texto en 
Inglés (pdf)
Artículo en XML
Referencias del artículo
Traducción automática
Enviar artículo por email
Citado por SciELO 
Similares en
SciELO 
Permalink
INTRODUCTION
Symbiodinium Gert Hansen & Daugbjerg is a photosynthetic genus with opportunistic and free-living clades and subclades, which is distributed in tropical and subtropical areas (Takabayashi et al. 2012, Granados-Cifuentes et al. 2015, Hoppenrath et al. 2023). The genus name is considered neutral and means “living together” and “whirling” (LaJeunesse et al. 2018). These organisms have also been called Zooxanthella K. Brandt (Guiry and Andersen 2018). This refers to the mutualistic symbiosis shown by the alga, which in this case is the symbiosis of the dinoflagellate with invertebrates such as cnidarians, clams, copepods, flatworms, sponges, and some protozoans, such as foraminiferans, radiolarians, and ciliates (Baker 2003, Hirose et al. 2008, LaJeunesse et al. 2018). In addition, they are classified as cytobionts or intracellular symbionts (Taylor and Harrison 1983).
Species of the Symbiodiniaceae family (e.g., Symbiodinium microadriaticum LaJeunesse and Symbiodinium pilosum Trench & R.J. Blank ex La Jeunesse) show different photosynthetic responses under identical laboratory conditions. These photosynthetic differences explain the presence of this family in the diverse niches they can occupy (Iglesias-Prieto and Trench 1994). Host-symbiont specificity demonstrates the ability of a symbiont to be specialized for a certain host that inhabits a specific region; in addition, variations in said specificity are shown over wide geographic ranges. Physical (temperature and irradiance) and biological (host diversity and abundance and symbiont diversity and abundance) variables modulate symbiont-host specificity (Iglesias-Prieto et al. 1992, 2004; Iglesias-Prieto and Trench 1994, 1997). In addition to their role as symbionts, extracts of strains of the Symbiodiniaceae family isolated from the anemone Stichodactyla haddoni (Saville-Kent) have been reported to be cytotoxic to the crustacean Artemia salina (Linnaeus) (Bigham-Soostani et al. 2021).
The Symbiodiniaceae family is a diverse group comprising several generic and subgeneric clades, each consisting of an unknown number of subspecies or subclades (Iglesias-Prieto et al. 2004, LaJeunesse et al. 2018). Phylogenetic reconstructions with ribosomal (28S and 23S) and chloroplast (psbA) genes have revealed 9 clades (A to I) (Hirose et al. 2008, Hansen and Daugbjerg 2009, Pochon and Gates 2010, Yamashita and Koike 2013, LaJeunesse et al. 2018). New genera have been proposed with respect to phylogenetic groupings: Symbiodinium Gert Hansen & Daugbjerg (clade A), Breviolum J.E. Parkinson & LaJeunesse (clade B), Cladocopium (clade C), Durusdinium LaJeunesse (clade D), Effrenium LaJeunesse & H.J. Jeong (clade E), Fugacium LaJeunesse (clade F), and Gerakladium LaJeunesse (clade G) (LaJeunesse et al. 2018). Few clades show apparent morphological differences: clade B has cell sizes of 6 to 12 µm, and clade C has an apical groove called acrobase (LaJeunesse et al. 2018).
The strains analyzed in this study belong to clade A, corresponding to the oldest lineage of the Symbiodiniaceae family, which is made up of S. microadriaticum, Symbiodinium necroappetens LaJeunesse, S. Y. Lee, Knowlton & H. J. Jeong, Symbiodinium tridacnidarum S. Y. Lee, H. J. Jeong, N. S. Kang & LaJeunesse, Symbiodinium natans Gert Hansen & Daugbjerg, and Symbiodinium linucheae (Hansen and Daugbjerg 2009, LaJeunesse et al. 2018). Clade A includes free-living species, such as S. pilosum and S. natans (Yamashita and Koike 2013, LaJeunesse et al. 2015), in addition to groups with transient, opportunistic, free-living, and symbiotic forms that can be found associated with invertebrate hosts or protists (LaJeunesse et al. 2018). It can also inhabit different substrates, such as sand (Carlos et al. 1999, Hoppenrath et al. 2023), and form symbioses with soft corals (e.g., Stereonephthya cundabiluensis Verseveldt), stony corals, or hard corals, such as Orbicella faveolata (Ellis & Solander) (Hirose et al. 2008, Kemp et al. 2014).
MATERIALS AND METHODS
Isolation and growing conditions
Cells were isolated from the column of the solitary anemone Actinostella sp. (Hexacorallia: Actinaria: Actiniidae; Fig. 1) that was collected by Ana E Ramos-Santiago on August 9, 2018, at the CETMAR beach (4°08′39.2″ N, 110°20′41.0″ W; Fig. 2), Bahía de La Paz, in the southwestern Gulf of California, Mexico. Non-motile cells were isolated on an AXIO Vert.A1 inverted microscope (Carl Zeiss, Oberkochen, Germany), using capillaries with reduced tips. A progressive escalation was carried out until we obtained 25-mL cultures in flat 50-mL tubes. The SNCETMAR-1 and SNCETMAR-2 strains were kept in GSe medium with vermicompost extract (Bustillos-Guzmán et al. 2015) and K medium (Keller et al. 1987) modified with vermicompost extract at 34 salinity, 24 °C ± 1 °C temperature, continuous 150-μmol E·m-2·s-1 illumination, and a 12 h light:12 h dark cycle.
Figure 1 Symbiodinium-clade A and the host anemone Actinostella sp. Specimen of the anemone Actinostella sp. preserved in 4% formalin, the top view of the collar and oral disc with retracted tentacles is observed (red arrow); lateral view of the spine (blue arrow) (a). Details of live Actinostella sp., coloration is observed, lower detail of the collar with small aligned warts (gray arrow) (b). Lateral view of the spine with large and numerous warts (white arrows) (c). Freshly isolated cells of Symbiodinium sp. (SNCETMAR-2 strain), with green-yellow chloroplasts (ch) and pyrenoid (py) (black arrows) (d).
Morphological identification
Strain identification was done with the help of specialized literature (LaJeunesse et al. 2015, 2018; Hoppenrath et al. 2023). We evaluated morphometry and obtained micrographs of live cells on the Axio Vert.A1 inverted photonic microscope (Carl Zeiss) in bright field. In addition, we used the Axio Scope.A1 epifluorescence microscope (Carl Zeiss) with a 6-megapixel Axiocam 506 color digital camera to observe cells stained with the fluorescent marker DAPI (4',6-diamidino-2-phenylindole; Sigma) and visualize the position and size of the nucleus. To process samples for scanning electron microscopy (SEM), we followed the protocol used by Ramos-Santiago (2023) for naked dinoflagellates. Broadly, the methodology consisted of a prefixation process with 4% glutaraldehyde, postfixation with 2% osmium tetroxide (OsO4), in-between washes to eliminate fixative residues, dehydration with an ethanol gradient (EtOH) at 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and twice at 99%, and a drying process of the samples with hexamethyldisilazane (HMDS). The samples were sent to the Academic Service of Scanning Electron Microscopy (SAMEB) of the Instituto de Ciencias del Mar y Limnología (ICMyL), Universidad Nacional Autónoma de México (UNAM), Mexico City, Mexico.
Molecular identification
DNA extraction and amplification
DNA extraction from the SNCETMAR-1 and SNCETMAR-2 strains was performed using the Quick-DNA Miniprep Plus Universal kit (Zymo Research, USA). For amplification, a mixture of 6.25 µL of DreamTaq Green PCR 2X (Thermo Scientific, USA), 2 µL of milli-Q H2O, 1 µL of each primer (F and R), and 1 µL of DNA was used. Primers for 28S rDNA were used (Hosoi-Tanabe et al. 2006). Amplification conditions consisted of a denaturation step at 95 °C for 5 min, followed by 35 cycles at 95 °C for 1 min, at an annealing temperature of 52 °C for 1 min, 72 °C for 2 min, and, finally, an extension at 72 °C for 7 min. The PCR samples were verified by 1.5% agarose gel electrophoresis with 1X TBE Buffer. The image was digitized under the Bio-Rad ChemiDoc XRS+ system. The PCR products were sent for purification and sequencing to the company Macrogen (Seoul, South Korea). It is important to note that DNA extraction was successful in both strains; however, amplification was only achieved with the SNCETMAR-2 strain.
Phylogenetic analysis
The sequence was edited with the Sequencher program v. 4.1.4. Sequences from the GenBank database were used to perform a BLAST analysis of the consensus sequence (Forward + Reverse). Sequences from the different clades of the Symbiodiniaceae family from the 28S rDNA region were selected for the reconstruction of the phylogenetic trees. Sequence alignment was performed with the MEGA v. 10.0.5 program, with the MUSCLE alignment algorithm. The model that best described the nucleotide substitution rates of the sequences was selected with the JModel test v. 2.1.10, using the general time reversible (GTR) model for the phylogenetic analysis, which was done with the maximum likelihood (ML) and maximum parsimony (MP) algorithms in the MEGA v. 10.0.5 program and Bayesian inference (BI) with the MrBayes program v. 3.2.7a. Trees were constructed with a bootstrap of 1,000 replicates for ML and 3 × 106 generations for BI. The trees were edited with Photoshop CS6 v. 13.1.2.
RESULTS
Symbiodinium-clade A morphology
Solitary cells, reddish in color, with rotating movement, which are distributed at the bottom and on the walls of the culture tube (Fig. 3a, b). Reticulated, brownish, and peripheral chloroplasts (Fig. 3c). The pyrenoid is located in the central area of the cell and has a diameter of 2.65 ± 0.52 µm (Fig. 3c-e). The mastigote (motile) cells are mushroom-shaped, laterally compressed, with the epicone slightly larger than the hypocone (Fig. 3c-i). Motile cells measure 10.93 ± 0.98 µm long and 7.79 ± 1.06 µm wide (mean ± SD; n = 30) in strain SNCETMAR-1 (Table S1), and 11.78 ± 1.05 µm long and 8.02 ± 1.38 µm wide (mean ± SD; n = 30) in the SNCETMAR-2 strain (Table S1). The nucleus is located in the anterior part of the cell (Fig. 3h, l). Another morphotype observed were coccoid non-motile cells, with a diameter of 12.01 ± 0.83 µm (mean ± SD; n = 30; Fig. 3j-l). The non-motile morphotype lacks morphological characteristics typical of motile cells.
Figure 3 Subclade Symbiodinium natans-clade A from Bahía de La Paz. SNCETMAR-2 strain in GSe medium (a). SNCETMAR-1 strain (c-f). SNCETMAR-2 strain (b, g-l). Coccoid cells, non-motile, grouped together by mucilage (b). Ventral view of motile cells with the typical mushroom shape (c-e). Rounded and elongated reproductive cysts, they show reserve substances or an eye spot (white arrows) (f). Non-motile mushroom-shaped cells and coccoid state (g, j). DAPI staining shows the position and shape of the nucleus (n) in the 2 phases of the life cycle of Symbiodinium sp. (h, l). Epifluorescence images of chloroplasts (i, k). ch = chloroplasts, py = pyrenoid, lf = longitudinal flagellum.
We documented 2 life forms in both strains of Symbiodinium, motile cells and non-motile cells in the coccoid state, the latter being the dominant phase. Non-motile cells were observed in pre-division (Fig. 4a-c). Different phases of the division processes were documented: dyads with duplicated pyrenoids and the presence of one accumulation body and, on the other hand, cells with 2 accumulation bodies, which could indicate the fusion of 2 gametes (Fig. 4c-f). Cells forming clusters in triads and tetrads (Fig. 3g-j) and the presence of zygotes formed by fusion (syngamy) of gametes (Fig. 4k, l) were also observed. In coccoid cells in the process of reproduction, red-orange accumulation bodies were observed; these are probably lipids used as reserve substances or an eyespot (Fig. 5).
Figure 4 Symbiodinium natans-clade A cysts from Bahía de La Paz. SNCETMAR-1 strain (e, h, j-l). SNCETMAR-2 strain (a, b, c, d, f, g, i). Non-motile predivision cyst or coccoid stage (a-c). Bicellular division cyst (dyad phase) (d-f). Meiosis I, triad phase (g-i). Meiosis II, tetrad phase, where 4 haploid cells are formed (j). Zygote (diploid) (k, l). White arrow with margin = reserve substances (lipids) or an eyespot, asterisk (*) = binary fission process, py = pyrenoid.
Figure 5 Symbiodinium-clade A cells (SNCETMAR-2 strain) in epifluorescence microscope. Optical micrographs of non-motile cysts (a, d, g, i, k). Chloroplast autofluorescence in ventral view (b, c, f, j). DAPI staining of the nucleus (n) (e, h, l). White arrow with margin = lipids as storage products or eyespot, red arrow = division plane.
With SEM, the morphology of motile cells and the aforementioned reproductive stages were corroborated, as well as the cingulum, which is descending and displaced approximately one width of the cingulum (Fig. 6). We were unable to observe the arrangement of amphiesmal plates with this technique.
Figure 6 Vegetative cells of Symbiodinium natans-clade A observed under a scanning electron microscope. SNCETMAR-1 strain (b, c, g, h). SNCETMAR-2 strain (a, d, e, f, i, j). Vegetative cysts, the dominant phase of Symbiodinium sp. (a). Ventral view (b). Dorsal view (c, e). Zoosporangium with cingulum housing the transverse flagellum (d). Binary fission process (f, g). Meiosis I (h, i). Meiosis II (j). ci = cingulum, su = sulcus, ep = epicone, hy = hypocone.
Molecular identification
We obtained a 514 bp sequence of the 28S rDNA region of strain SNCETMAR-2 (PP563703). In BLAST analyses performed in this study, the sequence showed 100% identity with the OQ449283 sequence, identified as S. natans. However, efforts were made to ensure that the selected sequences came from published studies or subsequent research in which their molecular identification has been corroborated. For the phylogenetic reconstruction of this taxonomic group, we included sequences from the 9 clades accepted for the Symbiodiniaceae family (Table 1). The sequence from this study clustered within clade A, with bootstrap support values of 91 and 97 for MP and ML, and posterior probability of 0.98 with BI (Fig. 7). The SNCETMAR-2 strain sequence formed a subclade with S. natans sequences with bootstrap support values between 70 and 89 (Fig. 7). We compared 2 S. natans sequences (AB704055 and AB704058) with the SNCETMAR-2 sequence; the AB704055 sequence showed a transition (C to T) at position 416, whereas in the AB704058 sequence there is a transition (A to G) at position 461. The analysis of the pairwise genetic divergences between the sequences identified as clade A revealed low values, ranging between 0.025 and 0.032. In contrast, divergences with sequences from other clades were greater than 0.141 (Table 2).
Table 1 Sequences of the Symbiodiniaceae family included in the phylogenetic analysis of the 28S rDNA region and Gymnodinium catenatum, which was used as an outgroup.
| GenBank ID | Taxon | Strain | Isolation origin | Site | Reference |
| PP563703 | Symbiodinium natans | SNCETMAR-2 | Actinostella sp. | Bahía de La Paz, Mexico | This study |
| AB704058 | Symbiodinium natans | FGS-D6-Sy | Sand | Gahi Island, Okinawa, Japan | Yamashita and Koike (2013) |
| AB704055 | Symbiodinium natans | GTP-A6-Sy | Tide pool | Gahi Island, Okinawa, Japan | Yamashita and Koike (2013) |
| EU315917 | Symbiodinium natans | CAT2393 | ND | Tenerife, Spain | Hansen and Daugbjerg (2009) |
| KT634312 | Symbiodinium sp. | zs12xd | Zoanthus sp. | Tavernier, Florida Keys, USA | Graham et al. (2015) |
| LK934674 | Symbiodinium sp. | CCMP2456 | Alveopora japonica Eguchi, 1965 | Jeju, South Korea | Not published |
| AB778578 | Symbiodinium sp. | KMG004-a-02 | Pteraeolidia ianthina (Angas, 1864) | Chiba, Japan | Yorifuji et al. (2015) |
| KF740671 | Symbiodinium pilosum | rt-185 | Zoanthus sociatus (Ellis, 1768) | Jamaica | Jeong et al. (2014) |
| ON263282 | Symbiodinium necroappetens | A13 | ND | ND | Not published |
| MK692538 | Symbiodinium linucheae | SSA01 | ND | ND | Not published |
| KM972549 | Symbiodinium microadriaticum | rt-061 | Cassiopea xamachana Bigelow, 1892 | Florida, USA | Lee et al. (2015) |
| LK934669 | Symbiodinium microadriaticum | CCMP2467 | Alveopora japonica | Jeju, South Korea | Not published |
| KT149349 | Symbiodinium minutum | Mf1.05b | ND | Caribbean | Parkinson et al. (2015) |
| LK934670 | Symbiodinium minutum | CCMP830 | Alveopora japonica | Jeju, South Korea | Not published |
| KT149351 | Symbiodinium psygmophilum | PurPFlex | ND | Caribbean | Parkinson et al. (2015) |
| KF364606 | Symbiodinium sp. | RCC 1521 | Surface net trawl | Blanes, Mediterranean Sea | Jeong et al. (2014) |
| AF060899 | Gymnodinium varians | CCMP 421 | ND | ND | Wilcox (1998) |
| AJ291539 | Symbiodinium sp. | 1584 | Amphisorus sp. | Guam | Pawlowski et al. (2001) |
| AJ291536 | Symbiodinium sp. | 1643 | Marginopora | Luminao, Guam | Pawlowski et al. (2001) |
| KF740689 | Symbiodinium sp. | MTB4 | Orbicella faveolata (Ellis & Solander, 1786) | USA | Jeong et al. (2014) |
| KF740686 | Symbiodinium sp. | Tha09-57 | Oulastrea crispata (Lamarck, 1816) | Thailand | Jeong et al. (2014) |
| FN561562 | Symbiodinium sp. | nr-i4 | Foraminífera subfamily Soritinae Ehrenberg | Oahu, Hawaii, USA | Pochon and Gates (2010) |
| AJ291513 | Symbiodinium sp. | 751 | Sorites sp. | Florida, USA | Pawlowski et al. (2001) |
| KF740682 | Symbiodinium sp. | Zam03-3m-83 | Millepora Linnaeus, 1758 | Japan | Jeong et al. (2014) |
| FJ529530 | Symbiodinium sp. | C3nt | Seriatopora hystrix Dana, 1846 | Australia | Sampayo et al. (2009) |
| AJ830916 | Symbiodinium sp. | MS26_5244x | Amphisorus hemprichii Ehrenberg, 1965 | Guam Island | Not published |
| AJ291525 | Symbiodinium sp. | 1635 | Marginopora sp. | Piti, Guam | Pawlowski et al. (2001) |
| KU359161 | Symbiodinium kawagutii | symka | ND | ND | Not published |
| AF360577 | Symbiodinium kawagutii | Clade C | Montipora verrucosa (Lamarck, 1816) | Hawaii, USA | Santos et al. (2001) |
| SD | Gymnodinium catenatum | BAPAZ 16 | ND | Bahía de La Paz, BCS, Mexico | Not published |
| +ND: No data. | |||||
Table 2 Pairwise genetic distances based on 13 Symbiodiniaceae family sequences selected from the 28S rDNA region. The genetic divergences observed between the sequence of this study (*) belonging to clade A and the other clades are shown in bold. The letters A-I indicate the clades of the family Symbiodiniaceae.
| A | B | C | D | E | F | G | H | I | Symbiodinium kawagutii | |||||
| KF740671 | PP563703* | KT634312 | AB778578 | KT149349 | FJ529530 | KF740689 | AF060899 | AJ830916 | AJ291539 | AJ291513 | FN561562 | AF360577 | ||
| A | KF740671 | |||||||||||||
| PP563703* | 0.025 | |||||||||||||
| KT634312 | 0.028 | 0.014 | ||||||||||||
| AB778578 | 0.032 | 0.018 | 0.004 | |||||||||||
| B | KT149349 | 0.237 | 0.240 | 0.240 | 0.244 | |||||||||
| C | FJ529530 | 0.208 | 0.208 | 0.212 | 0.216 | 0.134 | ||||||||
| D | KF740689 | 0.198 | 0.219 | 0.219 | 0.216 | 0.208 | 0.170 | |||||||
| E | AF060899 | 0.155 | 0.141 | 0.155 | 0.159 | 0.216 | 0.187 | 0.194 | ||||||
| F | AJ830916 | 0.212 | 0.212 | 0.216 | 0.219 | 0.120 | 0.092 | 0.198 | 0.201 | |||||
| G | AJ291539 | 0.265 | 0.265 | 0.265 | 0.261 | 0.226 | 0.184 | 0.177 | 0.233 | 0.216 | ||||
| H | AJ291513 | 0.187 | 0.191 | 0.194 | 0.198 | 0.124 | 0.049 | 0.159 | 0.180 | 0.085 | 0.184 | |||
| I | FN561562 | 0.205 | 0.212 | 0.219 | 0.219 | 0.159 | 0.134 | 0.177 | 0.205 | 0.141 | 0.201 | 0.127 | ||
| Symbiodinium kawagutii | AF360577 | 0.449 | 0.452 | 0.449 | 0.452 | 0.484 | 0.470 | 0.473 | 0.466 | 0.491 | 0.516 | 0.459 | 0.456 | |
Figure 7 Symbiodinium phylogenetic tree of the 28S rDNA region. The sequence of the SNCETMAR-2 strain (PP563703) from this study is shown in bold. The analysis was deduced using the method of maximum parsimony (MP), maximum likelihood (ML), and Bayesian inference (BI). The percentage of bootstrap support values and posterior probability of the clades and subclades are shown in the nodes. The analysis included 30 sequences and analyzed partial sequences of 558 bp. Sequences selected for genetic divergence analysis in Table 2 are indicated with asterisks. ND = no data.
DISCUSSION
The size of motile S. natans-clade A cells can range from 9.5 to 11.5 µm in length and 7.4 to 9 µm in width (Hansen and Daugbjerg 2009, LaJeunesse et al. 2015, Lee et al. 2015, Guiry and Guiry 2024), which agrees with what was reported in this study. The subclades S. necroappetens and S. microadriaticum show similar morphologies; nonetheless, these have cell sizes ranging from 9 to 12 µm (larger than S. natans) and from 7 to 10 µm (smaller than S. natans), respectively (LaJeunesse et al. 2015).
Currently, morphological and molecular identification within the Symbiodiniaceae family is complex. The number, shape, and position of the amphiesmal plates are used as descriptive morphological characteristics for the group; however, these are not sufficient for specific identification, as they may be similar or different within and between the groups that make up the clades (Lee et al. 2015, LaJeunesse et al. 2018). On the other hand, the morphological information of the amphiesmal plates in the motile stage (mastigote) of distantly related clades can yield different morphological information, as in clades A and E (Lee et al. 2015). The shape and size of the pyrenoid, chloroplasts, and nucleus have been used as morphological characters; however, in the Symbiodiniaceae family these characters are shared and cannot be used to differentiate clades or subclades (Lee et al. 2015). There is only one autapomorphy character, which is the reduction of a pronounced elongated apical vesicle (acrobase or apical groove), a characteristic observed in clade C (LaJeunesse et al. 2018). Nevertheless, members belonging to clade A lack this character, so this structure is not relevant in the strains analyzed in this study.
The morphology of the group can vary depending on the phase, with an observable coccoid phase, typical of asexual reproduction, and ellipsoidal to mushroom shapes (motile phase). Coccoid cells can measure 8-10 µm in diameter (LaJeunesse et al. 2018), whereas motile cells can average 6-12 µm in length (Hansen and Daugbjerg 2009, LaJeunesse et al. 2018). In culture, cells were observed forming groups in triads and tetrads, corresponding to meiosis I and late meiosis II of sexual reproduction, respectively. Figueroa et al. (2021) reported these reproductive phases in a Symbiodinium strain of clade C (Cladocopium latusorum Turnham, Sampayo & LaJeunesse) from Moorea in French Polynesia, South Pacific Ocean.
Phylogenetic reconstructions with ribosomal (28S and 23S) and chloroplast (psbA) genes reveal 9 evolutionarily divergent clades (A to I) (seeTable 2, Pochon and Gates 2010, LaJeunesse et al. 2018). The high and low pairwise genetic divergence values in this study were very similar to those of Pochon and Gates (2010) because we included some long subunit sequences identified by these authors as clade E (AF060899), clade F3 (AJ830916, AJ291525), clade G (AJ291539), clade H (AJ291513), and clade I (FN561562). The different clades of the Symbiodiniaceae family have different genetic, physiological, and ecological attributes; therefore, these clades can be subdivided into an unknown number of phylospecies (Hirose et al. 2008, De Palmas et al. 2015, LaJeunesse et al. 2018). The 9 clades accepted for the group were obtained with the analysis of the 28S region. The sequence from this study had greater genetic affinity with sequences from clade A; specifically, with sequences from the free-living species S. natans from Japan and Spain (Yamashita and Koike 2013, LaJeunesse et al. 2015). Although this clade shows high genetic diversity (around 15 subclades), the 28S marker helped us identify clade A and the presence of 4 subclades within it; however, a highly variable marker such as the ITS set has been observed to help identify specificities with respect to the environments where these microorganisms develop (pelagic and benthic) and their symbiotic associations to understand the divergence within the same clade or group (Mordret et al. 2016).
Clade A is widely distributed in the Atlantic, Pacific, and Indian oceans and in the Red Sea. Nevertheless, some subclades may be limited to certain ocean basins, for example, the Caribbean Sea, which has the highest number of reports (LaJeunesse et al. 2015). The clade has been reported in Callao Salvaje, Tenerife, in the Canary Islands (Hansen and Daugbjerg 2009, Guiry and Guiry 2024); Japan and Hawaii (Carlos et al. 1999, Hirose et al. 2008, Yamashita and Koike 2013); Florida Keys in the USA (Lee et al. 2015); and Puerto Morelos in the Mexican Caribbean (Kemp et al. 2014).
In the southern Gulf of California, studies report clade C (abundant and widely distributed) associated with Pavona gigantea (Verrill) and clade D (extremophiles, its distribution centered in the Indo-West Pacific) with Pocillopora verrucosa (Ellis & Solander) (Iglesias-Prieto et al. 2004, LaJeunesse et al. 2018, Méndez-Méndez 2020). This study expands the list of Symbiodinium clades in the Gulf of California and describes the presence of clade A associated with the anemone Actinostella sp.
Usually, Symbiodinium clade A is associated with shallow water corals in the Caribbean and tidal pools, which generated the hypothesis that this clade is adapted to shallow areas (less than 1 m) due to the presence of photoacclimation and photoprotection pathways that counteract the high irradiance and high temperatures that can occur in these environments (Iglesias-Prieto and Trench 1997, Hirose et al. 2008, Takahashi et al. 2009, Yamashita and Koike 2013, Kemp et al. 2014). The results of the present study agree with this hypothesis, since the dinoflagellate was isolated in Bahía de La Paz, at a depth of less than 1 m (benthic habitat), in an area with high irradiance and high temperatures, which can reach 27 to 32 °C in the summer (Sea temperature 2024).
Clade A has been shown to be one of the easiest to culture due to its physiological and ecological characteristics and can occur in non-symbiotic, free-living form. This study provides information on the laboratory culture of symbiotic dinoflagellates from clade A from Bahía de La Paz; these strains have been maintained since 2018 to the present and can be cultured in specialized media (e.g., ASP-8A) and conventional media used for planktonic and benthic dinoflagellates, such as IMK, L1, modified GSe, and modified K media (this study, Hirose et al. 2008, LaJeunesse et al. 2015, Lee et al. 2015).
CONCLUSIONS
The results of the morphometric analyses combined with the phylogenetic analysis are conclusive for the Symbiodinium-clade A taxon. The phylogenetic analysis of the 28S region showed 9 clades currently accepted for the Symbiodiniaceae family. Clade A was divided into different subclades of phylospecies, showing that the PP563703 sequence of strain SNCETMAR-2 has phylogenetic affinity with sequences from S. natans (currently accepted taxon). This study reported the first detailed description of S. natans-clade A for Bahía de La Paz, Gulf of California; this dinoflagellate alternates between 2 life phases, a free-living (planktonic) phase and a non-obligate symbiotic phase (benthic) that showed an association with the sea anemone Actinostella sp.
