marine species, reported for the Caribbean coast. For now, our new records provide further data to strengthen the baseline of the floristic richness of Costa Rica. As such, this baseline can be used as a guideline for future ecological or economic studies or can act as a tool to inform policy makers in the development of conservation methods.