# Morphological and Molecular Characters Differentiate Common Morphotypes of Atlantic Holopelagic Sargassum

Amy Siuda, Aurélie Blanfuné, Skye Dibner, Marc Verlaque, Charles-François Boudouresque, Solène Connan, Deborah Goodwin, Valérie Stiger-Pouvreau, Frédérique Viard, Florence Rousseau, et al.

► To cite this version:

Amy Siuda, Aurélie Blanfuné, Skye Dibner, Marc Verlaque, Charles-François Boudouresque, et al. Morphological and Molecular Characters Differentiate Common Morphotypes of Atlantic Holopelagic Sargassum. Journal of Phycology, 2024, 4 (2), pp.256-275. 10.3390/phycology4020014. mnhn-04572926

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# Morphological and Molecular Characters Differentiate Common Morphotypes of Atlantic Holopelagic Sargassum

Amy Siuda, Aurélie Blanfuné, Skye Dibner, Marc Verlaque, Charles-François Boudouresque, Solène Connan, Deborah Goodwin, Valérie Stiger-Pouvreau, Frédérique Viard, Florence Rousseau, et al.

<figure><img src="image_1.png" /></figure>

## ► To cite this version:

Amy Siuda, Aurélie Blanfuné, Skye Dibner, Marc Verlaque, Charles-François Boudouresque, et al. Morphological and Molecular Characters Differentiate Common Morphotypes of Atlantic Holopelagic Sargassum. Journal of Phycology, 2024, 4 (2), pp.256-275. 10.3390/phycology4020014. mnhn-04572926

HAL Id: mnhn-04572926

https://mnhn.hal.science/mnhn-04572926v1

Submitted on 16 May 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

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Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_2.png" /></figure>

Article

# Morphological and Molecular Characters Differentiate Common
## Morphotypes of Atlantic Holopelagic *Sargassum*

Amy N. S. Siuda <fnref n="1" />,* ¹D, Aurélie Blanfuné ²D, Skye Dibner <fnref n="1" />, Marc Verlaque <fnref n="2" />, Charles-François Boudouresque <fnref n="2" />, Solène Connan ³D, Deborah S. Goodwin ⁴D, Valérie Stiger-Pouvreau <fnref n="3" />, Frédérique Viard ⁵D, Florence Rousseau ⁶D, Valérie Michotey ²D, Jeffrey M. Schell <fnref n="7" />, Thomas Changeaux <fnref n="2" />, Didier Aurelle <fnref n="2" /> and Thierry Thibaut ²D

<figure><img src="image_3.png" /></figure>

check for
updates

**Citation:** Siuda, A.N.S.; Blanfuné, A.; Dibner, S.; Verlaque, M.; Boudouresque, C.-F.; Connan, S.; Goodwin, D.S.; Stiger-Pouvreau, V.; Viard, F.; Rousseau, F.; et al.
Morphological and Molecular
Characters Differentiate Common
Morphotypes of Atlantic Holopelagic
*Sargassum*. *Phycology* **2024**, *4*, 256–275.
https://doi.org/10.3390/phycology4020014

Academic Editor: John
James Milledge

Received: 28 February 2024

Revised: 25 April 2024

Accepted: 27 April 2024

Published: 7 May 2024

<figure><img src="image_4.png" /></figure>

**Copyright:** © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).

**Abstract:** Since 2011, massive new strandings of holopelagic *Sargassum* have been reported on the coasts of the Caribbean, northern Brazil, Guiana, and West Africa, causing severe economic and ecological damage. Three common morphotypes (*S. fluitans* III, *S. natans* I, and *S. natans* VIII) were identified as responsible for these catastrophic events, with dominance shifts between them over time. However, the taxonomic status of these holopelagic *Sargassum* morphotypes remains unclear. Using an integrative taxonomy framework, combining a morphological study and molecular analyses, this study aimed to clarify their taxonomic status. Morphological analyses of 54 characters revealed no intermediate form between the three morphotypes, with the overall shape, nature of the axis, and size and shape of blades and vesicles being the most discriminating. An analysis of mitochondrial (IGS, *cox2*, *cox3*, *mt16S rRNA*, and *nad6*) and plastid (*rbcL*) markers confirmed the genetic divergence among the three morphotypes, with a lower level of divergence between the two *S. natans* morphotypes. Without additional molecular characterization, these morphotypes cannot be classified as three distinct species. However, due to their distinct morphological characteristics and sympatry within drifting aggregations, a revision of holopelagic species names is proposed, with *Sargassum fluitans* var. *fluitans* (for *S. fluitans* III), *Sargassum natans* var. *natans* (for *S. natans* I), and *S. natans* var. *wingei* (for *S. natans* VIII). This revision provides necessary clarity on the species involved in inundations of the tropical Atlantic.

**Keywords:** holopelagic *Sargassum*; North Atlantic Ocean; phylogeny; *Sargassum natans* var. *wingei*

**1. Introduction**

Bloom-forming macroalgae are of global concern, causing ecological, economic, and health issues [1–5]. Among the most widely proliferating seaweeds, several species of the genus *Sargassum* C. Agardh (Phaeophyceae, Fucales) are threatening coastal ecosystems in temperate [6–8] and tropical [9–14] areas. Most *Sargassum* species are benthic, colonizing

<footnote n="1">¹ Eckerd College, Marine Science Discipline, 4200 54th Ave. S, St. Petersburg, FL 33711, USA;
skyedibner@gmail.com</footnote>

<footnote n="2">² Aix Marseille University, CNRS, IRD, MIO (Mediterranean Institute of Oceanography), UM 110, 13288 Marseille, France; aurelie.blanfune-thibaut@mio.osupytheas.fr (A.B.); marclouis.verlaque@gmail.com (M.V.); charles-francois.boudouresque@mio.osupytheas.fr (C.-F.B.); valerie.michotey@univ-amu.fr (V.M.); thomas.changeux@ird.fr (T.C.); didier.aurelle@mio.osupytheas.fr (D.A.); thierry.thibaut@univ-amu.fr (T.T.)</footnote>

<footnote n="3">³ Univ Brest, CNRS, IRD, Ifremer, LEMAR, IUEM, 29280 Plouzané, France; solene.connan@univ-brest.fr (S.C.); valerie.stiger@univ-brest.fr (V.S.-P.)</footnote>

<footnote n="4">⁴ Independent Researcher, Bozeman, MT 59718, USA; goodwinds@gmail.com</footnote>

<footnote n="5">5 ISEM, University of Montpellier, CNRS, EPHE, IRD, 34095 Montpellier, France;
frederique.viard@umontpellier.fr</footnote>

<footnote n="6">⁶ Institut de Systématique, Evolution, Biodiversité (ISYEB), Sorbonne Université, Muséum national d'Histoire naturelle (MNHN), CNRS, EPHE, Université des Antilles, CP 39, 57 Rue Cuvier, 75005 Paris, France;
florence.rousseau@mnhn.fr</footnote>

<footnote n="7">⁷ Sea Education Association, P.O. Box 6, Woods Hole, MA 02543, USA; jschell@sea.edu</footnote>

<footnote>* Correspondence: siudaan@eckerd.edu; Tel.: +1-727-864-7726</footnote>
Article

# Morphological and Molecular Characters Differentiate Common
## Morphotypes of Atlantic Holopelagic *Sargassum*

Amy N. S. Siuda <fnref n="1" />,* ¹D, Aurélie Blanfuné ²D, Skye Dibner <fnref n="1" />, Marc Verlaque <fnref n="2" />, Charles-François Boudouresque <fnref n="2" />, Solène Connan ³D, Deborah S. Goodwin ⁴D, Valérie Stiger-Pouvreau <fnref n="3" />, Frédérique Viard ⁵D, Florence Rousseau ⁶D, Valérie Michotey ²D, Jeffrey M. Schell <fnref n="7" />, Thomas Changeaux <fnref n="2" />, Didier Aurelle <fnref n="2" /> and Thierry Thibaut ²D

<figure><img src="image_3.png" /></figure>

check for
updates

**Citation:** Siuda, A.N.S.; Blanfuné, A.; Dibner, S.; Verlaque, M.; Boudouresque, C.-F.; Connan, S.; Goodwin, D.S.; Stiger-Pouvreau, V.; Viard, F.; Rousseau, F.; et al.
Morphological and Molecular
Characters Differentiate Common
Morphotypes of Atlantic Holopelagic
*Sargassum*. *Phycology* **2024**, *4*, 256-275.
https://doi.org/10.3390/phycology4020014

Academic Editor: John
James Milledge

Received: 28 February 2024
Revised: 25 April 2024
Accepted: 27 April 2024
Published: 7 May 2024

<figure><img src="image_4.png" /></figure>

**Copyright:** © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).

**Abstract:** Since 2011, massive new strandings of holopelagic *Sargassum* have been reported on the coasts of the Caribbean, northern Brazil, Guiana, and West Africa, causing severe economic and ecological damage. Three common morphotypes (*S. fluitans* III, *S. natans* I, and *S. natans* VIII) were identified as responsible for these catastrophic events, with dominance shifts between them over time. However, the taxonomic status of these holopelagic *Sargassum* morphotypes remains unclear. Using an integrative taxonomy framework, combining a morphological study and molecular analyses, this study aimed to clarify their taxonomic status. Morphological analyses of 54 characters revealed no intermediate form between the three morphotypes, with the overall shape, nature of the axis, and size and shape of blades and vesicles being the most discriminating. An analysis of mitochondrial (IGS, *cox2*, *cox3*, *mt16S rRNA*, and *nad6*) and plastid (*rbcL*) markers confirmed the genetic divergence among the three morphotypes, with a lower level of divergence between the two *S. natans* morphotypes. Without additional molecular characterization, these morphotypes cannot be classified as three distinct species. However, due to their distinct morphological characteristics and sympatry within drifting aggregations, a revision of holopelagic species names is proposed, with *Sargassum fluitans* var. *fluitans* (for *S. fluitans* III), *Sargassum natans* var. *natans* (for *S. natans* I), and *S. natans* var. *wingei* (for *S. natans* VIII). This revision provides necessary clarity on the species involved in inundations of the tropical Atlantic.

**Keywords:** holopelagic *Sargassum*; North Atlantic Ocean; phylogeny; *Sargassum natans* var. *wingei*

1. Introduction

Bloom-forming macroalgae are of global concern, causing ecological, economic, and health issues [1–5]. Among the most widely proliferating seaweeds, several species of the genus *Sargassum* C. Agardh (Phaeophyceae, Fucales) are threatening coastal ecosystems in temperate [6–8] and tropical [9–14] areas. Most *Sargassum* species are benthic, colonizing

<footnote n="1">¹ Eckerd College, Marine Science Discipline, 4200 54th Ave. S, St. Petersburg, FL 33711, USA;
skyedibner@gmail.com</footnote>

<footnote n="2">² Aix Marseille University, CNRS, IRD, MIO (Mediterranean Institute of Oceanography), UM 110, 13288 Marseille, France; aurelie.blanfune-thibaut@mio.osupytheas.fr (A.B.); marclouis.verlaque@gmail.com (M.V.); charles-francois.boudouresque@mio.osupytheas.fr (C.-F.B.); valerie.michotey@univ-amu.fr (V.M.); thomas.changeux@ird.fr (T.C.); didier.aurelle@mio.osupytheas.fr (D.A.); thierry.thibaut@univ-amu.fr (T.T.)</footnote>

<footnote n="3">³ Univ Brest, CNRS, IRD, Ifremer, LEMAR, IUEM, 29280 Plouzané, France; solene.connan@univ-brest.fr (S.C.); valerie.stiger@univ-brest.fr (V.S.-P.)</footnote>

<footnote n="4">⁴ Independent Researcher, Bozeman, MT 59718, USA; goodwinds@gmail.com</footnote>

<footnote n="5">5 ISEM, University of Montpellier, CNRS, EPHE, IRD, 34095 Montpellier, France;
frederique.viard@umontpellier.fr</footnote>

<footnote n="6">⁶ Institut de Systématique, Evolution, Biodiversité (ISYEB), Sorbonne Université, Muséum national d'Histoire naturelle (MNHN), CNRS, EPHE, Université des Antilles, CP 39, 57 Rue Cuvier, 75005 Paris, France;
florence.rousseau@mnhn.fr</footnote>

<footnote n="7">⁷ Sea Education Association, P.O. Box 6, Woods Hole, MA 02543, USA; jschell@sea.edu</footnote>

<footnote>* Correspondence: siudaan@eckerd.edu; Tel.: +1-727-864-7726</footnote>
rocky reefs, pebbles, detritic sediments, or coral reefs from the sea surface to several dozen
meters in depth [15–17]. In the Atlantic Ocean, however, two holopelagic species have
been characterized: *Sargassum natans* (Linnaeus) Gaillon and *Sargassum fluitans* (Børgesen)
Børgesen [18,19]. Winge [20] described several morphotypes of these species, and their
delineation was refined by Parr [21], although still absent from current taxonomic keys.

Holopelagic *Sargassum* species, historically most abundant in the Sargasso Sea and open Gulf of Mexico [21,22], float and grow at the sea surface during their entire lifetime, as they have no benthic stage. Individual thalli decay and sink to depth [23] or can wash ashore, removing them from the pelagic system. At the sea surface, *Sargassum* thalli tend to aggregate and form mats of different structure and density [24], which host a diverse community of marine organisms [25–28]. These drifting aggregations can travel long distances under the action of winds, waves, and currents [22,24,29]. Since 2011, unprecedented massive strandings of holopelagic *Sargassum* have been reported on the coasts of the eastern and western Caribbean, northern Brazil, Guiana, and West Africa (reviewed in Fidai et al. [30]). These recurrent strandings are associated with extensive *Sargassum* aggregations in the tropical North Atlantic, as reported by satellite imagery [31], hydrographic modeling [32], and field campaigns [24,33]. Satellite images and direct observations show that the Great Atlantic *Sargassum* Belt (GASB) phenomenon has been annually variable in magnitude [31]. Thus, the absence of reliable growth and dispersion models for holopelagic *Sargassum* complicates bloom forecasting efforts [34–37] and the establishment of suitable management procedures.

Species identification adds another level of complexity to understanding the origins and composition of these recent massive standings. The growth response [38–43] and morphology-induced windage [44,45] indeed differ between *Sargassum* morphotypes. In addition, in the aggregations, the most abundant morphotype has shifted across annual blooms. Schell et al. [33] reported, in the North Atlantic Ocean between 10 and 30° N, three dominant morphotypes described by Parr [21], namely, *Sargassum natans* VIII, *Sargassum natans* I, and *Sargassum fluitans* III, with *S. natans* VIII noted as the predominant form inundating eastern Caribbean coasts. Later, *S. fluitans* III dominated beached *Sargassum* in Mexico during 2016 and *S. natans* I contributed substantially to the community composition in the same location in 2018 [46]. On a finer temporal scale, the morphotype composition of beached holopelagic *Sargassum* in Barbados switched from *S. fluitans* III dominating during spring/summer 2021 to *S. natans* VIII-dominated during autumn 2021/winter 2022 [27].

Integrative taxonomy [47], combining a morphological study with molecular analyses, can clarify whether the different holopelagic *Sargassum* morphotypes observed in recent years correspond to different species or to intra-specific variants. As Winge [20] and Parr's [21] morphotype descriptions did not follow the Botanical Code (International Code of Nomenclature for algae, fungi, and plants; [48]) for species description and the subsequent discontinuity in classification based on inconsistent character selection [49], a comprehensive morphological description of each common morphotype is warranted. Moreover, molecular analysis challenged traditional taxonomic divisions within the genus and questioned the use of some morphological or chemical characters for species delineation and taxonomy [50,51]. Even the monophyly of the *Sargassum* genus has been disputed [52].

Early sequencing efforts revealed no divergence between holopelagic species nor be-
tween holopelagic and some benthic species [53–56]. Such results may have arisen from the
selection of markers that, while standard for macroalgal studies, did not capture the regions
where polymorphisms among holopelagic *Sargassum* were located [53–56]. Moreover, some
included the incorrect identification of the specimens sequenced [54,56]. Subsequent com-
parisons of full-length mitogenomes, with a documented correct morphotype identification,
indicated a closer proximity between *S. natans* I and *S. natans* VIII when compared to *S.
fluitans* III; the divergence levels were around 0.02% at the mitogenome level between *S.
natans* I and *S. natans* VIII and 0.3% between *S. natans* I/VIII and *S. fluitans* III [57]. In a more
extended temporal and geographic analysis of holopelagic *Sargassum*, targeting multiple
mitochondrial gene segments each with multiple polymorphisms, genetic distances among
rocky reefs, pebbles, detritic sediments, or coral reefs from the sea surface to several dozen meters in depth [15–17]. In the Atlantic Ocean, however, two holopelagic species have been characterized: *Sargassum natans* (Linnaeus) Gaillon and *Sargassum fluitans* (Børgesen) Børgesen [18,19]. Winge [20] described several morphotypes of these species, and their delineation was refined by Parr [21], although still absent from current taxonomic keys.

Holopelagic *Sargassum* species, historically most abundant in the Sargasso Sea and open Gulf of Mexico [21,22], float and grow at the sea surface during their entire lifetime, as they have no benthic stage. Individual thalli decay and sink to depth [23] or can wash ashore, removing them from the pelagic system. At the sea surface, *Sargassum* thalli tend to aggregate and form mats of different structure and density [24], which host a diverse community of marine organisms [25–28]. These drifting aggregations can travel long distances under the action of winds, waves, and currents [22,24,29]. Since 2011, unprecedented massive strandings of holopelagic *Sargassum* have been reported on the coasts of the eastern and western Caribbean, northern Brazil, Guiana, and West Africa (reviewed in Fidai et al. [30]). These recurrent strandings are associated with extensive *Sargassum* aggregations in the tropical North Atlantic, as reported by satellite imagery [31], hydrographic modeling [32], and field campaigns [24,33]. Satellite images and direct observations show that the Great Atlantic *Sargassum* Belt (GASB) phenomenon has been annually variable in magnitude [31]. Thus, the absence of reliable growth and dispersion models for holopelagic *Sargassum* complicates bloom forecasting efforts [34–37] and the establishment of suitable management procedures.

Species identification adds another level of complexity to understanding the origins and composition of these recent massive standings. The growth response [38–43] and morphology-induced windage [44,45] indeed differ between *Sargassum* morphotypes. In addition, in the aggregations, the most abundant morphotype has shifted across annual blooms. Schell et al. [33] reported, in the North Atlantic Ocean between 10 and 30° N, three dominant morphotypes described by Parr [21], namely, *Sargassum natans* VIII, *Sargassum natans* I, and *Sargassum fluitans* III, with *S. natans* VIII noted as the predominant form inundating eastern Caribbean coasts. Later, *S. fluitans* III dominated beached *Sargassum* in Mexico during 2016 and *S. natans* I contributed substantially to the community composition in the same location in 2018 [46]. On a finer temporal scale, the morphotype composition of beached holopelagic *Sargassum* in Barbados switched from *S. fluitans* III dominating during spring/summer 2021 to *S. natans* VIII-dominated during autumn 2021/winter 2022 [27].

Integrative taxonomy [47], combining a morphological study with molecular analyses, can clarify whether the different holopelagic *Sargassum* morphotypes observed in recent years correspond to different species or to intra-specific variants. As Winge [20] and Parr's [21] morphotype descriptions did not follow the Botanical Code (International Code of Nomenclature for algae, fungi, and plants; [48]) for species description and the subsequent discontinuity in classification based on inconsistent character selection [49], a comprehensive morphological description of each common morphotype is warranted. Moreover, molecular analysis challenged traditional taxonomic divisions within the genus and questioned the use of some morphological or chemical characters for species delineation and taxonomy [50,51]. Even the monophyly of the *Sargassum* genus has been disputed [52].

Early sequencing efforts revealed no divergence between holopelagic species nor between holopelagic and some benthic species [53–56]. Such results may have arisen from the selection of markers that, while standard for macroalgal studies, did not capture the regions where polymorphisms among holopelagic *Sargassum* were located [53–56]. Moreover, some included the incorrect identification of the specimens sequenced [54,56]. Subsequent comparisons of full-length mitogenomes, with a documented correct morphotype identification, indicated a closer proximity between *S. natans* I and *S. natans* VIII when compared to *S. fluitans* III; the divergence levels were around 0.02% at the mitogenome level between *S. natans* I and *S. natans* VIII and 0.3% between *S. natans* I/VIII and *S. fluitans* III [57]. In a more extended temporal and geographic analysis of holopelagic *Sargassum*, targeting multiple mitochondrial gene segments each with multiple polymorphisms, genetic distances among
holopelagic morphotypes were in the range of those between pelagic and benthic species and on the same order as divergence among some benthic species [58].

The present study aimed to clarify the taxonomic status of holopelagic *Sargassum* morphotypes presently found in the Atlantic Ocean. A broad suite of morphological characters and multi-gene molecular analyses were conducted across a large sample of individuals representing all three common holopelagic morphotypes. With this deeper understanding of *Sargassum* taxonomy, the scientific record can be rectified and an appropriate level of differentiation can be incorporated into future modeling, forecasting, and ecological studies.

**2. Materials and Methods**

**2.1. Sample Collection**

Holopelagic *Sargassum* specimens were randomly collected during two scientific cruises in the North and tropical Atlantic Ocean in 2017, with the goal of studying the composition and characteristics of *Sargassum* aggregations. The Caribbean expedition (http://dx.doi.org/10.17600/17004300, accessed on 28 March 2024) used the R/V Antea from 19th June to 13th July 2017, and extended from the area of extensive *Sargassum* bloom development off northeast Brazil up to the Sargasso Sea (25° N), with a focus along French Caribbean islands. The Transatlantic expedition (http://dx.doi.org/10.17600/17016900, accessed on 28 March 2024) used the M/Y Yersin from 6th to 24th October 2017, and crossed the Atlantic between 8° and 12° N, from Cape Verde to Martinique (see [24] for details).

Holopelagic *Sargassum* aggregations were randomly sampled at 31 of 49 stations during the two oceanographic cruises (Figure 1). The three morphotypes were simultaneously present at all 31 sampling stations, but proportions differed by site. *Sargassum fluitans* III was dominant nearly everywhere (around 70%), with ~15% each for *S. natans* VIII and *S. natans* I. However, at two stations (18 and 20) in the south Sargasso Sea, *S. natans* VIII dominated (~70%) and *S. natans* I and *S. fluitans* III contributed ~15% each.

<figure><img src="image_5.png" /><figcaption>**Figure 1.** Sampling locations of holopelagic *Sargassum* during the two 2017 expeditions. S: Caribbean expedition; Y: Transatlantic expedition.</figcaption></figure>

At each station, collected *Sargassum* thalli were identified and sorted by morphotype. For each *Sargassum* morphotype, two to three replicate fronds were randomly selected for analysis. A total of 264 individuals of holopelagic *Sargassum* were collected to carry out joint morphological and genetic analyses. All specimens have been kept as vouchers with a corresponding fragment stored in silica gel at room temperature for further molecular analysis. Eight specimens of benthic *Sargassum*, five of *S. hystrix*, two of *S. furcatum*, and one of *S. polyceratium* var. *ovatum* were also collected by scuba-diving and snorkeling in
holopelagic morphotypes were in the range of those between pelagic and benthic species and on the same order as divergence among some benthic species [58].

The present study aimed to clarify the taxonomic status of holopelagic *Sargassum* morphotypes presently found in the Atlantic Ocean. A broad suite of morphological characters and multi-gene molecular analyses were conducted across a large sample of individuals representing all three common holopelagic morphotypes. With this deeper understanding of *Sargassum* taxonomy, the scientific record can be rectified and an appropriate level of differentiation can be incorporated into future modeling, forecasting, and ecological studies.

**2. Materials and Methods**

**2.1. Sample Collection**

Holopelagic *Sargassum* specimens were randomly collected during two scientific cruises in the North and tropical Atlantic Ocean in 2017, with the goal of studying the composition and characteristics of *Sargassum* aggregations. The Caribbean expedition (http://dx.doi.org/10.17600/17004300, accessed on 28 March 2024) used the R/V Antea from 19th June to 13th July 2017, and extended from the area of extensive *Sargassum* bloom development off northeast Brazil up to the Sargasso Sea (25° N), with a focus along French Caribbean islands. The Transatlantic expedition (http://dx.doi.org/10.17600/17016900, accessed on 28 March 2024) used the M/Y Yersin from 6th to 24th October 2017, and crossed the Atlantic between 8° and 12° N, from Cape Verde to Martinique (see [24] for details).

Holopelagic *Sargassum* aggregations were randomly sampled at 31 of 49 stations during the two oceanographic cruises (Figure 1). The three morphotypes were simultaneously present at all 31 sampling stations, but proportions differed by site. *Sargassum fluitans* III was dominant nearly everywhere (around 70%), with ~15% each for *S. natans* VIII and *S. natans* I. However, at two stations (18 and 20) in the south Sargasso Sea, *S. natans* VIII dominated (~70%) and *S. natans* I and *S. fluitans* III contributed ~15% each.

<figure><img src="image_5.png" /><figcaption>**Figure 1.** Sampling locations of holopelagic *Sargassum* during the two 2017 expeditions. S: Caribbean expedition; Y: Transatlantic expedition.</figcaption></figure>

At each station, collected *Sargassum* thalli were identified and sorted by morphotype. For each *Sargassum* morphotype, two to three replicate fronds were randomly selected for analysis. A total of 264 individuals of holopelagic *Sargassum* were collected to carry out joint morphological and genetic analyses. All specimens have been kept as vouchers with a corresponding fragment stored in silica gel at room temperature for further molecular analysis. Eight specimens of benthic *Sargassum*, five of *S. hystrix*, two of *S. furcatum*, and one of *S. polyceratium* var. *ovatum* were also collected by scuba-diving and snorkeling in
the French Caribbean islands of Guadeloupe and Martinique and used as out-groups in phylogenetic analyses. All specimens are kept in the Herbarium of the Mediterranean Institute of Oceanography at Aix-Marseille University, Marseille, France (HCOM). Herbarium abbreviations follow Thiers [59].

## 2.2. Morphological Analyses

Measurements were made on dried specimens for comparison with dried specimens stored in different herbaria housed in HCOM. Fifty-four characters were identified and selected based on analysis of all available descriptions from the literature of *Sargassum* identification keys from the North Atlantic Ocean [49,60–65] (Table S1). These 54 characters were observed and measured on 264 individuals; they correspond to all the specimens collected during the cruises.

Non-Multi-Dimensional Scaling (nMDS) was performed based on the resulting 54 characters (coding 0, 1) and 264 individual matrices (Table S1), using S17 Bray–Curtis similarity distance. PERMANOVA and pair-wise tests were applied to detect significant differences between the three examined morphotypes. P-values were obtained by 999 permutations of residuals under a reduced model [66–68].

## 2.3. DNA Processing and Phylogenetic Analyses

### 2.3.1. Amplification and Sequencing of Mitochondrial and Plastid Loci

Total DNA was extracted with the Qiagen DNeasy Plant Mini Kit or Macherey-Nagel NucleoSpin 96 Plant II kit according to manufacturer's instructions. Before extraction, samples were disrupted with a FastPrep®-24 by 2.381 mm using inox beads (CIMAP). Using 1/50 dilution of DNA solutions, five mitochondrial markers (mt spacer IGS, *cox2*, *cox3*, *mt16S rRNA*, and *nad6*) and two plastid markers (*psbA* and *rbcL*) were amplified. Primer names, sequences, and PCR programs for all loci are presented in Table 1.

<table><caption>**Table 1.** Primer names and sequences, annealing temperature and time, elongation time, expected size of the PCR product, and references for all loci studied in the present study.</caption><thead><tr><th>Locus</th><th>Primer</th><th>Sequence</th><th>Annealing</th><th>Elongation</th><th>Size (bp)</th><th>Reference</th></tr></thead><tbody><tr><td>mt spacer IGS</td><td>tRNALys-F<br>tRNALys-R</td><td>CGTTTGGTGAGAACCTTACC<br>TACCACTGAGTTATTGCTCCC</td><td>48 °C, 1 min</td><td>1 min</td><td>250</td><td>[52]</td></tr><tr><td>cox2</td><td>cox2-370F<br>cox2-776R</td><td>CAAAGATGGATTCGACGGTTGG<br>CCGGTATCAAACTCGCCCTT</td><td>57 °C, 1 min</td><td>1 min</td><td>406</td><td>[57]</td></tr><tr><td>cox3</td><td>cox3-467F<br>cox3-901R</td><td>GGTTCACGACACCCATTT<br>TAGCGTGATGAGCCCATG</td><td>50 °C, 1 min</td><td>1 min</td><td>434</td><td>[57]</td></tr><tr><td>16S rRNA</td><td>Sarg-mt16S-F<br>Sarg-mt16S-R</td><td>GTAGTCGGTTGGGTTAGGCC<br>GTTTGAACCCCCGCCAATTC</td><td>60 °C, 1 min</td><td>1 min</td><td>616</td><td>[58]</td></tr><tr><td>nad6</td><td>Sarg-nad6-F<br>(External)<br>Sarg-nad6-R<br>(External)</td><td>TATGATTCTTGGGGCTGGT<br>GGGATCATTCAAAGCAGAAGA</td><td>56 °C, 1 min</td><td>1 min</td><td>431</td><td>[58]</td></tr><tr><td>psbA</td><td>psbA-F<br>psbA-R1</td><td>ATGACTGCTACTTTAGAAAGACG<br>GCTAAATCTARWGGGAAGTTGTG</td><td>49 °C, 1 min</td><td>1 min 30 s</td><td>970</td><td>[69]</td></tr><tr><td>rbcL</td><td>rbcL-F<br>rbcL-R</td><td>TATGATTGATTT AGTGGTTGG<br>GTTCGTCAC TTAAATCTGGTA</td><td>51 °C, 1 min 30 s</td><td>2 min</td><td>820</td><td>[70]</td></tr></tbody></table>

The final number of sequences obtained for each marker depended on PCR and sequencing success, and on the observed levels of variability between *Sargassum* morphotypes; fewer samples for *psbA* were sequenced, because that marker exhibited the lowest level of variability. The numbers of sequences analyzed per marker and morphotype are presented in the resulting phylogenetic trees and haplotype networks (see Results). The PCR reaction mix for all loci was prepared for a final volume of 20 µL with GoTaq® Flexi Buffer 1X, GoTaq® DNA Polymerase 0.02 U/µL, dNTP 0.2 mM, MgCl₂ 5 mM, forward
the French Caribbean islands of Guadeloupe and Martinique and used as out-groups in phylogenetic analyses. All specimens are kept in the Herbarium of the Mediterranean Institute of Oceanography at Aix-Marseille University, Marseille, France (HCOM). Herbarium abbreviations follow Thiers [59].

## 2.2. Morphological Analyses

Measurements were made on dried specimens for comparison with dried specimens stored in different herbaria housed in HCOM. Fifty-four characters were identified and selected based on analysis of all available descriptions from the literature of *Sargassum* identification keys from the North Atlantic Ocean [49,60–65] (Table S1). These 54 characters were observed and measured on 264 individuals; they correspond to all the specimens collected during the cruises.

Non-Multi-Dimensional Scaling (nMDS) was performed based on the resulting 54 characters (coding 0, 1) and 264 individual matrices (Table S1), using S17 Bray–Curtis similarity distance. PERMANOVA and pair-wise tests were applied to detect significant differences between the three examined morphotypes. P-values were obtained by 999 permutations of residuals under a reduced model [66–68].

## 2.3. DNA Processing and Phylogenetic Analyses

### 2.3.1. Amplification and Sequencing of Mitochondrial and Plastid Loci

Total DNA was extracted with the Qiagen DNeasy Plant Mini Kit or Macherey-Nagel NucleoSpin 96 Plant II kit according to manufacturer's instructions. Before extraction, samples were disrupted with a FastPrep®-24 by 2.381 mm using inox beads (CIMAP). Using 1/50 dilution of DNA solutions, five mitochondrial markers (mt spacer IGS, *cox2*, *cox3*, *mt16S rRNA*, and *nad6*) and two plastid markers (*psbA* and *rbcL*) were amplified. Primer names, sequences, and PCR programs for all loci are presented in Table 1.

<table><caption>**Table 1.** Primer names and sequences, annealing temperature and time, elongation time, expected size of the PCR product, and references for all loci studied in the present study.</caption><thead><tr><th>Locus</th><th>Primer</th><th>Sequence</th><th>Annealing</th><th>Elongation</th><th>Size (bp)</th><th>Reference</th></tr></thead><tbody><tr><td>mt spacer IGS</td><td>tRNALys-F<br>tRNALys-R</td><td>CGTTTGGTGAGAACCTTACC<br>TACCACTGAGTTATTGCTCCC</td><td>48 °C, 1 min</td><td>1 min</td><td>250</td><td>[52]</td></tr><tr><td>cox2</td><td>cox2-370F<br>cox2-776R</td><td>CAAAGATGGATTCGACGGTTGG<br>CCGGTATCAAACTCGCCCTT</td><td>57 °C, 1 min</td><td>1 min</td><td>406</td><td>[57]</td></tr><tr><td>cox3</td><td>cox3-467F<br>cox3-901R</td><td>GGTTCAACGACACCCATTT<br>TAGCGTGATGAGCCCATG</td><td>50 °C, 1 min</td><td>1 min</td><td>434</td><td>[57]</td></tr><tr><td>16S rRNA</td><td>Sarg-mt16S-F<br>Sarg-mt16S-R</td><td>GTAGTCGGTTGGGTTAGGCC<br>GTTTGAACCCCCGCCAATTC</td><td>60 °C, 1 min</td><td>1 min</td><td>616</td><td>[58]</td></tr><tr><td>nad6</td><td>Sarg-nad6-F<br>(External)<br>Sarg-nad6-R<br>(External)</td><td>TATGATTCTTGGGGCTGGT<br>GGGATCATTCAAAGCAGAAGA</td><td>56 °C, 1 min</td><td>1 min</td><td>431</td><td>[58]</td></tr><tr><td>psbA</td><td>psbA-F<br>psbA-R1</td><td>ATGACTGCTACTTTAGAAAGACG<br>GCTAAATCTARWGGGAAGTTGTG</td><td>49 °C, 1 min</td><td>1 min 30 s</td><td>970</td><td>[69]</td></tr><tr><td>rbcL</td><td>rbcL-F<br>rbcL-R</td><td>TATGATTGATTT AGTGGTTGG<br>GTTCGTCAC TTAAATCTGGTA</td><td>51 °C, 1 min 30 s</td><td>2 min</td><td>820</td><td>[70]</td></tr></tbody></table>

The final number of sequences obtained for each marker depended on PCR and sequencing success, and on the observed levels of variability between *Sargassum* morphotypes; fewer samples for *psbA* were sequenced, because that marker exhibited the lowest level of variability. The numbers of sequences analyzed per marker and morphotype are presented in the resulting phylogenetic trees and haplotype networks (see Results). The PCR reaction mix for all loci was prepared for a final volume of 20 µL with GoTaq® Flexi Buffer 1X, GoTaq® DNA Polymerase 0.02 U/µL, dNTP 0.2 mM, MgCl₂ 5 mM, forward
and reverse primers 0.5 µM each, BSA 0.8 mg/mL, and 2 µL of diluted DNA. All PCR programs included 3 min at 94 °C, 35 cycles with [1 min at 94 °C, 1 min or 1 min 30 s at annealing temperature (Table 1), and 1 to 2 min at 72 °C], and 5 min at 72 °C. The PCR products were sent for Sanger sequencing at Eurofins Genomics. Each unique sequence (i.e., haplotype) obtained in this study is available in GenBank under accession numbers MT422788 to MT422805, and the final sequence alignments have been submitted in Zenodo.

### 2.3.2. Analysis of Sequence Data

Sequences were visualized and aligned with UGENE v 1.31.1 [71]. For each marker, all *Sargassum* sequences available were retrieved from Genbank and aligned with the new sequences obtained here. Sequence alignments were performed with MUSCLE [72] in UGENE. Markers were first analyzed separately, then some were concatenated for tree or network construction. Concatenation was not always possible because of the very unbalanced distribution of *Sargassum* species across markers in Genbank.

For phylogenetic analyses, two datasets were used: a *psbA* alignment allowing a comparison with distant Phaeophyceae taxa, and a *rbcL-cox3* concatenated alignment for the *Sargassum* genus. Other markers were not used because of the lack of corresponding sequences in Genbank or difficulty aligning evolutionary distant taxa (as in the case of mitochondrial IGS). A Maximum Likelihood (ML) phylogenetic reconstruction was performed with IQ-TREE 1.6.8 [73] and a Bayesian Inference (BI) with MrBayes 3.2 [74,75]. With IQ-TREE, 1000 ultrafast bootstraps were used, along with the bnni option to reduce risk of overestimating branch support with ultrafast bootstrap [76]. In the case of combined datasets, the ModelFinder option of IQ-TREE was used to find the best partition scheme [77]. For BI, a sample frequency of 100 over 10⁶ generations (burnin 250,000) was used for *rbcL-cox3* and 2·10⁶ generations (burnin 10⁶) was used for *psbA*. Convergence of the MCMC analysis was assumed when the average standard of split frequency was below 0.01, when the log probability of the data did not show any trend, and when the potential scale reduction factor was close to 1.0. Trees were visualized with FigTree 1.4.4 [78]. For the analyses based on *psbA* sequences, *Cutleria* and *Bachelotia* sequences were used as outgroups [79]. For *rbcL-cox3*, according to the present results and to Mattio and Payri [51], the tree was rooted with the clade containing two species, *Sargassum ringgoldianum* Harvey and *Sargassum thunbergii* (Mertens ex Roth) Kuntze, belonging to the subgenus *Bactrophycus*. Because of different representation in Genbank, the *psbA* and *rbcL-cox3* dataset did not lead to the same species lists; previous *S. fluitans* and *S. natans* *rbcL* sequences were not included, as available sequences for these taxa were only partially aligned with the ones obtained in this present study.

As a complementary approach, haplotype networks were built with the TCS 1.21 software [80], in order to study relationships among the most closely related sequences, to include indels in the analysis, and to visualize the frequency of the different haplotypes. Indels longer than one base pair were transformed into a single base-pair indel before analysis. Networks were built independently for five datasets: mt spacer IGS, a concatenation of partial *cox2* and *cox3*, *nad6*, and *16S rRNA* mitochondrial sequences, as well as part of the plastidial *rbcL*.

## 3. Results

### 3.1. *Morphological Analyses*

According to the morphological analysis of the 54 characters (Table S1), all individuals of *Sargassum* examined during the oceanographic cruises unambiguously belonged to one of three dominant morphotypes described by Parr [21], namely, *Sargassum fluitans* III, *Sargassum natans* I, and *Sargassum natans* VIII. Importantly, no intermediate morphology was observed. Analysis of all collected individuals (n = 264) based on all characters showed three distinct, clearly separated clusters, matching with the three morphotypes (Figure 2). In addition, these clusters differed significantly, as a whole and by pairs (Table 2).
and reverse primers 0.5 µM each, BSA 0.8 mg/mL, and 2 µL of diluted DNA. All PCR programs included 3 min at 94 °C, 35 cycles with [1 min at 94 °C, 1 min or 1 min 30 s at annealing temperature (Table 1), and 1 to 2 min at 72 °C], and 5 min at 72 °C. The PCR products were sent for Sanger sequencing at Eurofins Genomics. Each unique sequence (i.e., haplotype) obtained in this study is available in GenBank under accession numbers MT422788 to MT422805, and the final sequence alignments have been submitted in Zenodo.

### 2.3.2. Analysis of Sequence Data

Sequences were visualized and aligned with UGENE v 1.31.1 [71]. For each marker, all *Sargassum* sequences available were retrieved from Genbank and aligned with the new sequences obtained here. Sequence alignments were performed with MUSCLE [72] in UGENE. Markers were first analyzed separately, then some were concatenated for tree or network construction. Concatenation was not always possible because of the very unbalanced distribution of *Sargassum* species across markers in Genbank.

For phylogenetic analyses, two datasets were used: a *psbA* alignment allowing a comparison with distant Phaeophyceae taxa, and a *rbcL-cox3* concatenated alignment for the *Sargassum* genus. Other markers were not used because of the lack of corresponding sequences in Genbank or difficulty aligning evolutionary distant taxa (as in the case of mitochondrial IGS). A Maximum Likelihood (ML) phylogenetic reconstruction was performed with IQ-TREE 1.6.8 [73] and a Bayesian Inference (BI) with MrBayes 3.2 [74,75]. With IQ-TREE, 1000 ultrafast bootstraps were used, along with the bnni option to reduce risk of overestimating branch support with ultrafast bootstrap [76]. In the case of combined datasets, the ModelFinder option of IQ-TREE was used to find the best partition scheme [77]. For BI, a sample frequency of 100 over 10⁶ generations (burnin 250,000) was used for *rbcL-cox3* and 2·10⁶ generations (burnin 10⁶) was used for *psbA*. Convergence of the MCMC analysis was assumed when the average standard of split frequency was below 0.01, when the log probability of the data did not show any trend, and when the potential scale reduction factor was close to 1.0. Trees were visualized with FigTree 1.4.4 [78]. For the analyses based on *psbA* sequences, *Cutleria* and *Bachelotia* sequences were used as outgroups [79]. For *rbcL-cox3*, according to the present results and to Mattio and Payri [51], the tree was rooted with the clade containing two species, *Sargassum ringgoldianum* Harvey and *Sargassum thunbergii* (Mertens ex Roth) Kuntze, belonging to the subgenus *Bactrophycus*. Because of different representation in Genbank, the *psbA* and *rbcL-cox3* dataset did not lead to the same species lists; previous *S. fluitans* and *S. natans* *rbcL* sequences were not included, as available sequences for these taxa were only partially aligned with the ones obtained in this present study.

As a complementary approach, haplotype networks were built with the TCS 1.21 software [80], in order to study relationships among the most closely related sequences, to include indels in the analysis, and to visualize the frequency of the different haplotypes. Indels longer than one base pair were transformed into a single base-pair indel before analysis. Networks were built independently for five datasets: mt spacer IGS, a concatenation of partial *cox2* and *cox3*, *nad6*, and *16S rRNA* mitochondrial sequences, as well as part of the plastidial *rbcL*.

## 3. Results

### 3.1. *Morphological Analyses*

According to the morphological analysis of the 54 characters (Table S1), all individuals of *Sargassum* examined during the oceanographic cruises unambiguously belonged to one of three dominant morphotypes described by Parr [21], namely, *Sargassum fluitans* III, *Sargassum natans* I, and *Sargassum natans* VIII. Importantly, no intermediate morphology was observed. Analysis of all collected individuals (n = 264) based on all characters showed three distinct, clearly separated clusters, matching with the three morphotypes (Figure 2). In addition, these clusters differed significantly, as a whole and by pairs (Table 2).
<figure><img src="image_6.png" /><figcaption>**Figure 2.** Two-dimensional nMDS ordination plot using S17 Bray-Curtis similarity based on 54 discriminant morphological characters of 264 holopelagic specimens of *Sargassum* collected during the two 2017 campaigns.</figcaption></figure>

<table><caption>**Table 2.** Permutation multivariate analysis of variance (PERMANOVA) and pair-wise test, based on the morphological characters of holopelagic specimens of *Sargassum* collected during the two 2017 campaigns (after a square root transformation of the data, and using S17 Bray-Curtis similarity).</caption><thead><tr><th>Source</th><th>df</th><th>SS</th><th>MS</th><th>Pseudo-F</th><th>P.perm)</th><th>Unique perms</th></tr></thead><tbody><tr><td>Morphotype</td><td>2</td><td>77,756</td><td>38,878</td><td>175.73</td><td>0.001</td><td>998</td></tr><tr><td>Residual</td><td>252</td><td>55,752</td><td>221.24</td><td></td><td></td><td></td></tr><tr><td>Total</td><td>254</td><td>1.3351 × 10<sup>5</sup></td><td></td><td></td><td></td><td></td></tr><tr><th>Groups</th><th>t</th><th>P.perm)</th><th>Unique perms</th><th>P(MC)</th><th></th><th></th></tr><tr><td><i>S. natans</i> VIII, <i>S. natans</i> I</td><td>14.31</td><td>0.001</td><td>999</td><td>0.001</td><td></td><td></td></tr><tr><td><i>S. natans</i> VIII, <i>S. fluitans</i> III</td><td>10.59</td><td>0.001</td><td>999</td><td>0.001</td><td></td><td></td></tr><tr><td><i>S. natans</i> I, <i>S. fluitans</i> III</td><td>14.93</td><td>0.001</td><td>999</td><td>0.001</td><td></td><td></td></tr></tbody></table>

Among the 54 characters analyzed, several were particularly discriminating: the overall shape, the nature of the axis (thorny or smooth), the blades (size and shape), and the vesicles (air bladders; size, presence or absence of apical spine-like appendage; length of pedicel) (Figures 3 and 4). Briefly, *Sargassum fluitans* III was characterized by (1) a short to loose, globular or cylindrical, open, and stringy shape, (2) a thorny axis, (3) blades that are lanceolate with an asymmetrical base, and (4) short pedicellate, oblong vesicles without an apical spine-like appendage but frequently with a foliaceous margin. *Sargassum natans* I was characterized by (1) a mostly cylindrical, sometimes pyramidal shape, (2) a smooth axis, (3) linear blades that are narrow with a symmetrical base, and (4) vesicles, frequently with a long pedicel, a foliaceous margin, and an apical spine-like or reduced foliaceous appendage. *S. natans* VIII was well-characterized by (1) its measurements larger than those of the two other morphotypes and a mostly pyramidal shape, (2) a smooth axis, (3) lanceolate and broad blades with a symmetrical base, and (4) vesicles that are shortly pedicellate, usually without an apical spine-like appendage (Figures 3 and 4; see key for identification).
<figure><img src="image_6.png" /><figcaption>**Figure 2.** Two-dimensional nMDS ordination plot using S17 Bray–Curtis similarity based on 54 discriminant morphological characters of 264 holopelagic specimens of *Sargassum* collected during the two 2017 campaigns.</figcaption></figure>

<table><caption>**Table 2.** Permutation multivariate analysis of variance (PERMANOVA) and pair-wise test, based on the morphological characters of holopelagic specimens of *Sargassum* collected during the two 2017 campaigns (after a square root transformation of the data, and using S17 Bray–Curtis similarity).</caption><thead><tr><th>Source</th><th>df</th><th>SS</th><th>MS</th><th>Pseudo-F</th><th>P(perm)</th><th>Unique perms</th></tr></thead><tbody><tr><td>Morphotype</td><td>2</td><td>77,756</td><td>38,878</td><td>175.73</td><td>0.001</td><td>998</td></tr><tr><td>Residual</td><td>252</td><td>55,752</td><td>221.24</td><td></td><td></td><td></td></tr><tr><td>Total</td><td>254</td><td>1.3351 × 10<sup>5</sup></td><td></td><td></td><td></td><td></td></tr><tr><th>Groups</th><th>t</th><th>P(perm)</th><th>Unique perms</th><th>P(MC)</th><th></th><th></th></tr><tr><td><i>S. natans</i> VIII, <i>S. natans</i> I</td><td>14.31</td><td>0.001</td><td>999</td><td>0.001</td><td></td><td></td></tr><tr><td><i>S. natans</i> VIII, <i>S. fluitans</i> III</td><td>10.59</td><td>0.001</td><td>999</td><td>0.001</td><td></td><td></td></tr><tr><td><i>S. natans</i> I, <i>S. fluitans</i> III</td><td>14.93</td><td>0.001</td><td>999</td><td>0.001</td><td></td><td></td></tr></tbody></table>

Among the 54 characters analyzed, several were particularly discriminating: the overall shape, the nature of the axis (thorny or smooth), the blades (size and shape), and the vesicles (air bladders; size, presence or absence of apical spine-like appendage; length of pedicel) (Figures 3 and 4). Briefly, *Sargassum fluitans* III was characterized by (1) a short to loose, globular or cylindrical, open, and stringy shape, (2) a thorny axis, (3) blades that are lanceolate with an asymmetrical base, and (4) short pedicellate, oblong vesicles without an apical spine-like appendage but frequently with a foliaceous margin. *Sargassum natans* I was characterized by (1) a mostly cylindrical, sometimes pyramidal shape, (2) a smooth axis, (3) linear blades that are narrow with a symmetrical base, and (4) vesicles, frequently with a long pedicel, a foliaceous margin, and an apical spine-like or reduced foliaceous appendage. *S. natans* VIII was well-characterized by (1) its measurements larger than those of the two other morphotypes and a mostly pyramidal shape, (2) a smooth axis, (3) lanceolate and broad blades with a symmetrical base, and (4) vesicles that are shortly pedicellate, usually without an apical spine-like appendage (Figures 3 and 4; see key for identification).
<figure><img src="image_7.png" /><figcaption>**Figure 3.** Dried specimens and detail of holopelagic *Sargassum* morphotypes collected at station S10. (A) *Sargassum fluitans* III (S. fluitans var. fluitans, ref. HCOM S10-S3-1). (B) *Sargassum natans* I (S. natans ref. HCOM S10-S2-2). (C) *Sargassum natans* VIII (S. natans var. *wingei* nov. var., holotype, ref. HCOM S10-S1-3).</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>**Figure 4.** Morphological comparison between *S. fluitans* III, *S. natans* I, and *S. natans* VIII morphotypes of holopelagic *Sargassum* collected during the two expeditions of 2017. (A) Habitus, axis, and vesicles of *S. fluitans* III morphotype (*S. fluitans* var. *fluitans*). Scale bars = 1 cm. (B) Habitus, axis, and vesicles of *S. natans* I morphotype (*S. natans* var. *natans*). (C) Habitus, axis, and vesicles of *S. natans* VIII morphotype (*S. natans* var. *wingei* nov. var.). Each vesicle is attached to the axis by a pedicel. Vesicles have an apical spine-like appendage only for *Sargassum natans* var. *natans*.</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>**Figure 3.** Dried specimens and detail of holopelagic *Sargassum* morphotypes collected at station S10. (A) *Sargassum fluitans* III (S. fluitans var. fluitans, ref. HCOM S10-S3-1). (B) *Sargassum natans* I (S. natans ref. HCOM S10-S2-2). (C) *Sargassum natans* VIII (S. natans var. *wingei* nov. var., holotype, ref. HCOM S10-S1-3).</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>**Figure 4.** Morphological comparison between *S. fluitans* III, *S. natans* I, and *S. natans* VIII morphotypes of holopelagic *Sargassum* collected during the two expeditions of 2017. (A) Habitus, axis, and vesicles of *S. fluitans* III morphotype (*S. fluitans* var. *fluitans*). Scale bars = 1 cm. (B) Habitus, axis, and vesicles of *S. natans* I morphotype (*S. natans* var. *natans*). (C) Habitus, axis, and vesicles of *S. natans* VIII morphotype (*S. natans* var. *wingei* nov. var.). Each vesicle is attached to the axis by a pedicel. Vesicles have an apical spine-like appendage only for *Sargassum natans* var. *natans*.</figcaption></figure>
## 3.2. Genetic Analyses

Molecular phylogenetic analyses were conducted for *pbsA* and *rbcL-cox3* concatenated sequences. The phylogenetic tree made with *psbA* did not reveal any variation among the three morphotypes, and the sequences were identical to benthic species such as *Sargassum vulgare* (Figure S2). Thus, the tree did not clarify the phylogenetic position of the holopelagic samples but confirmed that they belong to the *Sargassum* genus. Identical *psbA* sequences were also observed between close species within the Sargassaceae family, like between *Polycladia indica* (Schiffner) Draisma, Ballesteros, F. Rousseau & T. Thibaut and *P. heinii* [52] or *Ericaria amentacea* (C.Agardh) Molinari & Guiry and *E. selaginoides* (Hudson) Molinari & Guiry. With the concatenated fragment *rbcL-cox3*, sequences from the holopelagic samples were placed, with good support, in the same monophyletic group as *Sargassum yendoi* Okamura & Yamada, *S. vachellianum* Greville and *S. piluliferum* (Turner) C. Agardh (Figure 5). The separation of holopelagic morphotypes *S. natans* VIII and *S. natans* I was well-supported (bootstraps of 96%). The morphotype *S. fluitans* III appeared in the sister position to this group, though with lower support (76/0.89).

<figure><img src="image_9.png" /><figcaption>**Figure 5.** Phylogenetic tree based on the concatenation of *rbcL* and *cox3* sequences. The topology shown here corresponds to the output of the Maximum Likelihood analysis. Bootstrap values higher than 80%, and posterior probabilities higher than 0.9 are indicated at the left of the corresponding group. Newly generated sequences are in red. For the sequences retrieved from GenBank, the accession number is indicated together with the species name (corrected with current taxonomic status).</figcaption></figure>

The molecular identification of the three holopelagic *Sargassum* morphotypes, among each other and in comparison with benthic species, was dependent upon the marker used for analysis. The new mt spacer IGS for the *S. fluitans* III morphotype was identical to previous sequences from *S. fluitans* III. New mt spacer IGS sequences for the *S. natans* VIII and *S. natans* I morphotypes were identical to each other and to *S. natans* VIII and *S. natans* I sequences from Amaral-Zettler et al. [57]. The TCS network showed a divergence of six mutations between the *S. natans* complex and *S. fluitans* III, including two indels of two base pairs (bps) each (Figure 6). For plastidial *rbcL*, the fragment sequenced did not completely match with the ones previously sequenced for *S. natans* and *S. fluitans*. Therefore, no comparisons could be made beyond this dataset. The morphotypes *S. natans* VIII and *S. natans* I shared the same haplotype with the benthic *Sargassum* analyzed in this study. This haplotype was separated by two substitutions in *rbcL* and a 9 bp deletion in the
## 3.2. Genetic Analyses

Molecular phylogenetic analyses were conducted for *pbsA* and *rbcL-cox3* concatenated sequences. The phylogenetic tree made with *psbA* did not reveal any variation among the three morphotypes, and the sequences were identical to benthic species such as *Sargassum vulgare* (Figure S2). Thus, the tree did not clarify the phylogenetic position of the holopelagic samples but confirmed that they belong to the *Sargassum* genus. Identical *psbA* sequences were also observed between close species within the Sargassaceae family, like between *Polycladia indica* (Schiffner) Draisma, Ballesteros, F. Rousseau & T. Thibaut and *P. heinii* [52] or *Ericaria amentacea* (C.Agardh) Molinari & Guiry and *E. selaginoides* (Hudson) Molinari & Guiry. With the concatenated fragment *rbcL-cox3*, sequences from the holopelagic samples were placed, with good support, in the same monophyletic group as *Sargassum yendoi* Okamura & Yamada, *S. vachellianum* Greville and *S. piluliferum* (Turner) C. Agardh (Figure 5). The separation of holopelagic morphotypes *S. natans* VIII and *S. natans* I was well-supported (bootstraps of 96%). The morphotype *S. fluitans* III appeared in the sister position to this group, though with lower support (76/0.89).

<figure><img src="image_9.png" /><figcaption>**Figure 5.** Phylogenetic tree based on the concatenation of *rbcL* and *cox3* sequences. The topology shown here corresponds to the output of the Maximum Likelihood analysis. Bootstrap values higher than 80%, and posterior probabilities higher than 0.9 are indicated at the left of the corresponding group. Newly generated sequences are in red. For the sequences retrieved from GenBank, the accession number is indicated together with the species name (corrected with current taxonomic status).</figcaption></figure>

The molecular identification of the three holopelagic *Sargassum* morphotypes, among each other and in comparison with benthic species, was dependent upon the marker used for analysis. The new mt spacer IGS for the *S. fluitans* III morphotype was identical to previous sequences from *S. fluitans* III. New mt spacer IGS sequences for the *S. natans* VIII and *S. natans* I morphotypes were identical to each other and to *S. natans* VIII and *S. natans* I sequences from Amaral-Zettler et al. [57]. The TCS network showed a divergence of six mutations between the *S. natans* complex and *S. fluitans* III, including two indels of two base pairs (bps) each (Figure 6). For plastidial *rbcL*, the fragment sequenced did not completely match with the ones previously sequenced for *S. natans* and *S. fluitans*. Therefore, no comparisons could be made beyond this dataset. The morphotypes *S. natans* VIII and *S. natans* I shared the same haplotype with the benthic *Sargassum* analyzed in this study. This haplotype was separated by two substitutions in *rbcL* and a 9 bp deletion in the
spacer (not shown) from the morphotype *S. fluitans* III haplotype (Figure 6). One haplotype of morphotype *S. fluitans* III (station 20) was separated from other morphotype *S. fluitans* III samples by four mutations, and from the other multi-species haplotypes by one mutation and a 6 bp indel. The mitochondrial *16S rRNA*, *cox2-cox3* concatenated, and *nad6* sequences separated the three morphotypes and confirmed matches between the sequences obtained in this study and earlier published sequences for all three holopelagic morphotypes. The *16S rRNA* sequences for *S. fluitans* III differed from the benthic species and from *S. natans* I by a single substitution and from *S. natans* VIII by two substitutions (Figure 6). Two *S. natans* VIII sequences from the current dataset matched the *S. natans* I sequences, and one *S. hystrix* sequence matched the *S. fluitans* III sequences. In the *cox2-cox3* network, haplotypes from morphotypes *S. natans* VIII and *S. natans* I were only separated by one substitution in the *cox3* partial fragment (Figure 6). Haplotypes from morphotypes *S. natans* VIII and *S. fluitans* III were separated by four substitutions, one in *cox3* and three in *cox2*. The *nad6* network was composed of multiple grouped haplotypes representing each holopelagic *Sargassum* morphotype, with one dominant haplotype in each group (Figure 6). Two *S. natans* VIII and one *S. fluitans* III matched the dominant *S. natans* I haplotype, which differed from the dominant *S. fluitans* III haplotype by two nucleotide substitutions and from the dominant *S. natans* VIII by one nucleotide substitution.

<figure><img src="image_10.png" /><figcaption>**Figure 6.** Sequence networks for mt spacer IGS, *rbcL*, *16S rRNA* gene, concatenation of *cox2* and *cox3*, and *nad6*. Sequences of *S. polyceratium*, *S. vachellianum*, *S. spinuligerum*, and *Sargassum* sp., as well as dot-filled wedges correspond to sequences retrieved from GenBank. Number of sequences per haplotype is noted.</figcaption></figure>
spacer (not shown) from the morphotype *S. fluitans* III haplotype (Figure 6). One haplotype of morphotype *S. fluitans* III (station 20) was separated from other morphotype *S. fluitans* III samples by four mutations, and from the other multi-species haplotypes by one mutation and a 6 bp indel. The mitochondrial *16S rRNA*, *cox2-cox3* concatenated, and *nad6* sequences separated the three morphotypes and confirmed matches between the sequences obtained in this study and earlier published sequences for all three holopelagic morphotypes. The *16S rRNA* sequences for *S. fluitans* III differed from the benthic species and from *S. natans* I by a single substitution and from *S. natans* VIII by two substitutions (Figure 6). Two *S. natans* VIII sequences from the current dataset matched the *S. natans* I sequences, and one *S. hystrix* sequence matched the *S. fluitans* III sequences. In the *cox2-cox3* network, haplotypes from morphotypes *S. natans* VIII and *S. natans* I were only separated by one substitution in the *cox3* partial fragment (Figure 6). Haplotypes from morphotypes *S. natans* VIII and *S. fluitans* III were separated by four substitutions, one in *cox3* and three in *cox2*. The *nad6* network was composed of multiple grouped haplotypes representing each holopelagic *Sargassum* morphotype, with one dominant haplotype in each group (Figure 6). Two *S. natans* VIII and one *S. fluitans* III matched the dominant *S. natans* I haplotype, which differed from the dominant *S. fluitans* III haplotype by two nucleotide substitutions and from the dominant *S. natans* VIII by one nucleotide substitution.

<figure><img src="image_10.png" /><figcaption>**Figure 6.** Sequence networks for mt spacer IGS, *rbcL*, *16S rRNA* gene, concatenation of *cox2* and *cox3*, and *nad6*. Sequences of *S. polyceratium*, *S. vachellianum*, *S. spinuligerum*, and *Sargassum* sp., as well as dot-filled wedges correspond to sequences retrieved from GenBank. Number of sequences per haplotype is noted.</figcaption></figure>
## 4. Discussion

Morphology strongly supports the delineation between the three holopelagic *Sargassum* morphotypes (S. fluitans III, S. natans I, and S. natans VIII). No individuals displayed intermediate characters, and individuals defined by their morphotype were clearly separated into groups specific to each (Figure 2). S. fluitans III, described by Parr [21], morphologically corresponds to the description of S. fluitans (Børgesen) Børgesen [18,19]. Consequently, S. fluitans III can be renamed as *Sargassum fluitans* var. *fluitans*. S. fluitans X, the other described S. fluitans morphotype [21], is rare; upon further analysis, it should be assigned the appropriate nomenclature. Moreover, S. natans I and S. natans VIII are syntopic, i.e., living together in the same habitat at each station, which ruled out the hypothesis that the observed morphological differences were related to their habitat differences. These two taxa, namely, S. natans I and S. natans VIII, could be in an early stage of speciation (i.e., the gray zone following de Queiroz's terminology [81]). Thus, we conservatively propose their formal separation into varieties as *Sargassum natans* var. *natans* (S. natans I) and *Sargassum natans* var. *wingei* (S. natans VIII).

Molecular results confirmed the phylogenetic positioning of Atlantic holopelagic *Sargassum* species, in close proximity to each other and among benthic species within a group corresponding to the section *Sargassum* of the subgenus *Sargassum*, as previously suggested (e.g., [51,54]). Nevertheless, within the section *Sargassum*, the low divergence of the sequences used as well as the reduced taxonomic sampling were not sufficient to unambiguously resolve the phylogenetic affinities of holopelagic species and, therefore, do not allow the discussion of the question of a single or multiple evolutionary origin of holopelagic *Sargassum*. The difficulty in resolving the phylogeny of this group could be due to a combination of a relatively recent evolution of the pelagic lifestyle, along with the slow molecular evolution of mitochondrial and plastidial genes considered.

In other *Sargassum* species, mitochondrial *cox3* or plastidial *rbcL* showed intraspecific variation, sometimes useful for phylogeographic studies [50,70,82]. Therefore, the lack of diversity inside holopelagic *Sargassum* species and observed with our marker set may not be a fixed rule. The discrepancy between morphological differentiation and low molecular divergence may be explained by this slow rate of molecular evolution of cytoplasmic genomes in *Sargassum* species. More generally, the observation of morphological differences can usually be explained by the interaction between genetic and environmental factors, but not in this case. There are also cases where species that are very similar genetically may exhibit significant morphological differences due to environmental influences or other factors (e.g., [83,84]), but holopelagic *Sargassum* taxa live in sympatry and are always found mixed together, even if their abundance can vary from year to year and/or place to place [27].

Regarding species/morphotype delineation using molecular markers, the examined genes exhibited only a few mutations separating the three morphotypes, as reported by Amaral-Zettler et al. [57] and Dibner et al. [58] and demonstrated in previous studies on nuclear, mitochondrial, and chloroplastic markers dedicated to the genus *Sargassum* [85-87]. The present study confirmed weak but clear genetic differences (three to six mutations) for the mitochondrial *IGS*, *cox2*, *cox3*, and plastid *rbcL* genes, between morphotypes *S. natans* I and VIII, and *S. fluitans* III [57]. An additional analysis of the mitochondrial 16S rRNA and *nad6* genes also confirmed weak but clear genetic differences between *S. natans* I and *S. natans* VIII [58]. In all cases, genetic data from these 2017 samples were consistent with previous sampling efforts between 2012 and 2016 [57] and between 2015 and 2019 [58], which seems to indicate a relative stability of composition at that scale. The fact that observed genetic differences between *S. natans* I and *S. natans* VIII were low may be the result of the aforementioned low rate of evolution for cytoplasmic genomes. In future studies, other more polymorphic markers may reveal a higher divergence between the two morphotypes.

Within the genus *Sargassum*, similarities in molecular marker sequences between distinct species are frequent. Yoshida et al. [86] demonstrated similarities in the nuclear
**4. Discussion**

Morphology strongly supports the delineation between the three holopelagic *Sargassum* morphotypes (S. fluitans III, S. natans I, and S. natans VIII). No individuals displayed intermediate characters, and individuals defined by their morphotype were clearly separated into groups specific to each (Figure 2). S. fluitans III, described by Parr [21], morphologically corresponds to the description of S. fluitans (Børgesen) Børgesen [18,19]. Consequently, S. fluitans III can be renamed as *Sargassum fluitans* var. *fluitans*. S. fluitans X, the other described S. fluitans morphotype [21], is rare; upon further analysis, it should be assigned the appropriate nomenclature. Moreover, S. natans I and S. natans VIII are syntopic, i.e., living together in the same habitat at each station, which ruled out the hypothesis that the observed morphological differences were related to their habitat differences. These two taxa, namely, S. natans I and S. natans VIII, could be in an early stage of speciation (i.e., the gray zone following de Queiroz's terminology [81]). Thus, we conservatively propose their formal separation into varieties as *Sargassum natans* var. *natans* (S. natans I) and *Sargassum natans* var. *wingei* (S. natans VIII).

Molecular results confirmed the phylogenetic positioning of Atlantic holopelagic *Sargassum* species, in close proximity to each other and among benthic species within a group corresponding to the section *Sargassum* of the subgenus *Sargassum*, as previously suggested (e.g., [51,54]). Nevertheless, within the section *Sargassum*, the low divergence of the sequences used as well as the reduced taxonomic sampling were not sufficient to unambiguously resolve the phylogenetic affinities of holopelagic species and, therefore, do not allow the discussion of the question of a single or multiple evolutionary origin of holopelagic *Sargassum*. The difficulty in resolving the phylogeny of this group could be due to a combination of a relatively recent evolution of the pelagic lifestyle, along with the slow molecular evolution of mitochondrial and plastidial genes considered.

In other *Sargassum* species, mitochondrial *cox3* or plastidial *rbcL* showed intraspecific variation, sometimes useful for phylogeographic studies [50,70,82]. Therefore, the lack of diversity inside holopelagic *Sargassum* species and observed with our marker set may not be a fixed rule. The discrepancy between morphological differentiation and low molecular divergence may be explained by this slow rate of molecular evolution of cytoplasmic genomes in *Sargassum* species. More generally, the observation of morphological differences can usually be explained by the interaction between genetic and environmental factors, but not in this case. There are also cases where species that are very similar genetically may exhibit significant morphological differences due to environmental influences or other factors (e.g., [83,84]), but holopelagic *Sargassum* taxa live in sympatry and are always found mixed together, even if their abundance can vary from year to year and/or place to place [27].

Regarding species/morphotype delineation using molecular markers, the examined genes exhibited only a few mutations separating the three morphotypes, as reported by Amaral-Zettler et al. [57] and Dibner et al. [58] and demonstrated in previous studies on nuclear, mitochondrial, and chloroplastic markers dedicated to the genus *Sargassum* [85-87]. The present study confirmed weak but clear genetic differences (three to six mutations) for the mitochondrial *IGS*, *cox2*, *cox3*, and plastid *rbcL* genes, between morphotypes *S. natans* I and VIII, and *S. fluitans* III [57]. An additional analysis of the mitochondrial 16S rRNA and *nad6* genes also confirmed weak but clear genetic differences between *S. natans* I and *S. natans* VIII [58]. In all cases, genetic data from these 2017 samples were consistent with previous sampling efforts between 2012 and 2016 [57] and between 2015 and 2019 [58], which seems to indicate a relative stability of composition at that scale. The fact that observed genetic differences between *S. natans* I and *S. natans* VIII were low may be the result of the aforementioned low rate of evolution for cytoplasmic genomes. In future studies, other more polymorphic markers may reveal a higher divergence between the two morphotypes.

Within the genus *Sargassum*, similarities in molecular marker sequences between distinct species are frequent. Yoshida et al. [86] demonstrated similarities in the nuclear
ITS2 sequences of four allied *Sargassum* species belonging to the subgenus *Bactrophycus*: *Sargassum boreale*, *S. confusum*, *S. microceratium*, and *S. pallidum*. In that work, *S. boreale* was identified as a new species because of the presence of a large deletion of 28 bp, which was stable in all specimens of *S. boreale* studied [86]. No nucleotide difference was observed in the ITS2 sequences of *Sargassum mcclurei* and *S. quinhonense* belonging to the subgenus *Sargassum* although their morphology is totally different [85]. The separation of the two species was not supported by the ITS2 phylogeny; nevertheless, *S. mcclurei* and *S. quinhonense* are distinct species as no intermediate morphological form has been reported [85]. On another Sargassaceae genus, Rohfritsch et al. [88] demonstrated that a divergence of almost 17 mutations on 2102 concatenated base pairs was enough to confirm that *Turbinaria ornata* and *T. conoides* were separate species.

The proposed revision is particularly critical at this time, as considerable confusion exists regarding the morphological identification of holopelagic *Sargassum* species in the literature. To address and resolve these inconsistencies, a thorough historic review of holopelagic *Sargassum* taxonomy is presented here. Early taxonomic classification of holopelagic *Sargassum* recognized two species, *Sargassum natans* (L.) and *S. hystrix* (Agardh) var. *fluitans*, based on several morphological traits that clearly distinguished one species from the other [18,19]. In addition to differences in the shape of blades and vesicles, *S. hystrix* var. *fluitans* was notable for branches that are "provided with short spinules." Winge [20], based on hundreds of preserved samples collected from numerous cruises across the North Atlantic between 1911-1922, identified "8 different drifting *Sargassum*-species." Parr [21], based on several cruises in the North Atlantic from 1933-1935, advanced Winge's [20] *Sargassum* taxonomy and provided the first dichotomous key for drifting *Sargassum* species, as well as detailing their unique distributional patterns. Notably, the second couplet in Parr's [21] key addresses the nature of *Sargassum* branches as 'thorny' for all *S. fluitans* morphological types (III and X) versus branches as 'smooth' for all *S. natans* morphotypes (I, II, VIII, and IX). *S. natans* morphotypes are further distinguished by the presence (*S. natans* I) or absence (*S. natans* II, VIII, and IX) of an apical spine on the vesicles, and then the size and shape of blades.

Parr [21] is recognized as the definitive source on holopelagic *Sargassum* taxonomy. Indeed, early reviews of Sargasso Sea science always referenced Parr's [21] taxonomic and distribution studies [89-91]. Investigations examining the changing distribution and abundance of holopelagic *Sargassum* [22,92,93] compared their findings to Parr [21]. Investigations of the epibionts [94-97] and motile fauna [98-100] that live on different types of holopelagic *Sargassum* looked to Parr [21] for proper species identification. Studies examining the growth and production of different holopelagic *Sargassum* species [101,102] also reference Parr [21]. Even though these studies were familiar with the true diversity of holopelagic *Sargassum* morphotypes, in practice, *S. natans* I and *S. fluitans* III were consistently the most abundant, and the need to consider rare morphotypes (*S. natans* II, VIII, and IX, and *S. fluitans* X) waned over time. Eventually, Parr's [21] numeric system was dropped and *S. natans* I and *S. fluitans* III were simply referred to as *S. natans* and *S. fluitans*.

The contemporary definitive source for benthic algae taxonomy in the western North Atlantic and Caribbean has been Taylor [49], which naturally includes a thorough account of benthic *Sargassum* species. However, Taylor's [49] approach to the taxonomy of holopelagic *Sargassum* was overly simplified and only recognized two species: *S. natans* and *S. fluitans*, making no mention of the other morphotypes. Critically, Taylor [49] did not distinguish the two *Sargassum* species in his dichotomous key based on the presence or absence of thorns on the branches, the well-established historical precedent, but instead used the blade and vesicle shape. In fact, Taylor [49] describes *S. fluitans* as having a "...stem smooth or very sparingly spinulose," which contradicts the historical descriptions of *S. fluitans* III [18-21], and the accompanying illustration of *S. fluitans* (see Plate 39, Figure 2) shows smooth branches and spherical vesicles that more closely resembles Winge's [20] and Parr's [21] descriptions and illustrations of *S. natans* VIII. A more recent authority on benthic algae in the Caribbean, Wynne's [103] checklist, also adopted Taylor's [49] simplified view of
ITS2 sequences of four allied *Sargassum* species belonging to the subgenus *Bactrophycus*: *Sargassum boreale*, *S. confusum*, *S. microceratium*, and *S. pallidum*. In that work, *S. boreale* was identified as a new species because of the presence of a large deletion of 28 bp, which was stable in all specimens of *S. boreale* studied [86]. No nucleotide difference was observed in the ITS2 sequences of *Sargassum mcclurei* and *S. quinhonense* belonging to the subgenus *Sargassum* although their morphology is totally different [85]. The separation of the two species was not supported by the ITS2 phylogeny; nevertheless, *S. mcclurei* and *S. quinhonense* are distinct species as no intermediate morphological form has been reported [85]. On another Sargassaceae genus, Rohfritsch et al. [88] demonstrated that a divergence of almost 17 mutations on 2102 concatenated base pairs was enough to confirm that *Turbinaria ornata* and *T. conoides* were separate species.

The proposed revision is particularly critical at this time, as considerable confusion exists regarding the morphological identification of holopelagic *Sargassum* species in the literature. To address and resolve these inconsistencies, a thorough historic review of holopelagic *Sargassum* taxonomy is presented here. Early taxonomic classification of holopelagic *Sargassum* recognized two species, *Sargassum natans* (L.) and *S. hystrix* (Agardh) var. *fluitans*, based on several morphological traits that clearly distinguished one species from the other [18,19]. In addition to differences in the shape of blades and vesicles, *S. hystrix* var. *fluitans* was notable for branches that are "provided with short spinules." Winge [20], based on hundreds of preserved samples collected from numerous cruises across the North Atlantic between 1911-1922, identified "8 different drifting *Sargassum*-species." Parr [21], based on several cruises in the North Atlantic from 1933-1935, advanced Winge's [20] *Sargassum* taxonomy and provided the first dichotomous key for drifting *Sargassum* species, as well as detailing their unique distributional patterns. Notably, the second couplet in Parr's [21] key addresses the nature of *Sargassum* branches as 'thorny' for all *S. fluitans* morphological types (III and X) versus branches as 'smooth' for all *S. natans* morphotypes (I, II, VIII, and IX). *S. natans* morphotypes are further distinguished by the presence (*S. natans* I) or absence (*S. natans* II, VIII, and IX) of an apical spine on the vesicles, and then the size and shape of blades.

Parr [21] is recognized as the definitive source on holopelagic *Sargassum* taxonomy. Indeed, early reviews of Sargasso Sea science always referenced Parr's [21] taxonomic and distribution studies [89-91]. Investigations examining the changing distribution and abundance of holopelagic *Sargassum* [22,92,93] compared their findings to Parr [21]. Investigations of the epibionts [94-97] and motile fauna [98-100] that live on different types of holopelagic *Sargassum* looked to Parr [21] for proper species identification. Studies examining the growth and production of different holopelagic *Sargassum* species [101,102] also reference Parr [21]. Even though these studies were familiar with the true diversity of holopelagic *Sargassum* morphotypes, in practice, *S. natans* I and *S. fluitans* III were consistently the most abundant, and the need to consider rare morphotypes (*S. natans* II, VIII, and IX, and *S. fluitans* X) waned over time. Eventually, Parr's [21] numeric system was dropped and *S. natans* I and *S. fluitans* III were simply referred to as *S. natans* and *S. fluitans*.

The contemporary definitive source for benthic algae taxonomy in the western North Atlantic and Caribbean has been Taylor [49], which naturally includes a thorough account of benthic *Sargassum* species. However, Taylor's [49] approach to the taxonomy of holopelagic *Sargassum* was overly simplified and only recognized two species: *S. natans* and *S. fluitans*, making no mention of the other morphotypes. Critically, Taylor [49] did not distinguish the two *Sargassum* species in his dichotomous key based on the presence or absence of thorns on the branches, the well-established historical precedent, but instead used the blade and vesicle shape. In fact, Taylor [49] describes *S. fluitans* as having a "...stem smooth or very sparingly spinulose," which contradicts the historical descriptions of *S. fluitans* III [18-21], and the accompanying illustration of *S. fluitans* (see Plate 39, Figure 2) shows smooth branches and spherical vesicles that more closely resembles Winge's [20] and Parr's [21] descriptions and illustrations of *S. natans* VIII. A more recent authority on benthic algae in the Caribbean, Wynne's [103] checklist, also adopted Taylor's [49] simplified view of
holopelagic *Sargassum* diversity and did not reference Winge [20] or Parr [21]. Schneider and Searles [104] and Littler and Littler [63] also described two species, making no mention of additional holopelagic *Sargassum* morphotypes, and simplified their dichotomous keys by removing any mention of thorns along the branches. Notably, Littler and Littler [63] provide an accurate photograph of *S. fluitans* III with thorns evident on the branches.

When Wynne [103] and subsequent revisions are consulted as the authoritative catalog of currently accepted nomenclature for species in the region and Taylor [49], Schneider and Searles [104], or Littler and Littler [63] are the only source material used for holopelagic *Sargassum* identification, specimens of *S. natans* VIII, with smooth branches and large, spherical vesicles lacking an apical spine-like appendage, can be mistakenly identified as *S. fluitans* III. de Szechy et al. [105] collected drift *Sargassum* off the coast of Brazil during the first GASB bloom and misidentified *S. fluitans* as *S. natans* (see Table 1, Figures 4 and 5). The extensive morphological and molecular assessment of benthic *Sargassum* in the Caribbean by Camacho et al. [54] appears to have used a specimen of *S. natans* VIII for their analysis of *S. fluitans* (see Figures 16 and 17 in [54]). Other studies at the time provided photographs or illustrations of *S. natans* VIII specimens but identified them as *S. fluitans* [106,107]. This is not surprising, considering sample collection occurred during the 2014–15 GASB event, when the composition of the bloom was dominated by *S. natans* VIII [33]. Sissini et al. [55] in their analysis of *Sargassum* washing up on Brazilian beaches in 2014–15 used Taylor [49], de Szechy et al. [105], and Camacho et al. [54] to make their morphological determinations (*S. natans* and *S. fluitans*), thus calling into question their morphological identification of *Sargassum* specimens and the validity of their molecular results. Similarly, Gonzalez-Nieto et al. [56], in their molecular assessment of *Sargassum* in the Gulf of Mexico and Caribbean region, included a misidentification of *S. natans* VIII, labeled as *S. fluitans* (see Figure 4, Panel D; open habitus, spherical vesicles, and low vesicle-to-blade ratio indicate *S. natans* VIII). Given the likelihood that their holopelagic *Sargassum* samples have been improperly categorized, the merits of their molecular analyses cannot be critically assessed.

These errors in identification have persisted without much cause for concern due to the rarity of several of Parr's [21] *Sargassum* morphotypes and, thus, had little meaningful consequence for misidentification. However, the landscape has changed since 2011, with the development of the GASB and re-emergence of *S. natans* VIII [33]. The improper identification of holopelagic *Sargassum* samples now has significant consequences and limits our ability to understand the driving mechanisms of the GASB, as recent studies have observed annual and seasonal fluctuations in the composition of holopelagic *Sargassum* morphotypes [27,33,46]. For example, the proposal that ten species with their varieties should be synonymized under *S. cymosum* [56], a recognized benthic species, was accepted by Wynne [108], resulting in the removal of *S. fluitans* from the checklist.

We strongly reject the conclusions of Gonzalez-Nieto et al. [56] and Wynne [108] that holopelagic *Sargassum* are in a polytomy with benthic *Sargassum* species. First, all holopelagic *Sargassum* morphotypes are considered sterile, as no receptacles have ever been observed; thus, grouping them with sexually reproductive benthic species with well-documented receptacles is nonsensical. Second, the three most common holopelagic *Sargassum* morphotypes (*S. fluitans* III, and *S. natans* I and VIII) co-occur across broad geographic and environmental ranges and, yet, their morphological and molecular differences are conserved as demonstrated in the present study and Dibner et al. [58]. Given the new research presented in this study, we maintain the continued species delineation between *S. fluitans* III and *S. natans* I and now formally recognize *S. natans* VIII as a separate variety of *S. natans*.

Recognition of a greater, not less, amount of diversity in holopelagic *Sargassum* is further supported by the research across ecological, biochemical, and physiological fields of study. Govindarajan et al. [109] observed population-level differences in the hydroid *Aglaophenia latecarinata* among *S. fluitans* III and *S. natans* VIII samples, despite the significant geographic overlap in their distribution. Martin et al. [110] observed differences in motile fauna abundance, diversity, and species composition across the three morpho-
holopelagic *Sargassum* diversity and did not reference Winge [20] or Parr [21]. Schneider and Searles [104] and Littler and Littler [63] also described two species, making no mention of additional holopelagic *Sargassum* morphotypes, and simplified their dichotomous keys by removing any mention of thorns along the branches. Notably, Littler and Littler [63] provide an accurate photograph of *S. fluitans* III with thorns evident on the branches.

When Wynne [103] and subsequent revisions are consulted as the authoritative catalog of currently accepted nomenclature for species in the region and Taylor [49], Schneider and Searles [104], or Littler and Littler [63] are the only source material used for holopelagic *Sargassum* identification, specimens of *S. natans* VIII, with smooth branches and large, spherical vesicles lacking an apical spine-like appendage, can be mistakenly identified as *S. fluitans* III. de Szechy et al. [105] collected drift *Sargassum* off the coast of Brazil during the first GASB bloom and misidentified *S. fluitans* as *S. natans* (see Table 1, Figures 4 and 5). The extensive morphological and molecular assessment of benthic *Sargassum* in the Caribbean by Camacho et al. [54] appears to have used a specimen of *S. natans* VIII for their analysis of *S. fluitans* (see Figures 16 and 17 in [54]). Other studies at the time provided photographs or illustrations of *S. natans* VIII specimens but identified them as *S. fluitans* [106,107]. This is not surprising, considering sample collection occurred during the 2014–15 GASB event, when the composition of the bloom was dominated by *S. natans* VIII [33]. Sissini et al. [55] in their analysis of *Sargassum* washing up on Brazilian beaches in 2014–15 used Taylor [49], de Szechy et al. [105], and Camacho et al. [54] to make their morphological determinations (*S. natans* and *S. fluitans*), thus calling into question their morphological identification of *Sargassum* specimens and the validity of their molecular results. Similarly, Gonzalez-Nieto et al. [56], in their molecular assessment of *Sargassum* in the Gulf of Mexico and Caribbean region, included a misidentification of *S. natans* VIII, labeled as *S. fluitans* (see Figure 4, Panel D; open habitus, spherical vesicles, and low vesicle-to-blade ratio indicate *S. natans* VIII). Given the likelihood that their holopelagic *Sargassum* samples have been improperly categorized, the merits of their molecular analyses cannot be critically assessed.

These errors in identification have persisted without much cause for concern due to the rarity of several of Parr's [21] *Sargassum* morphotypes and, thus, had little meaningful consequence for misidentification. However, the landscape has changed since 2011, with the development of the GASB and re-emergence of *S. natans* VIII [33]. The improper identification of holopelagic *Sargassum* samples now has significant consequences and limits our ability to understand the driving mechanisms of the GASB, as recent studies have observed annual and seasonal fluctuations in the composition of holopelagic *Sargassum* morphotypes [27,33,46]. For example, the proposal that ten species with their varieties should be synonymized under *S. cymosum* [56], a recognized benthic species, was accepted by Wynne [108], resulting in the removal of *S. fluitans* from the checklist.

We strongly reject the conclusions of Gonzalez-Nieto et al. [56] and Wynne [108] that holopelagic *Sargassum* are in a polytomy with benthic *Sargassum* species. First, all holopelagic *Sargassum* morphotypes are considered sterile, as no receptacles have ever been observed; thus, grouping them with sexually reproductive benthic species with well-documented receptacles is nonsensical. Second, the three most common holopelagic *Sargassum* morphotypes (*S. fluitans* III, and *S. natans* I and VIII) co-occur across broad geographic and environmental ranges and, yet, their morphological and molecular differences are conserved as demonstrated in the present study and Dibner et al. [58]. Given the new research presented in this study, we maintain the continued species delineation between *S. fluitans* III and *S. natans* I and now formally recognize *S. natans* VIII as a separate variety of *S. natans*.

Recognition of a greater, not less, amount of diversity in holopelagic *Sargassum* is further supported by the research across ecological, biochemical, and physiological fields of study. Govindarajan et al. [109] observed population-level differences in the hydroid *Aglaophenia latecarinata* among *S. fluitans* III and *S. natans* VIII samples, despite the significant geographic overlap in their distribution. Martin et al. [110] observed differences in motile fauna abundance, diversity, and species composition across the three morpho-
types. Garcia-Sanchez et al. [46] observed shifts in the abundance of holopelagic *Sargassum* morphotypes reaching beaches in Mexico over several years. Similarly, seasonal shifts in *Sargassum* morphotype abundance were observed in Barbados [27]. McGillicuddy et al. [111] observed differences in the nutrient and arsenic biogeochemistry among *S. fluitans* III and *S. natans* I morphotypes. Finally, several studies have documented the different growth and environmental tolerance among holopelagic *Sargassum* morphotypes to varying nutrient, temperature, and salinity conditions [39–43].

Taxonomy is not an end in itself, but a tool allowing researchers to propose species hypotheses [47,112]. Most often, accepted species are distinguished by a set of morphological characters and/or specific characteristics. In addition to the need for name agreement among scientists to help in research communications, environmental managers also need definitive and consistent names to designate the populations they administer, even if the biodiversity is generally more complex than nomenclatural partitioning, notably because speciation processes are gradual. Terrestrial botany, especially studies carried out on Magnoliophytes, offers thousands of examples of taxa that are distinguished even though intermediate forms, in particular hybrids, unite them. In the case of *S. natans* VIII and *S. natans* I, we showed that no intermediate forms exist. In addition, hybridization is, a priori, irrelevant insofar as sexual reproduction has never been observed in these holopelagic macroalgae, despite the major sampling effort that has occurred. Nevertheless, the discord between the genetic differentiation (i.e., very low) and the morphological differences (i.e., high) for *S. natans* I and *S. natans* VIII makes it challenging to propose that the two morphotypes correspond to different species.

Current morphotype naming, associated with the lack of a formal description, leads to uncertainties in the description of the holopelagic *Sargassum*. We, therefore, propose a taxonomic framework, with the description of a new variety and an identification key. The type specimen of *Sargassum natans* belongs to the morphotype *Sargassum natans* I (Figure S1). The *Sargassum natans* VIII morphotype was well-identified by Winge [20] and Parr [21], but neither with a formal diagnosis nor a creation of different taxon. Although this name was taken up later (e.g., [33,57,58] and in most of the recent papers on *Sargassum* inundation since 2015), it remains undescribed according to the rules of botanical taxonomy (International Code of Nomenclature of algae, fungi, and plants [48]). It is, therefore, described below, under the name of *Sargassum natans* var. *wingei*, in tribute to Professor Ö. Winge who first recognized the originality of this form.

## Species description

We propose the following new name for the morphotype *Sargassum natans* VIII.

**Sargassum natans** var. *wingei* Thibaut, Blanfuné, Boudouresque, Siuda & Verlaque var. nov.

**Figures:** Figures 3 and 4.

**Holotype:** Specimen HCOM S10-S1-3 (Holotype deposited in HCOM Herbarium)

**Isotypes:** Specimens S10-S1-1 and S10-S1-2, deposited in HCOM Herbarium.

**Type locality:** Station 10, tropical Atlantic Ocean, geographic co-ordinates: 14°58.440′ N 59°07.230′ W.

**Etymology:** Named for Professor Öjvind Winge, who was the first scientist to distinguish this taxon (as *Sargassum* sp. VIII) among the pelagic *Sargassum* specimens.

**Representative material:** All samples of *Sargassum natans* var. *wingei* collected during the two expeditions (Caribbean and transatlantic expeditions). All samples are deposited in HCOM Herbarium (Table S2).

**Specimens sequenced and Genbank references:** New haplotypes produced here are available in GenBank under accession numbers MT422788 to MT422805, and final sequence alignments have been submitted in Zenodo.

**Other descriptions and illustrations:** Winge [20] (1923, Figure 13, as *Sargassum* sp. VIII); Parr [21] (1939, figs 9-10, 12-17, as *S. natans* (VIII) Winge); Schell et al. [33] (2015, Figure 1 in ref [33], as *S. natans* VIII Parr); Amaral-Zettler et al. [57] (2017, figs 2c and 2d, as *S. natans* form VIII); Martin (2016); Govindarajan et al. [109] (2019, Figure 1c, as *S. natans* VIII) (Table S1).
types. Garcia-Sanchez et al. [46] observed shifts in the abundance of holopelagic *Sargassum* morphotypes reaching beaches in Mexico over several years. Similarly, seasonal shifts in *Sargassum* morphotype abundance were observed in Barbados [27]. McGillicuddy et al. [111] observed differences in the nutrient and arsenic biogeochemistry among *S. fluitans* III and *S. natans* I morphotypes. Finally, several studies have documented the different growth and environmental tolerance among holopelagic *Sargassum* morphotypes to varying nutrient, temperature, and salinity conditions [39–43].

Taxonomy is not an end in itself, but a tool allowing researchers to propose species hypotheses [47,112]. Most often, accepted species are distinguished by a set of morphological characters and/or specific characteristics. In addition to the need for name agreement among scientists to help in research communications, environmental managers also need definitive and consistent names to designate the populations they administer, even if the biodiversity is generally more complex than nomenclatural partitioning, notably because speciation processes are gradual. Terrestrial botany, especially studies carried out on Magnoliophytes, offers thousands of examples of taxa that are distinguished even though intermediate forms, in particular hybrids, unite them. In the case of *S. natans* VIII and *S. natans* I, we showed that no intermediate forms exist. In addition, hybridization is, a priori, irrelevant insofar as sexual reproduction has never been observed in these holopelagic macroalgae, despite the major sampling effort that has occurred. Nevertheless, the discord between the genetic differentiation (i.e., very low) and the morphological differences (i.e., high) for *S. natans* I and *S. natans* VIII makes it challenging to propose that the two morphotypes correspond to different species.

Current morphotype naming, associated with the lack of a formal description, leads to uncertainties in the description of the holopelagic *Sargassum*. We, therefore, propose a taxonomic framework, with the description of a new variety and an identification key. The type specimen of *Sargassum natans* belongs to the morphotype *Sargassum natans* I (Figure S1). The *Sargassum natans* VIII morphotype was well-identified by Winge [20] and Parr [21], but neither with a formal diagnosis nor a creation of different taxon. Although this name was taken up later (e.g., [33,57,58] and in most of the recent papers on *Sargassum* inundation since 2015), it remains undescribed according to the rules of botanical taxonomy (International Code of Nomenclature of algae, fungi, and plants [48]). It is, therefore, described below, under the name of *Sargassum natans* var. *wingei*, in tribute to Professor Ö. Winge who first recognized the originality of this form.

## Species description

We propose the following new name for the morphotype *Sargassum natans* VIII.

**Sargassum natans** var. *wingei* Thibaut, Blanfuné, Boudouresque, Siuda & Verlaque var. nov.

**Figures:** Figures 3 and 4.

**Holotype:** Specimen HCOM S10-S1-3 (Holotype deposited in HCOM Herbarium)

**Isotypes:** Specimens S10-S1-1 and S10-S1-2, deposited in HCOM Herbarium.

**Type locality:** Station 10, tropical Atlantic Ocean, geographic co-ordinates: 14°58.440′ N 59°07.230′ W.

**Etymology:** Named for Professor Öjvind Winge, who was the first scientist to distinguish this taxon (as *Sargassum* sp. VIII) among the pelagic *Sargassum* specimens.

**Representative material:** All samples of *Sargassum natans* var. *wingei* collected during the two expeditions (Caribbean and transatlantic expeditions). All samples are deposited in HCOM Herbarium (Table S2).

**Specimens sequenced and Genbank references:** New haplotypes produced here are available in GenBank under accession numbers MT422788 to MT422805, and final sequence alignments have been submitted in Zenodo.

**Other descriptions and illustrations:** Winge [20] (1923, Figure 13, as *Sargassum* sp. VIII); Parr [21] (1939, figs 9-10, 12-17, as *S. natans* (VIII) Winge); Schell et al. [33] (2015, Figure 1 in ref [33], as *S. natans* VIII Parr); Amaral-Zettler et al. [57] (2017, figs 2c and 2d, as *S. natans* form VIII); Martin (2016); Govindarajan et al. [109] (2019, Figure 1c, as *S. natans* VIII) (Table S1).
**Websites:** as *S. natans* VIII: Sea Education Association, Woods Hole (https://www.sea.edu/sea_research/sargassum_ecosystem, accessed on 28 March 2024). Sailors for the Sea (https://sailorsforthesea.org/blog/conservation-heading-towards-bernuda/, accessed on 28 March 2024). Center for Resource Management and Environmental Studies (CERMES), University of West Indies Cave Hill, Barbados.

(https://www.cavehill.uwi.edu/cermes/research-projects/sargassum/tools-and-guidance.aspx, accessed on 28 March 2024)

**Diagnosis:** Plant holopelagic, brown to yellow-brown, without a holdfast or a distinct main axis; branches smooth without cryptostomata, 1–1.5 mm diam., ramified several times; blades simple, shortly pedicellate, flat, lanceolate to broadly lanceolate, without cryptostomata (13) 30–55 (–65) mm long and 3–10 mm wide (L/W ratio: 3–10, typically: 7–8), with coarsely irregularly serrated margins, symmetrical base, and midrib; vesicles (air bladders), more, less, or as much as the blades, smooth, without cryptostomata, spherical, rarely oblong, 2–5 (–7) mm diam., usually without foliaceous margin or apical spine-like appendage; pedicels short, 0.5–2.5 mm long, cylindrical, sometimes foliaceous with toothed margins; receptacles never observed.

**Distribution:** Tropical Atlantic Ocean, between 7° and 26° N.

**Habitat:** It is one of the three holopelagic taxa of the tropical Atlantic Ocean, with *S. fluitans* (Børgesen) Børgesen and *S. natans* (Linnaeus) Gaillon.

**Key for the identification of Atlantic *Sargassum* species**

1a. Plants fixed to the substrate; main axis distinct; cryptostomata and receptacles present...
Benthic species (not treated here)

1b. Plants floating, without holdfast and distinct main axis; receptacles absent and cryptostomata absent.
Pelagic species (2)

2a. Branches thorny; blades lanceolate, 3–8 mm wide and 11–60 mm long (L/W: 4–13), with asymmetrical base; vesicles oblong without apical spine-like or foliaceous appendage but frequently with foliaceous margin, usually more numerous than blades, 1–6 mm diam., with a short pedicel, 0.5–2.5 mm long, cylindrical to winged, sometimes with toothed margins
*S. fluitans* (Børgesen) Børgesen

2b. Branches smooth; blades with symmetrical base; vesicles more spherical, fewer or as numerous as blades
(3)

3a. Blades linear, narrow, 1–4 mm wide and 13–70 mm long (L/W: 5–22); vesicles, 2–5 mm diam., frequently with a foliaceous margin and an apical spine-like or reduced foliaceous appendage, and with a long pedicel, 1–5 mm long
*S. natans* var. *natans* (Linnaeus) Gaillon

3b. Blades lanceolate, broad, 3–10 mm wide and 13–65 mm long (L/W: 3–10); vesicles, 2–7 mm diam., usually without foliaceous margin and apical spine-like appendages, and with a short pedicel, 0.5–2.5 mm long
*S. natans* var. *wingei* nov. var.

The decision to place *S. natans* VIII at the level of variety, and not to elevate the morphotype to the status of a new species, is somewhat conservative. Beyond the question of renaming these taxa, the clear morphological differences between the three holopelagic *Sargassum* and the weak genetic differences raise significant doubts about the speciation stage of these taxa; their assignment to a species rank is even more complex as they most likely rely on asexual reproduction as their primary method of propagation, so that the biological species concept can hardly be used for delineation. Our genetic results on *S. natans* suggest that the two varieties could correspond to different fixed clones from a single species, where asexual lineages fixed morphological differences. However, the use of mitochondrial and plastidial DNA together is not sufficient to investigate this hypothesis. Fucales are oogamous [113], and, in oogamous brown algae, chloroplasts and mitochondria are thought to be maternally inherited [114]. Therefore, these two cytoplasmic genomes are not independently inherited, and, alone, they do not correspond to independent markers. Further investigation of the species' evolutionary history could be accomplished by using
**Websites:** as *S. natans* VIII: Sea Education Association, Woods Hole (https://www.sea.edu/sea_research/sargassum_ecosystem, accessed on 28 March 2024). Sailors for the Sea (https://sailorsforthesea.org/blog/conservation-heading-towards-bernuda/, accessed on 28 March 2024). Center for Resource Management and Environmental Studies (CERMES), University of West Indies Cave Hill, Barbados.

(https://www.cavehill.uwi.edu/cermes/research-projects/sargassum/tools-and-guidance.aspx, accessed on 28 March 2024)

**Diagnosis:** Plant holopelagic, brown to yellow-brown, without a holdfast or a distinct main axis; branches smooth without cryptostomata, 1–1.5 mm diam., ramified several times; blades simple, shortly pedicellate, flat, lanceolate to broadly lanceolate, without cryptostomata (13) 30–55 (–65) mm long and 3–10 mm wide (L/W ratio: 3–10, typically: 7–8), with coarsely irregularly serrated margins, symmetrical base, and midrib; vesicles (air bladders), more, less, or as much as the blades, smooth, without cryptostomata, spherical, rarely oblong, 2–5 (–7) mm diam., usually without foliaceous margin or apical spine-like appendage; pedicels short, 0.5–2.5 mm long, cylindrical, sometimes foliaceous with toothed margins; receptacles never observed.

**Distribution:** Tropical Atlantic Ocean, between 7° and 26° N.

**Habitat:** It is one of the three holopelagic taxa of the tropical Atlantic Ocean, with *S. fluitans* (Børgesen) Børgesen and *S. natans* (Linnaeus) Gaillon.

**Key for the identification of Atlantic *Sargassum* species**

1a. Plants fixed to the substrate; main axis distinct; cryptostomata and receptacles present...
Benthic species (not treated here)

1b. Plants floating, without holdfast and distinct main axis; receptacles absent and cryptostomata absent.
Pelagic species (2)

2a. Branches thorny; blades lanceolate, 3–8 mm wide and 11–60 mm long (L/W: 4–13), with asymmetrical base; vesicles oblong without apical spine-like or foliaceous appendage but frequently with foliaceous margin, usually more numerous than blades, 1–6 mm diam., with a short pedicel, 0.5–2.5 mm long, cylindrical to winged, sometimes with toothed margins
*S. fluitans* (Børgesen) Børgesen

2b. Branches smooth; blades with symmetrical base; vesicles more spherical, fewer or as numerous as blades
(3)

3a. Blades linear, narrow, 1–4 mm wide and 13–70 mm long (L/W: 5–22); vesicles, 2–5 mm diam., frequently with a foliaceous margin and an apical spine-like or reduced foliaceous appendage, and with a long pedicel, 1–5 mm long
*S. natans* var. *natans* (Linnaeus) Gaillon

3b. Blades lanceolate, broad, 3–10 mm wide and 13–65 mm long (L/W: 3–10); vesicles, 2–7 mm diam., usually without foliaceous margin and apical spine-like appendages, and with a short pedicel, 0.5–2.5 mm long
*S. natans* var. *wingei* nov. var.

The decision to place *S. natans* VIII at the level of variety, and not to elevate the morphotype to the status of a new species, is somewhat conservative. Beyond the question of renaming these taxa, the clear morphological differences between the three holopelagic *Sargassum* and the weak genetic differences raise significant doubts about the speciation stage of these taxa; their assignment to a species rank is even more complex as they most likely rely on asexual reproduction as their primary method of propagation, so that the biological species concept can hardly be used for delineation. Our genetic results on *S. natans* suggest that the two varieties could correspond to different fixed clones from a single species, where asexual lineages fixed morphological differences. However, the use of mitochondrial and plastidial DNA together is not sufficient to investigate this hypothesis. Fucales are oogamous [113], and, in oogamous brown algae, chloroplasts and mitochondria are thought to be maternally inherited [114]. Therefore, these two cytoplasmic genomes are not independently inherited, and, alone, they do not correspond to independent markers. Further investigation of the species' evolutionary history could be accomplished by using
nuclear markers, polymorphic within each of the two varieties. This approach would allow for a more in-depth analysis of the extent of clonality, as well as species delineation and speciation scenarios. For example, it would be interesting to test if the pelagic way of life originated once or several times in the *Sargassum* genus. Finally, the phylogeny of the genus *Sargassum* must be profoundly clarified considering that the holotype specimen defining the genus *Sargassum* was *Sargassum bacciferum* Turner (C. Agardh) currently named for the type species as *S. natans* (Agardh, 1820), a clonal holopelagic taxa lacking sexual reproductive apparatus.

**Supplementary Materials:** The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/phycology4020014/s1, Figure S1: *Sargassum natans* (L.) Gaillon. Type-specimen of *Fucus natans* L. from the Linnean Herbarium (Figure from Børgesen [18,19]); Figure S2: Phylogenetic tree obtained with *psbA*; Table S1: Morphological characters used to identify the North Atlantic *Sargassum* species applied to the holopelagic taxa; Table S2: All samples of *Sargassum wingei* collected during the two expeditions.

**Author Contributions:** Conceptualization, T.T. and A.N.S.S.; methodology, T.T., A.N.S.S., S.D. and D.A.; validation, T.T., M.V. and C.-F.B.; formal analysis, A.B. and D.A.; investigation, A.B., S.C., V.S.-P. and T.C.; writing—original draft preparation, T.T. and F.V.; writing—review and editing, T.T., A.N.S.S., A.B., S.D., C.-F.B., S.C., V.S.-P., F.V., D.S.G., V.M., F.R., J.M.S., T.C. and D.A.; visualization, T.T., A.N.S.S., S.D. and D.A.; supervision, T.T.; project administration, T.T.; funding acquisition, T.T. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the French Ministry for the Ecological and Inclusive Transition, agreement number 17-MOTEC-SARGASSES-1-CVS-001. The project leading to this publication has received funding from the Ministry for the Ecological and Inclusive Transition, agreement number 17-MOTEC-SARGASSES-1-CVS-001, European FEDER Fund under project 1166-39417 and ANR-19-SARG-0004-01, CNRS LEFE SAREDA-SWO, and TOSCA-CNES SAREDA_DA.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** New haplotypes produced here are available in GenBank under accession numbers MT422788 to MT422805, and final sequence alignments have been submitted in Zenodo.

**Acknowledgments:** We thank the *Flotte Oceanographique Française* (FOF), Captain Pierre Samuel and the crew of N/O ANTEA, Captain Jean Dumarais and the crew of Y/V YERSIN, Juerg Lichtenegger and Club Sentinel for helping routing the boats to the *Sargassum* rafts, M. Brossard and team from IRD Guyane, and C. Bouchon (UMR BOREA) who provided the logistics and implementation. The authors acknowledge the support of Monaco-Expéditions. We thank Jean Blanchot, Léo Berline, Cristele Chevalier, Mathilde Guéné, Anouck Ody, Sandrine Ruitton, and Dorian Guillemin for their precious help during the Antea and Yersin expeditions. We deeply thank François Bordeyne, Sarah Bouchemousse, and Quentin Langevin for their help on the morphological and genetics analysis. The Marine Biological Laboratory—Wood Hole Oceanographic Institution (MBL-WHOI) Library staff assisted with critical access to historical taxonomic references. The platforms Macrophytes and Omics of the MIO have been used in this work. This is publication ISEM 2024-098.

**Conflicts of Interest:** The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of the data; in the writing of the manuscript; or in the decision to publish the results.

## References

1. Liu, F.; Pang, S.; Chopin, T.; Gao, S.; Shan, T.; Zhao, X.; Li, J. Understanding the Recurrent Large-Scale Green Tide in the Yellow Sea: Temporal and Spatial Correlations between Multiple Geographical, Aquacultural and Biological Factors. *Mar. Environ. Res.* **2013**, *83*, 38–47. [CrossRef] [PubMed]

2. Smetacek, V.; Zingone, A. Green and Golden Seaweed Tides on the Rise. *Nature* **2013**, *504*, 84–88. [CrossRef] [PubMed]

3. Mineur, F.; Arenas, F.; Assis, J.; Davies, A.J.; Engelen, A.H.; Fernandes, F.; Malta, E.; Thibaut, T.; Van Nguyen, T.; Vaz-Pinto, F.; et al. European Seaweeds under Pressure: Consequences for Communities and Ecosystem Functioning. *J. Sea Res.* **2015**, *98*, 91–108. [CrossRef]
nuclear markers, polymorphic within each of the two varieties. This approach would allow for a more in-depth analysis of the extent of clonality, as well as species delineation and speciation scenarios. For example, it would be interesting to test if the pelagic way of life originated once or several times in the *Sargassum* genus. Finally, the phylogeny of the genus *Sargassum* must be profoundly clarified considering that the holotype specimen defining the genus *Sargassum* was *Sargassum bacciferum* Turner (C. Agardh) currently named for the type species as *S. natans* (Agardh, 1820), a clonal holopelagic taxa lacking sexual reproductive apparatus.

**Supplementary Materials:** The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/phycology4020014/s1, Figure S1: *Sargassum natans* (L.) Gaillon. Type-specimen of *Fucus natans* L. from the Linnean Herbarium (Figure from Børgesen [18,19]); Figure S2: Phylogenetic tree obtained with *psbA*; Table S1: Morphological characters used to identify the North Atlantic *Sargassum* species applied to the holopelagic taxa; Table S2: All samples of *Sargassum wingei* collected during the two expeditions.

**Author Contributions:** Conceptualization, T.T. and A.N.S.S.; methodology, T.T., A.N.S.S., S.D. and D.A.; validation, T.T., M.V. and C.-F.B.; formal analysis, A.B. and D.A.; investigation, A.B., S.C., V.S.-P. and T.C.; writing—original draft preparation, T.T. and F.V.; writing—review and editing, T.T., A.N.S.S., A.B., S.D., C.-F.B., S.C., V.S.-P., F.V., D.S.G., V.M., F.R., J.M.S., T.C. and D.A.; visualization, T.T., A.N.S.S., S.D. and D.A.; supervision, T.T.; project administration, T.T.; funding acquisition, T.T. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the French Ministry for the Ecological and Inclusive Transition, agreement number 17-MOTEC-SARGASSES-1-CVS-001. The project leading to this publication has received funding from the Ministry for the Ecological and Inclusive Transition, agreement number 17-MOTEC-SARGASSES-1-CVS-001, European FEDER Fund under project 1166-39417 and ANR-19-SARG-0004-01, CNRS LEFE SAREDA-SWO, and TOSCA-CNES SAREDA_DA.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** New haplotypes produced here are available in GenBank under accession numbers MT422788 to MT422805, and final sequence alignments have been submitted in Zenodo.

**Acknowledgments:** We thank the *Flotte Oceanographique Française* (FOF), Captain Pierre Samuel and the crew of N/O ANTEA, Captain Jean Dumarais and the crew of Y/V YERSIN, Juerg Lichtenegger and Club Sentinel for helping routing the boats to the *Sargassum* rafts, M. Brossard and team from IRD Guyane, and C. Bouchon (UMR BOREA) who provided the logistics and implementation. The authors acknowledge the support of Monaco-Expéditions. We thank Jean Blanchot, Léo Berline, Cristele Chevalier, Mathilde Guéné, Anouck Ody, Sandrine Ruitton, and Dorian Guillemin for their precious help during the Antea and Yersin expeditions. We deeply thank François Bordeyne, Sarah Bouchemousse, and Quentin Langevin for their help on the morphological and genetics analysis. The Marine Biological Laboratory—Wood Hole Oceanographic Institution (MBL-WHOI) Library staff assisted with critical access to historical taxonomic references. The platforms Macrophytes and Omics of the MIO have been used in this work. This is publication ISEM 2024-098.

**Conflicts of Interest:** The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of the data; in the writing of the manuscript; or in the decision to publish the results.

## References

1. Liu, F.; Pang, S.; Chopin, T.; Gao, S.; Shan, T.; Zhao, X.; Li, J. Understanding the Recurrent Large-Scale Green Tide in the Yellow Sea: Temporal and Spatial Correlations between Multiple Geographical, Aquacultural and Biological Factors. *Mar. Environ. Res.* **2013**, *83*, 38–47. [CrossRef] [PubMed]

2. Smetacek, V.; Zingone, A. Green and Golden Seaweed Tides on the Rise. *Nature* **2013**, *504*, 84–88. [CrossRef] [PubMed]

3. Mineur, F.; Arenas, F.; Assis, J.; Davies, A.J.; Engelen, A.H.; Fernandes, F.; Malta, E.; Thibaut, T.; Van Nguyen, T.; Vaz-Pinto, F.; et al. European Seaweeds under Pressure: Consequences for Communities and Ecosystem Functioning. *J. Sea Res.* **2015**, *98*, 91–108. [CrossRef]
4. Lapointe, B.E.; Burkholder, J.M.; Van Alstyne, K.L. Harmful Macroalgal Blooms in a Changing World: Causes, Impacts, and Management. In *Harmful Algal Blooms*; Shumway, S.E., Burkholder, J.M., Morton, S.L., Eds.; Wiley: Hoboken, NJ, USA, 2018; pp. 515–560. ISBN 978-1-118-99467-2.

5. Dominguez Almela, V.; Addo, K.A.; Corbett, J.; Cumberbatch, J.; Dash, J.; Marsh, R.; Oxenford, H.; Tonon, T.; Van Der Plank, S.; Webber, M.; et al. Science and Policy Lessons Learned from a Decade of Adaptation to the Emergent Risk of *Sargassum* Proliferation across the Tropical Atlantic. *Environ. Res. Commun.* **2023**, *5*, 061002. [CrossRef]

6. Le Lann, K.; Connan, S.; Stiger-Pouvreau, V. Phenology, TPC and Size-Fractioning Phenolics Variability in Temperate Sargassaceae (Phaeophyceae, Fucales) from Western Brittany: Native versus Introduced Species. *Mar. Environ. Res.* **2012**, *80*, 1–11. [CrossRef] [PubMed]

7. Engelen, A.H.; Serebryakova, A.; Ang, P.; Britton-Simmons, K.; Mineur, F.; Pedersen, M.F.; Arenas, F.; Fernandez, C.; Steen, H.; Svenson, R.; et al. Circumglobal Invasion by the Brown Seaweed Sargassum Muticum. *Oceanogr. Mar. Biol. Annu. Rev.* **2015**, *53*, 81–126.

8. Marks, L.; Salinas-Ruiz, P.; Reed, D.; Holbrook, S.; Culver, C.; Engle, J.; Kushner, D.; Caselle, J.; Freiwald, J.; Williams, J.; et al. Range Expansion of a Non-Native, Invasive Macroalga *Sargassum horneri* (Turner) C. Agardh, 1820 in the Eastern Pacific. *BioInvasions Rec.* **2015**, *4*, 243–248. [CrossRef]

9. Bouchon, C.; Bouchon-Navaro, Y.; Louis, M. A First Record of a *Sargassum* (Phaeophyta, Algae) Outbreak in a Caribbean Coral Reef Ecosystem. *Proc. Gulf Caribb. Fish. Inst.* **1992**, *41*, 171–188.

10. Done, T.J. Phase Shifts in Coral Reef Communities and Their Ecological Significance. *Hydrobiologia* **1992**, *247*, 131–132. [CrossRef]

11. Stiger, V.; Payri, C.E. Spatial and Seasonal Variations in the Biological Characteristics of Two Invasive Brown Algae, *Turbinaria ornata* (Turner) J. Agardh and *Sargassum mangarevense* (Grunow) Setchell (Sargassaceae, Fucales) Spreading on the Reefs of Tahiti (French Polynesia). *Bot. Mar.* **1999**, *42*, 295–306. [CrossRef]

12. De Ramon N'Yeurt, A.; Iese, V. The Proliferating Brown Alga *Sargassum polycystum* in Tuvalu, South Pacific: Assessment of the Bloom and Applications to Local Agriculture and Sustainable Energy. *J. Appl. Phycol.* **2015**, *27*, 2037–2045. [CrossRef]

13. Van Tussenbroek, B.I.; Hernández Arana, H.A.; Rodríguez-Martínez, R.E.; Espinoza-Avalos, J.; Canizales-Flores, H.M.; González-Godoy, C.E.; Barba-Santos, M.G.; Vega-Zepeda, A.; Collado-Vides, L. Severe Impacts of Brown Tides Caused by *Sargassum* spp. on near-Shore Caribbean Seagrass Communities. *Mar. Pollut. Bull.* **2017**, *122*, 272–281. [CrossRef] [PubMed]

14. Velázquez-Ochoa, R.; Enríquez, S. Environmental Degradation of the Mexican Caribbean Reef Lagoons. *Mar. Pollut. Bull.* **2023**, *191*, 114947. [CrossRef] [PubMed]

15. Stiger-Pouvreau, V.; Mattio, L.; De Ramon N'Yeurt, A.; Uwai, S.; Dominguez, H.; Flórez-Fernández, N.; Connan, S.; Critchley, A.T. A Concise Review of the Highly Diverse Genus *Sargassum* C. Agardh with Wide Industrial Potential. *J. Appl. Phycol.* **2023**, *35*, 1453–1483. [CrossRef]

16. Thibaut, T.; Blanfuné, A.; Verlaque, M.; Boudouresque, C.-F.; Ruitton, S. The *Sargassum* Conundrum: Very Rare, Threatened or Locally Extinct in the NW Mediterranean and Still Lacking Protection. *Hydrobiologia* **2016**, *781*, 3–23. [CrossRef]

17. Auissi, M.; Sellam, L.N.; Boudouresque, C.F.; Blanfuné, A.; Derbal, F.; Frihi, H.; Perret-Boudouresque, M.; Rebzani-Zahaf, C.; Verlaque, M.; Thibaut, T. Insights into the Species Diversity of the Genus *Sargassum* (Phaeophyceae) in the Mediterranean Sea, with a Focus on a Pre-Viously Unnoticed Taxon from Algeria. *Medit. Mar. Sci.* **2018**, *19*, 48. [CrossRef]

18. Børgesen, F. The Species of *Sargassum* Found Along the Coasts of the Danish West Indies With Remarks Upon the Floating Forms of the Sargasso Sea. In *Mindeskrift I Anledning af Hundredaaret for Japetus Steenstrups Fødsel*; Jungersen, F.E., Warming, E., Eds.; Bianco Luno Bogtrykkeri: Kobenhavn, Denmark, 1914; Volume 32, pp. 1–20.

19. Børgesen, F. Marine Algae of the Danish West Indies. Part II. Phaeophyceae.; Dansk Bot. Arkiv, Bianco Luno Copenhagen, Denmark, 1914; Volume 2.

20. Winge, Ø. *The Sargasso Sea, Its Boundaries and Vegetation. Report on the Danish Oceanographical Expeditions 1908–1910 to the Mediterranean and Adjacent Seas.*; A.F. Høst&son.: Copenhagen, Denmark, 1923.

21. Parr, A.E. Quantitative Observations on the Pelagic *Sargassum* Vegetation of the Western North Atlantic. *Bull. Bingham Oceanogr. Collect.* **1939**, *6*, 1–94.

22. Butler, J.N.; Morris, B.F.; Cadwallader, J.; Stoner, A.W. *Studies of Sargassum and the Sargassum Community*; Bermuda Biological Station for Research, Special Publication: Bermuda, UK, 1983; pp. 1–307.

23. Baker, P.; Minzlaff, U.; Schoenle, A.; Schwabe, E.; Hohlfeld, M.; Jeuck, A.; Brenke, N.; Prausse, D.; Rothenbeck, M.; Brix, S.; et al. Potential Contribution of Surface-Dwelling *Sargassum* Algae to Deep-Sea Ecosystems in the Southern North Atlantic. *Deep. Sea Res. Part II Top. Stud. Oceanogr.* **2018**, *148*, 21–34. [CrossRef]

24. Ody, A.; Thibaut, T.; Berline, L.; Changeux, T.; André, J.-M.; Chevalier, C.; Blanfuné, A.; Blanchot, J.; Ruitton, S.; Stiger-Pouvreau, V.; et al. From In Situ to Satellite Observations of Pelagic *Sargassum* Distribution and Aggregation in the Tropical North Atlantic Ocean. *PLoS ONE* **2019**, *14*, e0222584. [CrossRef]

25. Coston-Clements, L.; Settle, L.R.; Hoss, D.E.; Cross, F.A. *Utilization of the Sargassum Habitat by Marine Invertebrates and Vertebrates, a Review*; US Department of Commerce, National Oceanic and Atmospheric Administration, National Marine Fisheries Service, Southeast Fisheries Science Center, Beaufort Laboratory: St. Petersburg, FL, USA, 1991; Volume 296.

26. Huffard, C.L.; Von Thun, S.; Sherman, A.D.; Sealey, K.; Smith, K.L. Pelagic *Sargassum* Community Change over a 40-Year Period: Temporal and Spatial Variability. *Mar. Biol.* **2014**, *161*, 2735–2751. [CrossRef] [PubMed]
4. Lapointe, B.E.; Burkholder, J.M.; Van Alstyne, K.L. Harmful Macroalgal Blooms in a Changing World: Causes, Impacts, and Management. In *Harmful Algal Blooms*; Shumway, S.E., Burkholder, J.M., Morton, S.L., Eds.; Wiley: Hoboken, NJ, USA, 2018; pp. 515–560. ISBN 978-1-118-99467-2.

5. Dominguez Almela, V.; Addo, K.A.; Corbett, J.; Cumberbatch, J.; Dash, J.; Marsh, R.; Oxenford, H.; Tonon, T.; Van Der Plank, S.; Webber, M.; et al. Science and Policy Lessons Learned from a Decade of Adaptation to the Emergent Risk of *Sargassum* Proliferation across the Tropical Atlantic. *Environ. Res. Commun.* **2023**, *5*, 061002. [CrossRef]

6. Le Lann, K.; Connan, S.; Stiger-Pouvreau, V. Phenology, TPC and Size-Fractioning Phenolics Variability in Temperate Sargassaceae (Phaeophyceae, Fucales) from Western Brittany: Native versus Introduced Species. *Mar. Environ. Res.* **2012**, *80*, 1–11. [CrossRef] [PubMed]

7. Engelen, A.H.; Serebryakova, A.; Ang, P.; Britton-Simmons, K.; Mineur, F.; Pedersen, M.F.; Arenas, F.; Fernandez, C.; Steen, H.; Svenson, R.; et al. Circumglobal Invasion by the Brown Seaweed Sargassum Muticum. *Oceanogr. Mar. Biol. Annu. Rev.* **2015**, *53*, 81–126.

8. Marks, L.; Salinas-Ruiz, P.; Reed, D.; Holbrook, S.; Culver, C.; Engle, J.; Kushner, D.; Caselle, J.; Freiwald, J.; Williams, J.; et al. Range Expansion of a Non-Native, Invasive Macroalga *Sargassum horneri* (Turner) C. Agardh, 1820 in the Eastern Pacific. *BioInvasions Rec.* **2015**, *4*, 243–248. [CrossRef]

9. Bouchon, C.; Bouchon-Navaro, Y.; Louis, M. A First Record of a *Sargassum* (Phaeophyta, Algae) Outbreak in a Caribbean Coral Reef Ecosystem. *Proc. Gulf Caribb. Fish. Inst.* **1992**, *41*, 171–188.

10. Done, T.J. Phase Shifts in Coral Reef Communities and Their Ecological Significance. *Hydrobiologia* **1992**, *247*, 131–132. [CrossRef]

11. Stiger, V.; Payri, C.E. Spatial and Seasonal Variations in the Biological Characteristics of Two Invasive Brown Algae, *Turbinaria ornata* (Turner) J. Agardh and *Sargassum mangarevense* (Grunow) Setchell (Sargassaceae, Fucales) Spreading on the Reefs of Tahiti (French Polynesia). *Bot. Mar.* **1999**, *42*, 295–306. [CrossRef]

12. De Ramon N'Yeurt, A.; Iese, V. The Proliferating Brown Alga *Sargassum polycystum* in Tuvalu, South Pacific: Assessment of the Bloom and Applications to Local Agriculture and Sustainable Energy. *J. Appl. Phycol.* **2015**, *27*, 2037–2045. [CrossRef]

13. Van Tussenbroek, B.I.; Hernández Arana, H.A.; Rodríguez-Martínez, R.E.; Espinoza-Avalos, J.; Canizales-Flores, H.M.; González-Godoy, C.E.; Barba-Santos, M.G.; Vega-Zepeda, A.; Collado-Vides, L. Severe Impacts of Brown Tides Caused by *Sargassum* spp. on near-Shore Caribbean Seagrass Communities. *Mar. Pollut. Bull.* **2017**, *122*, 272–281. [CrossRef] [PubMed]

14. Velázquez-Ochoa, R.; Enríquez, S. Environmental Degradation of the Mexican Caribbean Reef Lagoons. *Mar. Pollut. Bull.* **2023**, *191*, 114947. [CrossRef] [PubMed]

15. Stiger-Pouvreau, V.; Mattio, L.; De Ramon N'Yeurt, A.; Uwai, S.; Dominguez, H.; Flórez-Fernández, N.; Connan, S.; Critchley, A.T. A Concise Review of the Highly Diverse Genus *Sargassum* C. Agardh with Wide Industrial Potential. *J. Appl. Phycol.* **2023**, *35*, 1453–1483. [CrossRef]

16. Thibaut, T.; Blanfuné, A.; Verlaque, M.; Boudouresque, C.-F.; Ruitton, S. The *Sargassum* Conundrum: Very Rare, Threatened or Locally Extinct in the NW Mediterranean and Still Lacking Protection. *Hydrobiologia* **2016**, *781*, 3–23. [CrossRef]

17. Auissi, M.; Sellam, L.N.; Boudouresque, C.F.; Blanfuné, A.; Derbal, F.; Frihi, H.; Perret-Boudouresque, M.; Rebzani-Zahaf, C.; Verlaque, M.; Thibaut, T. Insights into the Species Diversity of the Genus *Sargassum* (Phaeophyceae) in the Mediterranean Sea, with a Focus on a Pre-Viously Unnoticed Taxon from Algeria. *Medit. Mar. Sci.* **2018**, *19*, 48. [CrossRef]

18. Børgesen, F. The Species of *Sargassum* Found Along the Coasts of the Danish West Indies With Remarks Upon the Floating Forms of the Sargasso Sea. In *Mediteskrift I Anledning af Hundredaaret for Japetus Steenstrups Fødsel*; Jungersen, F.E., Warming, E., Eds.; Bianco Luno Bogtrykkeri: Kobenhavn, Denmark, 1914; Volume 32, pp. 1–20.

19. Børgesen, F. Marine Algae of the Danish West Indies. Part II. Phaeophyceae.; Dansk Bot. Arkiv, Bianco Luno Copenhagen, Denmark, 1914; Volume 2.

20. Winge, Ø. *The Sargasso Sea, Its Boundaries and Vegetation. Report on the Danish Oceanographical Expeditions 1908–1910 to the Mediterranean and Adjacent Seas.*; A.F. Høst&son.: Copenhagen, Denmark, 1923.

21. Parr, A.E. Quantitative Observations on the Pelagic *Sargassum* Vegetation of the Western North Atlantic. *Bull. Bingham Oceanogr. Collect.* **1939**, *6*, 1–94.

22. Butler, J.N.; Morris, B.F.; Cadwallader, J.; Stoner, A.W. *Studies of Sargassum and the Sargassum Community*; Bermuda Biological Station for Research, Special Publication: Bermuda, UK, 1983; pp. 1–307.

23. Baker, P.; Minzlaff, U.; Schoenle, A.; Schwabe, E.; Hohlfeld, M.; Jeuck, A.; Brenke, N.; Prausse, D.; Rothenbeck, M.; Brix, S.; et al. Potential Contribution of Surface-Dwelling *Sargassum* Algae to Deep-Sea Ecosystems in the Southern North Atlantic. *Deep. Sea Res. Part II Top. Stud. Oceanogr.* **2018**, *148*, 21–34. [CrossRef]

24. Ody, A.; Thibaut, T.; Berline, L.; Changeux, T.; André, J.-M.; Chevalier, C.; Blanfuné, A.; Blanchot, J.; Ruitton, S.; Stiger-Pouvreau, V.; et al. From In Situ to Satellite Observations of Pelagic *Sargassum* Distribution and Aggregation in the Tropical North Atlantic Ocean. *PLoS ONE* **2019**, *14*, e0222584. [CrossRef]

25. Coston-Clements, L.; Settle, L.R.; Hoss, D.E.; Cross, F.A. *Utilization of the Sargassum Habitat by Marine Invertebrates and Vertebrates, a Review*; US Department of Commerce, National Oceanic and Atmospheric Administration, National Marine Fisheries Service, Southeast Fisheries Science Center, Beaufort Laboratory: St. Petersburg, FL, USA, 1991; Volume 296.

26. Huffard, C.L.; Von Thun, S.; Sherman, A.D.; Sealey, K.; Smith, K.L. Pelagic *Sargassum* Community Change over a 40-Year Period: Temporal and Spatial Variability. *Mar. Biol.* **2014**, *161*, 2735–2751. [CrossRef] [PubMed]
27. Alleyne, K.S.T.; Johnson, D.; Neat, F.; Oxenford, H.A.; Vallès, H. Seasonal Variation in Morphotype Composition of Pelagic *Sargassum* Influx Events Is Linked to Oceanic Origin. *Sci. Rep.* **2023**, *13*, 3753. [CrossRef] [PubMed]

28. Theirlynck, T.; Mendonça, I.R.W.; Engelen, A.H.; Bolhuis, H.; Collado-Vides, L.; Van Tussenbroek, B.I.; García-Sánchez, M.; Zettler, E.; Muyzer, G.; Amaral-Zettler, L. Diversity of the Holopelagic *Sargassum* Microbiome from the Great Atlantic *Sargassum* Belt to Coastal Stranding Locations. *Harmful Algae* **2023**, *122*, 102369. [CrossRef] [PubMed]

29. Marmorino, G.O.; Miller, W.D.; Smith, G.B.; Bowles, J.H. Airborne Imagery of a Disintegrating *Sargassum* Drift Line. *Deep. Sea Res. Part I Oceanogr. Res. Pap.* **2011**, *58*, 316–321. [CrossRef]

30. Fidai, Y.A.; Dash, J.; Tompkins, E.L.; Tonon, T. A Systematic Review of Floating and Beach Landing Records of *Sargassum* beyond the Sargasso Sea. *Environ. Res. Commun.* **2020**, *2*, 122001. [CrossRef]

31. Wang, M.; Hu, C.; Barnes, B.B.; Mitchum, G.; Lapointe, B.; Montoya, J.P. The Great Atlantic *Sargassum* Belt. *Science* **2019**, *365*, 83–87. [CrossRef] [PubMed]

32. Franks, J.S.; Johnson, D.R.; Ko, D.S. Pelagic *Sargassum* in the Tropical North Atlantic. *GCR* **2016**, *27*, SC6–SC11. [CrossRef]

33. Schell, J.M.; Goodwin, D.S.; Siuda, A.N.S. Recent *Sargassum* Inundation Events in the Caribbean: Shipboard Observations Reveal Dominance of a Previously Rare Form. *Oceanography* **2015**, *28*, 8–10. [CrossRef]

34. Wang, M.; Hu, C. Predicting *Sargassum* Blooms in the Caribbean Sea from MODIS Observations. *Geophys. Res. Lett.* **2017**, *44*, 3265–3273. [CrossRef]

35. Marsh, R.; Addo, K.A.; Jayson-Quashigah, P.-N.; Oxenford, H.A.; Maxam, A.; Anderson, R.; Skliris, N.; Dash, J.; Tompkins, E.L. Seasonal Predictions of Holopelagic *Sargassum* Across the Tropical Atlantic Accounting for Uncertainty in Drivers and Processes: The SARTRAC Ensemble Forecast System. *Front. Mar. Sci.* **2021**, *8*, 722524.

36. Jouanno, J.; Moquet, J.-S.; Berline, L.; Radenac, M.-H.; Santini, W.; Changeux, T.; Thibaut, T.; Podlejski, W.; Ménard, F.; Martinez, J.-M.; et al. Evolution of the Riverine Nutrient Export to the Tropical Atlantic over the Last 15 Years: Is There a Link with *Sargassum* Proliferation? *Environ. Res. Lett.* **2021**, *16*, 034042. [CrossRef]

37. Trinanes, J.; Putman, N.F.; Goni, G.; Hu, C.; Wang, M. Monitoring Pelagic *Sargassum* Inundation Potential for Coastal Communities. *J. Oper. Oceanogr.* **2023**, *16*, 48–59. [CrossRef]

38. Hanisak, M.D.; Samuel, M.A. *Growth Rates in Culture of Several Species of Sargassum from Florida, USA*; Springer Netherlands: Sao Paulo, Brazil, 1987; pp. 399–404.

39. Changeux, T.; Berline, L.; Podlejski, W.; Guillot, T.; Stiger-Pouvreau, V.; Connan, S.; Thibaut, T. Variability in Growth and Tissue Composition (CNP, Natural Isotopes) of the Three Morphotypes of Holopelagic *Sargassum*. *Aquat. Bot.* **2023**, *187*, 103644. [CrossRef]

40. Corbin, M.; Oxenford, H.A. Assessing Growth of Pelagic *Sargassum* in the Tropical Atlantic. *Aquat. Bot.* **2023**, *187*, 103654. [CrossRef]

41. Magaña-Gallegos, E.; García-Sánchez, M.; Graham, C.; Olivos-Ortiz, A.; Siuda, A.N.S.; Van Tussenbroek, B.I. Growth Rates of Pelagic *Sargassum* Species in the Mexican Caribbean. *Aquat. Bot.* **2023**, *185*, 103614. [CrossRef]

42. Magaña-Gallegos, E.; Villegas-Muñoz, E.; Salas-Acosta, E.R.; Barba-Santos, M.G.; Silva, R.; Van Tussenbroek, B.I. The Effect of Temperature on the Growth of Holopelagic *Sargassum* Species. *Phycology* **2023**, *3*, 138–146. [CrossRef]

43. Schell, J.M.; Goodwin, D.S.; Volk, R.H.; Siuda, A.N.S. Preliminary Explorations of Environmental Tolerances and Growth Rates of Holopelagic *Sargassum* Morphotypes. *Aquat. Bot.* **2024**, *190*, 103723. [CrossRef]

44. Miron, P.; Olascoaga, M.J.; Beron-Vera, F.J.; Putman, N.F.; Triñanes, J.; Lumpkin, R.; Goni, G.J. Clustering of Marine-Debris- and *Sargassum* -Like Drifters Explained by Inertial Particle Dynamics. *Geophys. Res. Lett.* **2020**, *47*, e2020GL089874. [CrossRef]

45. Olascoaga, M.J.; Beron-Vera, F.J.; Beyea, R.T.; Bonner, G.; Castellucci, M.; Goni, G.J.; Guigand, C.; Putman, N.F. Physics-Informed Laboratory Estimation of *Sargassum* Windage. *Phys. Fluids* **2023**, *35*, 111702. [CrossRef]

46. García-Sánchez, M.; Graham, C.; Vera, E.; Escalante-Mancera, E.; Álvarez-Filip, L.; Van Tussenbroek, B.I. Temporal Changes in the Composition and Biomass of Beached Pelagic *Sargassum* Species in the Mexican Caribbean. *Aquat. Bot.* **2020**, *167*, 103275. [CrossRef]

47. Pante, E.; Puillandre, N.; Viricel, A.; Arnaud-Haond, S.; Aurelle, D.; Castelin, M.; Chenuil, A.; Destombe, C.; Forcioli, D.; Valero, M.; et al. Species Are Hypotheses: Avoid Connectivity Assessments Based on Pillars of Sand. *Mol. Ecol.* **2015**, *24*, 525–544. [CrossRef] [PubMed]

48. McNeill, J.; Barrie, F.R.; Buck, W.R.; Demoulin, V.; Greuter, W.; Hawksworth, D.L.; Herendeen, P.S.; Knapp, S.; Marhold, K.; Prado, J.; et al. (Eds.) *International Code of Nomenclature for Algae, Fungi, and Plants*; Regnum Vegetabile; Koeltz Botanical Books: Glashütten, Germany, 2018; Volume 159, ISBN 978-3-946583-16-5.

49. Taylor, W.R. *Marine Algae of the Eastern Tropical and Subtropical Coasts of the Americas*; The University of Michigan Press: Ann Arbor, MI, USA, 1960.

50. Uwai, S.; Kogame, K.; Yoshida, G.; Kawai, H.; Ajisaka, T. Geographical Genetic Structure and Phylogeography of the *Sargassum horneri/filicinum* Complex in Japan, Based on the Mitochondrial Cox3 Haplotype. *Mar. Biol.* **2009**, *156*, 901–911. [CrossRef]

51. Mattio, L.; Payri, C.E. 190 Years of *Sargassum* Taxonomy, Facing the Advent of DNA Phylogenies. *Bot. Rev.* **2011**, *77*, 31–70. [CrossRef]

52. Draisma, S.G.A.; Ballesteros, E.; Rousseau, F.; Thibaut, T. Dna Sequence Data Demonstrate The Polyphyly of The Genus *Cystoseira* and Other Sargassaceae Genera (Phaeophyceae) ¹. *J. Phycol.* **2010**, *46*, 1329–1345. [CrossRef]
27. Alleyne, K.S.T.; Johnson, D.; Neat, F.; Oxenford, H.A.; Vallès, H. Seasonal Variation in Morphotype Composition of Pelagic *Sargassum* Influx Events Is Linked to Oceanic Origin. *Sci. Rep.* **2023**, *13*, 3753. [CrossRef] [PubMed]

28. Theirlynck, T.; Mendonça, I.R.W.; Engelen, A.H.; Bolhuis, H.; Collado-Vides, L.; Van Tussenbroek, B.I.; García-Sánchez, M.; Zettler, E.; Muyzer, G.; Amaral-Zettler, L. Diversity of the Holopelagic *Sargassum* Microbiome from the Great Atlantic *Sargassum* Belt to Coastal Stranding Locations. *Harmful Algae* **2023**, *122*, 102369. [CrossRef] [PubMed]

29. Marmorino, G.O.; Miller, W.D.; Smith, G.B.; Bowles, J.H. Airborne Imagery of a Disintegrating *Sargassum* Drift Line. *Deep. Sea Res. Part I Oceanogr. Res. Pap.* **2011**, *58*, 316–321. [CrossRef]

30. Fidai, Y.A.; Dash, J.; Tompkins, E.L.; Tonon, T. A Systematic Review of Floating and Beach Landing Records of *Sargassum* beyond the Sargasso Sea. *Environ. Res. Commun.* **2020**, *2*, 122001. [CrossRef]

31. Wang, M.; Hu, C.; Barnes, B.B.; Mitchum, G.; Lapointe, B.; Montoya, J.P. The Great Atlantic *Sargassum* Belt. *Science* **2019**, *365*, 83–87. [CrossRef] [PubMed]

32. Franks, J.S.; Johnson, D.R.; Ko, D.S. Pelagic *Sargassum* in the Tropical North Atlantic. *GCR* **2016**, *27*, SC6–SC11. [CrossRef]

33. Schell, J.M.; Goodwin, D.S.; Siuda, A.N.S. Recent *Sargassum* Inundation Events in the Caribbean: Shipboard Observations Reveal Dominance of a Previously Rare Form. *Oceanography* **2015**, *28*, 8–10. [CrossRef]

34. Wang, M.; Hu, C. Predicting *Sargassum* Blooms in the Caribbean Sea from MODIS Observations. *Geophys. Res. Lett.* **2017**, *44*, 3265–3273. [CrossRef]

35. Marsh, R.; Addo, K.A.; Jayson-Quashigah, P.-N.; Oxenford, H.A.; Maxam, A.; Anderson, R.; Skliris, N.; Dash, J.; Tompkins, E.L. Seasonal Predictions of Holopelagic *Sargassum* Across the Tropical Atlantic Accounting for Uncertainty in Drivers and Processes: The SARTRAC Ensemble Forecast System. *Front. Mar. Sci.* **2021**, *8*, 722524.

36. Jouanno, J.; Moquet, J.-S.; Berline, L.; Radenac, M.-H.; Santini, W.; Changeux, T.; Thibaut, T.; Podlejski, W.; Ménard, F.; Martinez, J.-M.; et al. Evolution of the Riverine Nutrient Export to the Tropical Atlantic over the Last 15 Years: Is There a Link with *Sargassum* Proliferation? *Environ. Res. Lett.* **2021**, *16*, 034042. [CrossRef]

37. Trinanes, J.; Putman, N.F.; Goni, G.; Hu, C.; Wang, M. Monitoring Pelagic *Sargassum* Inundation Potential for Coastal Communities. *J. Oper. Oceanogr.* **2023**, *16*, 48–59. [CrossRef]

38. Hanisak, M.D.; Samuel, M.A. *Growth Rates in Culture of Several Species of Sargassum from Florida, USA*; Springer Netherlands: Sao Paulo, Brazil, 1987; pp. 399–404.

39. Changeux, T.; Berline, L.; Podlejski, W.; Guillot, T.; Stiger-Pouvreau, V.; Connan, S.; Thibaut, T. Variability in Growth and Tissue Composition (CNP, Natural Isotopes) of the Three Morphotypes of Holopelagic *Sargassum*. *Aquat. Bot.* **2023**, *187*, 103644. [CrossRef]

40. Corbin, M.; Oxenford, H.A. Assessing Growth of Pelagic *Sargassum* in the Tropical Atlantic. *Aquat. Bot.* **2023**, *187*, 103654. [CrossRef]

41. Magaña-Gallegos, E.; García-Sánchez, M.; Graham, C.; Olivos-Ortiz, A.; Siuda, A.N.S.; Van Tussenbroek, B.I. Growth Rates of Pelagic *Sargassum* Species in the Mexican Caribbean. *Aquat. Bot.* **2023**, *185*, 103614. [CrossRef]

42. Magaña-Gallegos, E.; Villegas-Muñoz, E.; Salas-Acosta, E.R.; Barba-Santos, M.G.; Silva, R.; Van Tussenbroek, B.I. The Effect of Temperature on the Growth of Holopelagic *Sargassum* Species. *Phycology* **2023**, *3*, 138–146. [CrossRef]

43. Schell, J.M.; Goodwin, D.S.; Volk, R.H.; Siuda, A.N.S. Preliminary Explorations of Environmental Tolerances and Growth Rates of Holopelagic *Sargassum* Morphotypes. *Aquat. Bot.* **2024**, *190*, 103723. [CrossRef]

44. Miron, P.; Olascoaga, M.J.; Beron-Vera, F.J.; Putman, N.F.; Triñanes, J.; Lumpkin, R.; Goni, G.J. Clustering of Marine-Debris- and *Sargassum* -Like Drifters Explained by Inertial Particle Dynamics. *Geophys. Res. Lett.* **2020**, *47*, e2020GL089874. [CrossRef]

45. Olascoaga, M.J.; Beron-Vera, F.J.; Beyea, R.T.; Bonner, G.; Castellucci, M.; Goni, G.J.; Guigand, C.; Putman, N.F. Physics-Informed Laboratory Estimation of *Sargassum* Windage. *Phys. Fluids* **2023**, *35*, 111702. [CrossRef]

46. García-Sánchez, M.; Graham, C.; Vera, E.; Escalante-Mancera, E.; Álvarez-Filip, L.; Van Tussenbroek, B.I. Temporal Changes in the Composition and Biomass of Beached Pelagic *Sargassum* Species in the Mexican Caribbean. *Aquat. Bot.* **2020**, *167*, 103275. [CrossRef]

47. Pante, E.; Puillandre, N.; Viricel, A.; Arnaud-Haond, S.; Aurelle, D.; Castelin, M.; Chenuil, A.; Destombe, C.; Forcioli, D.; Valero, M.; et al. Species Are Hypotheses: Avoid Connectivity Assessments Based on Pillars of Sand. *Mol. Ecol.* **2015**, *24*, 525–544. [CrossRef] [PubMed]

48. McNeill, J.; Barrie, F.R.; Buck, W.R.; Demoulin, V.; Greuter, W.; Hawksworth, D.L.; Herendeen, P.S.; Knapp, S.; Marhold, K.; Prado, J.; et al. (Eds.) *International Code of Nomenclature for Algae, Fungi, and Plants*; Regnum Vegetabile; Koeltz Botanical Books: Glashütten, Germany, 2018; Volume 159, ISBN 978-3-946583-16-5.

49. Taylor, W.R. *Marine Algae of the Eastern Tropical and Subtropical Coasts of the Americas*; The University of Michigan Press: Ann Arbor, MI, USA, 1960.

50. Uwai, S.; Kogame, K.; Yoshida, G.; Kawai, H.; Ajisaka, T. Geographical Genetic Structure and Phylogeography of the *Sargassum horneri/filicinum* Complex in Japan, Based on the Mitochondrial Cox3 Haplotype. *Mar. Biol.* **2009**, *156*, 901–911. [CrossRef]

51. Mattio, L.; Payri, C.E. 190 Years of *Sargassum* Taxonomy, Facing the Advent of DNA Phylogenies. *Bot. Rev.* **2011**, *77*, 31–70. [CrossRef]

52. Draisma, S.G.A.; Ballesteros, E.; Rousseau, F.; Thibaut, T. Dna Sequence Data Demonstrate The Polyphyly of The Genus *Cystoseira* and Other Sargassaceae Genera (Phaeophyceae) ¹. *J. Phycol.* **2010**, *46*, 1329–1345. [CrossRef]
53. Phillips, N.; Fredericq, S. Biogeographic and Phylogenetic Investigations of the Pantropical Genus *Sargassum* (Fucales, Phaeophyceae) with Respect to Gulf of Mexico Species. *Gulf Mex. Sci.* **2000**, *18*, 1. [CrossRef]

54. Camacho, O.; Mattio, L.; Draisma, S.; Fredericq, S.; Diaz-Pulido, G. Morphological and Molecular Assessment of *Sargassum* (Fucales, Phaeophyceae) from Caribbean Colombia, Including the Proposal of *Sargassum Giganteum* Sp. Nov., *Sargassum Schnetteri* Comb. Nov. and *Sargassum* Section *Cladophyllum* Sect. Nov. *Syst. Biodivers.* **2015**, *13*, 105–130. [CrossRef]

55. Sissini, M.N.; De Barros Barreto, M.B.B.; Széchy, M.T.M.; De Lucena, M.B.; Oliveira, M.C.; Gower, J.; Liu, G.; De Oliveira Bastos, E.; Milstein, D.; Gusmão, F.; et al. The Floating *Sargassum* (Phaeophyceae) of the South Atlantic Ocean—Likely Scenarios. *Phycologia* **2017**, *56*, 321–328. [CrossRef]

56. González-Nieto, D.; Oliveira, M.C.; Núñez Resendiz, M.L.; Dreckmann, K.M.; Mateo-Cid, L.E.; Sentíes, A. Molecular Assessment of the Genus *Sargassum* (Fucales, Phaeophyceae) from the Mexican Coasts of the Gulf of Mexico and Caribbean, with the Description of *S. xochitlae* Sp. Nov. *Phytotaxa* **2020**, *461*, 254–274. [CrossRef]

57. Amaral-Zettler, L.A.; Dragone, N.B.; Schell, J.; Slikas, B.; Murphy, L.G.; Morrall, C.E.; Zettler, E.R. Comparative Mitochondrial and Chloroplast Genomics of a Genetically Distinct Form of *Sargassum* Contributing to Recent "Golden Tides" in the Western Atlantic. *Ecol. Evol.* **2017**, *7*, 516–525. [CrossRef] [PubMed]

58. Dibner, S.; Martin, L.; Thibaut, T.; Aurelle, D.; Blanfuné, A.; Whittaker, K.; Cooney, L.; Schell, J.M.; Goodwin, D.S.; Siuda, A.N.S. Consistent Genetic Divergence Observed among Pelagic *Sargassum* Morphotypes in the Western North Atlantic. *Mar. Ecol.* **2022**, *43*, e12691. [CrossRef]

59. Thiers, B. Index Herbariorum: A Global Directory of Public Herbaria and Associated Staff. New York Botanical Garden's Virtual Herbarium. Available online: https://sweetgum.nybg.org/science/ih/ (accessed on 24 April 2024).

60. Taylor, W.R. *The Marine Algae of Florida with Special Reference to the Dry Tortugas Carnegie*; Institution of Washington: Washington, DC, USA, 1928; Volume 379.

61. Taylor, W.R. *Caribbean Marine Algae of the Allan Hancock Expedition, 1939*; The University of Southern California Press: Los Angeles, CA, USA, 1942.

62. Dawes, C.J. *Marine Algae of the West Coast of Florida*; University of Miami Press: Oxford, OH, USA, 1974.

63. Littler, D.S.; Littler, M.M. *Caribbean Reefs Plants*; Offshore Graphics, Inc.: Deerfield Beach, FL, USA, 2000.

64. John, D.M.; Lawson, G.W.; Ameka, G. The Marine Macroalgae of the Tropical Africa Sub-Region. *Nova Hedwig.* **2003**, *iv*, 1–217.

65. Dawes, C.J.; Mathieson, A.C. *The Seaweeds of Florida*; University Press of Florida: Gainesville, FL, USA, 2008.

66. Clarke, K.R.; Warwick, R.M. *Changes in Marine Communities: An Approach to Statistical Analysis and Interpretation*; Plymouth Marine Laboratory: Plymouth, UK, 1994.

67. Anderson, M.J. Permutation Tests for Univariate or Multivariate Analysis of Variance and Regression. *Can. J. Fish. Aquat. Sci.* **2001**, *58*, 626–639. [CrossRef]

68. Clarke, K.R.; Gorley, R.N. *Primer v6: User Manual/Tutorial*.; Primer-E Ltd.: Plymouth, UK, 2006.

69. Yoon, H.S.; Hackett, J.D.; Bhattacharya, D. A Single Origin of the Peridinin- and Fucoxanthin-Containing Plastids in Dinoflagellates through Tertiary Endosymbiosis. *Proc. Natl. Acad. Sci. USA* **2002**, *99*, 11724–11729. [CrossRef] [PubMed]

70. Hu, Z.; Uwai, S.; Yu, S.; Komatsu, T.; Ajisaka, T.; Duan, D. Phylogeographic Heterogeneity of the Brown Macroalga *Sargassum horneri* (Fucaceae) in the Northwestern Pacific in Relation to Late Pleistocene Glaciation and Tectonic Configurations. *Mol. Ecol.* **2011**, *20*, 3894–3909. [CrossRef] [PubMed]

71. Okonechnikov, K.; Golosova, O.; Fursov, M.; the UGENE team. Unipro UGENE: A Unified Bioinformatics Toolkit. *Bioinformatics* **2012**, *28*, 1166–1167. [CrossRef] [PubMed]

72. Edgar, R.C. MUSCLE: Multiple Sequence Alignment with High Accuracy and High Throughput. *Nucleic Acids Res.* **2004**, *32*, 1792–1797. [CrossRef] [PubMed]

73. Nguyen, L.-T.; Schmidt, H.A.; Von Haeseler, A.; Minh, B.Q. IQ-TREE: A Fast and Effective Stochastic Algorithm for Estimating Maximum-Likelihood Phylogenies. *Mol. Biol. Evol.* **2015**, *32*, 268–274. [CrossRef] [PubMed]

74. Huelsenbeck, J.P.; Ronquist, F. MRBAYES: Bayesian Inference of Phylogenetic Trees. *Bioinformatics* **2001**, *17*, 754–755. [CrossRef] [PubMed]

75. Ronquist, F.; Huelsenbeck, J.P. MrBayes 3: Bayesian Phylogenetic Inference under Mixed Models. *Bioinformatics* **2003**, *19*, 1572–1574. [CrossRef] [PubMed]

76. Hoang, D.T.; Chernomor, O.; Von Haeseler, A.; Minh, B.Q.; Vinh, L.S. UFBoot2: Improving the Ultrafast Bootstrap Approximation. *Mol. Biol. Evol.* **2018**, *35*, 518–522. [CrossRef] [PubMed]

77. Lanfear, R.; Calcott, B.; Ho, S.Y.W.; Guindon, S. PartitionFinder: Combined Selection of Partitioning Schemes and Substitution Models for Phylogenetic Analyses. *Mol. Biol. Evol.* **2012**, *29*, 1695–1701. [CrossRef]

78. Rambaut, A. FigTree v1. 4. 2012.

79. Silberfeld, T.; Leigh, J.W.; Verbruggen, H.; Cruaud, C.; De Reviers, B.; Rousseau, F. A Multi-Locus Time-Calibrated Phylogeny of the Brown Algae (Heterokonta, Ochrophyta, Phaeophyceae): Investigating the Evolutionary Nature of the "Brown Algal Crown Radiation". *Mol. Phylogenetics Evol.* **2010**, *56*, 659–674. [CrossRef] [PubMed]

80. Clement, M.; Posada, D.C.K.A.; Crandall, K.A. TCS: A Computer Program to Estimate Gene Genealogies. *Mol. Ecol.* **2000**, *9*, 1657–1659. [CrossRef] [PubMed]

81. De Queiroz, K. Species Concepts and Species Delimitation. *Syst. Biol.* **2007**, *56*, 879–886. [CrossRef] [PubMed]
53. Phillips, N.; Fredericq, S. Biogeographic and Phylogenetic Investigations of the Pantropical Genus *Sargassum* (Fucales, Phaeophyceae) with Respect to Gulf of Mexico Species. *Gulf Mex. Sci.* **2000**, *18*, 1. [CrossRef]

54. Camacho, O.; Mattio, L.; Draisma, S.; Fredericq, S.; Diaz-Pulido, G. Morphological and Molecular Assessment of *Sargassum* (Fucales, Phaeophyceae) from Caribbean Colombia, Including the Proposal of *Sargassum Giganteum* Sp. Nov., *Sargassum Schnetteri* Comb. Nov. and *Sargassum* Section *Cladophyllum* Sect. Nov. *Syst. Biodivers.* **2015**, *13*, 105–130. [CrossRef]

55. Sissini, M.N.; De Barros Barreto, M.B.B.; Széchy, M.T.M.; De Lucena, M.B.; Oliveira, M.C.; Gower, J.; Liu, G.; De Oliveira Bastos, E.; Milstein, D.; Gusmão, F.; et al. The Floating *Sargassum* (Phaeophyceae) of the South Atlantic Ocean—Likely Scenarios. *Phycologia* **2017**, *56*, 321–328. [CrossRef]

56. González-Nieto, D.; Oliveira, M.C.; Núñez Resendiz, M.L.; Dreckmann, K.M.; Mateo-Cid, L.E.; Sentíes, A. Molecular Assessment of the Genus *Sargassum* (Fucales, Phaeophyceae) from the Mexican Coasts of the Gulf of Mexico and Caribbean, with the Description of *S. xochitlae* Sp. Nov. *Phytotaxa* **2020**, *461*, 254–274. [CrossRef]

57. Amaral-Zettler, L.A.; Dragone, N.B.; Schell, J.; Slikas, B.; Murphy, L.G.; Morrall, C.E.; Zettler, E.R. Comparative Mitochondrial and Chloroplast Genomics of a Genetically Distinct Form of *Sargassum* Contributing to Recent "Golden Tides" in the Western Atlantic. *Ecol. Evol.* **2017**, *7*, 516–525. [CrossRef] [PubMed]

58. Dibner, S.; Martin, L.; Thibaut, T.; Aurelle, D.; Blanfuné, A.; Whittaker, K.; Cooney, L.; Schell, J.M.; Goodwin, D.S.; Siuda, A.N.S. Consistent Genetic Divergence Observed among Pelagic *Sargassum* Morphotypes in the Western North Atlantic. *Mar. Ecol.* **2022**, *43*, e12691. [CrossRef]

59. Thiers, B. Index Herbariorum: A Global Directory of Public Herbaria and Associated Staff. New York Botanical Garden's Virtual Herbarium. Available online: https://sweetgum.nybg.org/science/ih/ (accessed on 24 April 2024).

60. Taylor, W.R. *The Marine Algae of Florida with Special Reference to the Dry Tortugas Carnegie*; Institution of Washington: Washington, DC, USA, 1928; Volume 379.

61. Taylor, W.R. *Caribbean Marine Algae of the Allan Hancock Expedition, 1939*; The University of Southern California Press: Los Angeles, CA, USA, 1942.

62. Dawes, C.J. *Marine Algae of the West Coast of Florida*; University of Miami Press: Oxford, OH, USA, 1974.

63. Littler, D.S.; Littler, M.M. *Caribbean Reefs Plants*; Offshore Graphics, Inc.: Deerfield Beach, FL, USA, 2000.

64. John, D.M.; Lawson, G.W.; Ameka, G. The Marine Macroalgae of the Tropical Africa Sub-Region. *Nova Hedwig.* **2003**, *iv*, 1–217.

65. Dawes, C.J.; Mathieson, A.C. *The Seaweeds of Florida*; University Press of Florida: Gainesville, FL, USA, 2008.

66. Clarke, K.R.; Warwick, R.M. *Changes in Marine Communities: An Approach to Statistical Analysis and Interpretation*; Plymouth Marine Laboratory: Plymouth, UK, 1994.

67. Anderson, M.J. Permutation Tests for Univariate or Multivariate Analysis of Variance and Regression. *Can. J. Fish. Aquat. Sci.* **2001**, *58*, 626–639. [CrossRef]

68. Clarke, K.R.; Gorley, R.N. *Primer v6: User Manual/Tutorial*.; Primer-E Ltd.: Plymouth, UK, 2006.

69. Yoon, H.S.; Hackett, J.D.; Bhattacharya, D. A Single Origin of the Peridinin- and Fucoxanthin-Containing Plastids in Dinoflagellates through Tertiary Endosymbiosis. *Proc. Natl. Acad. Sci. USA* **2002**, *99*, 11724–11729. [CrossRef] [PubMed]

70. Hu, Z.; Uwai, S.; Yu, S.; Komatsu, T.; Ajisaka, T.; Duan, D. Phylogeographic Heterogeneity of the Brown Macroalga *Sargassum horneri* (Fucaceae) in the Northwestern Pacific in Relation to Late Pleistocene Glaciation and Tectonic Configurations. *Mol. Ecol.* **2011**, *20*, 3894–3909. [CrossRef] [PubMed]

71. Okonechnikov, K.; Golosova, O.; Fursov, M.; the UGENE team. Unipro UGENE: A Unified Bioinformatics Toolkit. *Bioinformatics* **2012**, *28*, 1166–1167. [CrossRef] [PubMed]

72. Edgar, R.C. MUSCLE: Multiple Sequence Alignment with High Accuracy and High Throughput. *Nucleic Acids Res.* **2004**, *32*, 1792–1797. [CrossRef] [PubMed]

73. Nguyen, L.-T.; Schmidt, H.A.; Von Haeseler, A.; Minh, B.Q. IQ-TREE: A Fast and Effective Stochastic Algorithm for Estimating Maximum-Likelihood Phylogenies. *Mol. Biol. Evol.* **2015**, *32*, 268–274. [CrossRef] [PubMed]

74. Huelsenbeck, J.P.; Ronquist, F. MRBAYES: Bayesian Inference of Phylogenetic Trees. *Bioinformatics* **2001**, *17*, 754–755. [CrossRef] [PubMed]

75. Ronquist, F.; Huelsenbeck, J.P. MrBayes 3: Bayesian Phylogenetic Inference under Mixed Models. *Bioinformatics* **2003**, *19*, 1572–1574. [CrossRef] [PubMed]

76. Hoang, D.T.; Chernomor, O.; Von Haeseler, A.; Minh, B.Q.; Vinh, L.S. UFBoot2: Improving the Ultrafast Bootstrap Approximation. *Mol. Biol. Evol.* **2018**, *35*, 518–522. [CrossRef] [PubMed]

77. Lanfear, R.; Calcott, B.; Ho, S.Y.W.; Guindon, S. PartitionFinder: Combined Selection of Partitioning Schemes and Substitution Models for Phylogenetic Analyses. *Mol. Biol. Evol.* **2012**, *29*, 1695–1701. [CrossRef]

78. Rambaut, A. FigTree v1. 4. 2012.

79. Silberfeld, T.; Leigh, J.W.; Verbruggen, H.; Cruaud, C.; De Reviers, B.; Rousseau, F. A Multi-Locus Time-Calibrated Phylogeny of the Brown Algae (Heterokonta, Ochrophyta, Phaeophyceae): Investigating the Evolutionary Nature of the "Brown Algal Crown Radiation". *Mol. Phylogenetics Evol.* **2010**, *56*, 659–674. [CrossRef] [PubMed]

80. Clement, M.; Posada, D.C.K.A.; Crandall, K.A. TCS: A Computer Program to Estimate Gene Genealogies. *Mol. Ecol.* **2000**, *9*, 1657–1659. [CrossRef] [PubMed]

81. De Queiroz, K. Species Concepts and Species Delimitation. *Syst. Biol.* **2007**, *56*, 879–886. [CrossRef] [PubMed]
82. Watanabe, K.; Homma, Y.; Karakisawa, H.; Ishikawa, R.; Uwai, S. Haplotypic Differentiation between Seasonal Populations of *Sargassum horneri* (Fucales, Phaeophyceae) in Japan. *Phycol. Res.* **2019**, *67*, 59–64. [CrossRef]

83. Sonsthagen, S.A.; Wilson, R.E.; Chesser, R.T.; Pons, J.-M.; Crochet, P.-A.; Driskell, A.; Dove, C. Recurrent Hybridization and Recent Origin Obscure Phylogenetic Relationships within the 'White-Headed' Gull (*Larus* Sp.) Complex. *Mol. Phylogenetics Evol.* **2016**, *103*, 41–54. [CrossRef] [PubMed]

84. Tuffisantos, L.; Meira, R.; Ferreira, F.; Santannasantos, B.; Ferreira, L. Morphological Responses of Different Eucalypt Clones Submitted to Glyphosate Drift. *Environ. Exp. Bot.* **2007**, *59*, 11–20. [CrossRef]

85. Stiger, V.; Horiguchi, T.; Yoshida, T.; Coleman, A.W.; Masuda, M. Phylogenetic Relationships of *Sargassum* (Sargassaceae, Phaeophyceae) with Reference to a Taxonomic Revision of the Section Phyllocystae Based on ITS-2 nrDNA Sequences. *Phycol. Res.* **2000**, *48*, 251–260. [CrossRef]

86. Yoshida, T.; Stiger, V.; Horiguchi, T. *Sargassum boreale* sp. Nov. (Fucales, Phaeophyceae) from Hokkaido, Japan. *Phycol. Res.* **2000**, *48*, 125–131. [CrossRef]

87. Mattio, L.; Payri, C.E.; Stiger-Pouvreau, V. Taxonomic Revision of *Sargassum* (Fucales, Phaeophyceae) from French Polynesia Based on Morphological And Molecular Analyses¹. *J. Phycol.* **2008**, *44*, 1541–1555. [CrossRef] [PubMed]

88. Rohfritsch, A.; Payri, C.; Stiger, V.; Bonhomme, F. Molecular and Morphological Relationships between Two Closely Related Species, *Turbinaria ornata* and *T. conoides* (Sargassaceae, Phaeophyceae). *Biochem. Syst. Ecol.* **2007**, *35*, 91–98. [CrossRef]

89. Deacon, G.E.R. The Sargasso Sea. *Geogr. J.* **1942**, *99*, 16–28. [CrossRef]

90. Ryther, J.H. The Sargasso Sea. *Sci. Am.* **1956**, *194*, 98–108. [CrossRef]

91. Woelkerling, W.J. On the Epibiotic and Pelagic Chlorophyceae, Phaeophyceae and Rhodophyceae of the Western Sargasso Sea. *Rhodora* **1975**, *77*, 1–40.

92. Butler, J.N.; Stoner, A.W. Pelagic *Sargassum*: Has its Biomass Changed in the Last 50 Years? *Deep. Sea Res.* **1984**, *31*, 1259–1264. [CrossRef]

93. Niermann, U.; Anders, H.-G.; John, H.-C. Distribution and Abundance of Pelagic *Sargassum* in Spring 1979. *Senckenberg. Marit* **1986**, *17*, 293–302.

94. Conover, J.T.; Sieburth, M.J. Effect of *Sargassum* Distribution on its Epibiota and Antibacterial Activity. *Bot. Mar.* **1964**, *6*, 147–156. [CrossRef]

95. Ryland, J.S. Observations on Some Epibionts of Gulf-Weed, *Sargassum natans* (I.) Meyen. *J. Exp. Mar. Biol. Ecol.* **1974**, *14*, 17–25. [CrossRef]

96. Niermann, U. Distribution of *Sargassum natans* and Some of its Epibionts in the Sargasso Sea. *Helgol. Meeresunters.* **1986**, *40*, 343–353. [CrossRef]

97. Calder, D.R. Hydroid Assemblages on Holopelagic *Sargassum* From the Sargasso Sea at Bermuda. *Bull. Mar. Sci.* **1995**, *56*, 537–546.

98. Weis, J.S. Fauna Associated with Pelagic *Sargassum* in the Gulf Stream. *Am. Midl. Nat.* **1968**, *80*, 554–558. [CrossRef]

99. Fine, M.L. Faunal Variation on Pelagic *Sargassum*. *Mar. Biol.* **1970**, *7*, 112–122. [CrossRef]

100. Stoner, A.W.; Greening, H.S. Geographic Variation in the Macrofauna Associates of Pelagic *Sargassum* and Some Biogeographic Implications. *Mar. Ecol. Prog. Ser.* **1984**, *20*, 185–192. [CrossRef]

101. Howard, K.L.; Menzies, R.J. Distribution and Production of *Sargassum* in the Waters off the Carolina Coast. *Bot. Mar.* **1967**, *12*, 244–254. [CrossRef]

102. Lapointe, B.E. Phosphorous-Limited Photosynthesis and Growth of *Sargassum natans* and *Sargassum fluitans* (Phaeophyceae) in the Western North Atlantic. *Deep. Sea Res.* **1986**, *33*, 391–399. [CrossRef]

103. Wynne, M.J. A Checklist of Benthic Marine Algae of the Tropical and Subtropical Western Atlantic. *Can. J. Bot.* **1986**, *64*, 2239–2281. [CrossRef]

104. Schneider, C.W.; Searles, R.B. Seaweeds of the Southeastern United States: Cape Hatteras to Cape Canaveral. Duke University Press: Durham, NC, USA, 1991; 584p.

105. de Szechy, M.T.M.; Guedes, P.M.; Baeta-Neves, M.H.; Oliveira, E.N. Verification of *Sargassum natans* (Linnaeus) Gaillon (Heterokontophyta: Phaeophyceae) from the Sargasso Sea Off the Coast of Brazil, Western Atlantic Ocean. *Checklist 2012*, *8*, 638–641. [CrossRef]

106. Lapointe, B.E.; West, L.E.; Sutton, T.T.; Hu, C. Ryther Revisited: Nutrient Excretions by Fishes Enhance Productivity of Pelagic *Sargassum* in the Western North Atlantic Ocean. *J. Exp. Mar. Biol. Ecol.* **2014**, *458*, 46–56. [CrossRef]

107. Doyle, E.; Franks, J. *Sargassum* Factsheet. Gulf and Caribbean Fisheries Institute: Guadeloupe, French, 2015; p. 4. Available online: https://www.gcfi.org/sargassum-influx/ (accessed on 27 February 2024).

108. Wynne, M.J. A Checklist of Benthic Marine Algae of the Tropical and Subtropical Western Atlantic: Fifth Revision Nova Hedwigia Beiheft, 153, Bomtraeger Science Publishers, Stuttgart, Germany; 2022. 180 pp.

109. Govindarajan, A.F.; Cooney, L.; Whittaker, K.; Bloch, D.; Burdorf, R.M.; Canning, S.; Carter, C.; Cellan, S.M.; Eriksson, F.A.A.; Freyer, H.; et al. The Distribution and Mitochondrial Genotype of the Hydroid *Aglaophenia Latecarinata* Is Correlated with Its Pelagic *Sargassum* Substrate Type in the Tropical and Subtropical Western Atlantic Ocean. *PeerJ* **2019**, *7*, e7814. [CrossRef] [PubMed]

110. Martin, L.M.; Taylor, M.; Huston, G.; Goodwin, D.S.; Schell, J.M.; Siuda, A.N.S. Pelagic *Sargassum* Morphotypes Support Different Rafting Motile Epifauna Communities. *Mar. Biol.* **2021**, *168*, 115. [CrossRef]

111. McGillicuddy, D.J.; Morton, P.L.; Brewton, R.A.; Hu, C.; Kelly, T.B.; Solow, A.R.; Lapointe, B.E. Nutrient and Arsenic Biogeochemistry of *Sargassum* in the Western Atlantic. *Nat. Commun.* **2023**, *14*, 6205. [CrossRef] [PubMed]
82. Watanabe, K.; Homma, Y.; Karakisawa, H.; Ishikawa, R.; Uwai, S. Haplotypic Differentiation between Seasonal Populations of *Sargassum horneri* (Fucales, Phaeophyceae) in Japan. *Phycol. Res.* **2019**, *67*, 59–64. [CrossRef]

83. Sonsthagen, S.A.; Wilson, R.E.; Chesser, R.T.; Pons, J.-M.; Crochet, P.-A.; Driskell, A.; Dove, C. Recurrent Hybridization and Recent Origin Obscure Phylogenetic Relationships within the 'White-Headed' Gull (*Larus* Sp.) Complex. *Mol. Phylogenetics Evol.* **2016**, *103*, 41–54. [CrossRef] [PubMed]

84. Tuffisantos, L.; Meira, R.; Ferreira, F.; Santannasantos, B.; Ferreira, L. Morphological Responses of Different Eucalypt Clones Submitted to Glyphosate Drift. *Environ. Exp. Bot.* **2007**, *59*, 11–20. [CrossRef]

85. Stiger, V.; Horiguchi, T.; Yoshida, T.; Coleman, A.W.; Masuda, M. Phylogenetic Relationships of *Sargassum* (Sargassaceae, Phaeophyceae) with Reference to a Taxonomic Revision of the Section Phyllocystae Based on ITS-2 nrDNA Sequences. *Phycol. Res.* **2000**, *48*, 251–260. [CrossRef]

86. Yoshida, T.; Stiger, V.; Horiguchi, T. *Sargassum boreale* sp. Nov. (Fucales, Phaeophyceae) from Hokkaido, Japan. *Phycol. Res.* **2000**, *48*, 125–131. [CrossRef]

87. Mattio, L.; Payri, C.E.; Stiger-Pouvreau, V. Taxonomic Revision of *Sargassum* (Fucales, Phaeophyceae) from French Polynesia Based on Morphological And Molecular Analyses¹. *J. Phycol.* **2008**, *44*, 1541–1555. [CrossRef] [PubMed]

88. Rohfritsch, A.; Payri, C.; Stiger, V.; Bonhomme, F. Molecular and Morphological Relationships between Two Closely Related Species, *Turbinaria ornata* and *T. conoides* (Sargassaceae, Phaeophyceae). *Biochem. Syst. Ecol.* **2007**, *35*, 91–98. [CrossRef]

89. Deacon, G.E.R. The Sargasso Sea. *Geogr. J.* **1942**, *99*, 16–28. [CrossRef]

90. Ryther, J.H. The Sargasso Sea. *Sci. Am.* **1956**, *194*, 98–108. [CrossRef]

91. Woelkerling, W.J. On the Epibiotic and Pelagic Chlorophyceae, Phaeophyceae and Rhodophyceae of the Western Sargasso Sea. *Rhodora* **1975**, *77*, 1–40.

92. Butler, J.N.; Stoner, A.W. Pelagic *Sargassum*: Has its Biomass Changed in the Last 50 Years? *Deep. Sea Res.* **1984**, *31*, 1259–1264. [CrossRef]

93. Niermann, U.; Anders, H.-G.; John, H.-C. Distribution and Abundance of Pelagic *Sargassum* in Spring 1979. *Senckenberg. Marit* **1986**, *17*, 293–302.

94. Conover, J.T.; Sieburth, M.J. Effect of *Sargassum* Distribution on its Epibiota and Antibacterial Activity. *Bot. Mar.* **1964**, *6*, 147–156. [CrossRef]

95. Ryland, J.S. Observations on Some Epibionts of Gulf-Weed, *Sargassum natans* (I.) Meyen. *J. Exp. Mar. Biol. Ecol.* **1974**, *14*, 17–25. [CrossRef]

96. Niermann, U. Distribution of *Sargassum natans* and Some of its Epibionts in the Sargasso Sea. *Helgol. Meeresunters.* **1986**, *40*, 343–353. [CrossRef]

97. Calder, D.R. Hydroid Assemblages on Holopelagic *Sargassum* From the Sargasso Sea at Bermuda. *Bull. Mar. Sci.* **1995**, *56*, 537–546.

98. Weis, J.S. Fauna Associated with Pelagic *Sargassum* in the Gulf Stream. *Am. Midl. Nat.* **1968**, *80*, 554–558. [CrossRef]

99. Fine, M.L. Faunal Variation on Pelagic *Sargassum*. *Mar. Biol.* **1970**, *7*, 112–122. [CrossRef]

100. Stoner, A.W.; Greening, H.S. Geographic Variation in the Macrofauna Associates of Pelagic *Sargassum* and Some Biogeographic Implications. *Mar. Ecol. Prog. Ser.* **1984**, *20*, 185–192. [CrossRef]

101. Howard, K.L.; Menzies, R.J. Distribution and Production of *Sargassum* in the Waters off the Carolina Coast. *Bot. Mar.* **1967**, *12*, 244–254. [CrossRef]

102. Lapointe, B.E. Phosphorous-Limited Photosynthesis and Growth of *Sargassum natans* and *Sargassum fluitans* (Phaeophyceae) in the Western North Atlantic. *Deep. Sea Res.* **1986**, *33*, 391–399. [CrossRef]

103. Wynne, M.J. A Checklist of Benthic Marine Algae of the Tropical and Subtropical Western Atlantic. *Can. J. Bot.* **1986**, *64*, 2239–2281. [CrossRef]

104. Schneider, C.W.; Searles, R.B. Seaweeds of the Southeastern United States: Cape Hatteras to Cape Canaveral. Duke University Press: Durham, NC, USA, 1991; 584p.

105. de Szechy, M.T.M.; Guedes, P.M.; Baeta-Neves, M.H.; Oliveira, E.N. Verification of *Sargassum natans* (Linnaeus) Gaillon (Heterokontophyta: Phaeophyceae) from the Sargasso Sea Off the Coast of Brazil, Western Atlantic Ocean. *Checklist 2012*, *8*, 638–641. [CrossRef]

106. Lapointe, B.E.; West, L.E.; Sutton, T.T.; Hu, C. Ryther Revisited: Nutrient Excretions by Fishes Enhance Productivity of Pelagic *Sargassum* in the Western North Atlantic Ocean. *J. Exp. Mar. Biol. Ecol.* **2014**, *458*, 46–56. [CrossRef]

107. Doyle, E.; Franks, J. *Sargassum* Factsheet. Gulf and Caribbean Fisheries Institute: Guadeloupe, French, 2015; p. 4. Available online: https://www.gcfi.org/sargassum-influx/ (accessed on 27 February 2024).

108. Wynne, M.J. A Checklist of Benthic Marine Algae of the Tropical and Subtropical Western Atlantic: Fifth Revision Nova Hedwigia Beiheft, 153, Bomtraeger Science Publishers, Stuttgart, Germany; 2022. 180 pp.

109. Govindarajan, A.F.; Cooney, L.; Whittaker, K.; Bloch, D.; Burdorf, R.M.; Canning, S.; Carter, C.; Cellan, S.M.; Eriksson, F.A.A.; Freyer, H.; et al. The Distribution and Mitochondrial Genotype of the Hydroid *Aglaoophenia Latecarinata* Is Correlated with Its Pelagic *Sargassum* Substrate Type in the Tropical and Subtropical Western Atlantic Ocean. *PeerJ* **2019**, *7*, e7814. [CrossRef] [PubMed]

110. Martin, L.M.; Taylor, M.; Huston, G.; Goodwin, D.S.; Schell, J.M.; Siuda, A.N.S. Pelagic *Sargassum* Morphotypes Support Different Rafting Motile Epifauna Communities. *Mar. Biol.* **2021**, *168*, 115. [CrossRef]

111. McGillicuddy, D.J.; Morton, P.L.; Brewton, R.A.; Hu, C.; Kelly, T.B.; Solow, A.R.; Lapointe, B.E. Nutrient and Arsenic Biogeochemistry of *Sargassum* in the Western Atlantic. *Nat. Commun.* **2023**, *14*, 6205. [CrossRef] [PubMed]
112. Samadi, S.; Barberousse, A. Species. In *Handbook of Evolution Thinking in the Sciences*; Heams, T., Huneman, P., Lecointre, G., Silberstein, M., Eds.; Springer: Berlin/Heidelberg, Germany, 2015.

113. Rousseau, F.; De Reviers, B. Phylogenetic Relationships within the Fucales (Phaeophyceae) Based on Combined Partial SSU+LSU rDNA Sequence Data. *Eur. J. Phycol.* **1999**, *34*, 53–64.

114. Motomura, T.; Nagasato, C.; Kimura, K. Cytoplasmic Inheritance of Organelles in Brown Algae. *J. Plant Res.* **2010**, *123*, 185–192.
[CrossRef] [PubMed]

**Disclaimer/Publisher's Note:** The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.
112. Samadi, S.; Barberousse, A. Species. In *Handbook of Evolution Thinking in the Sciences*; Heams, T., Huneman, P., Lecointre, G., Silberstein, M., Eds.; Springer: Berlin/Heidelberg, Germany, 2015.

113. Rousseau, F.; De Reviers, B. Phylogenetic Relationships within the Fucales (Phaeophyceae) Based on Combined Partial SSU+LSU rDNA Sequence Data. *Eur. J. Phycol.* **1999**, *34*, 53–64.

114. Motomura, T.; Nagasato, C.; Kimura, K. Cytoplasmic Inheritance of Organelles in Brown Algae. *J. Plant Res.* **2010**, *123*, 185–192.
[CrossRef] [PubMed]

**Disclaimer/Publisher's Note:** The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.